The PF4/PPBP/CXCL5 Gene Cluster Is Associated with Periodontitis.

Periodontitis is a common dysbiotic inflammatory disease with an estimated heritability of 50%. Due to the limited sample size of available periodontitis cohorts and the underlying trait heterogeneity, genome-wide association studies (GWAS) of chronic periodontitis (CP) have been unsuccessful in discovering susceptibility factors. A strategy that combines agnostic GWAS with a well-powered candidate-gene approach has the potential to discover novel loci. We combined RNA-seq data from gingival tissues with quantitative trait loci (QTLs) that were identified in a F2-cross of mice resistant and susceptible to infection with oral bacterial pathogens. Four genes, which were located within the mapped QTLs, showed differential expression. The chromosomal regions across the human orthologous were interrogated for putative periodontitis-associated variants using existing GWAS data from a German case-control sample of aggressive periodontitis (AgP; 651 cases, 4,001 controls), the most severe and early onset form of periodontitis. Two haplotype blocks, one upstream to the coding region of UGT2A1 (rs146712414, P = 9.1 × 10-5; odds ratio [OR], 1.34; 95% confidence interval [CI], 1.16-1.56) and one downstream of the genes PF4/PPBP/CXCL5 (rs1595009, P = 1.3 × 10-4; OR, 1.32; 95% CI, 1.15-1.52), were associated with AgP. The association of rs1595009 was validated in an independent cohort of CP of European Americans (1,961 cases and 1,864 controls; P = 0.03; OR, 1.45; 95% CI, 1.01-1.29). This association was further replicated in another sample of 399 German CP cases (disease onset <60 y of age) and 1,633 controls ( P = 0.03; OR, 1.75; 95% CI, 1.06-2.90). The combined estimates of association from all samples were P = 2.9 × 10-5 (OR, 1.2; 95% CI, 1.1-1.3). This study shows the strength of combining QTL mapping and RNA-Seq data from a mouse model with association studies in human case-control samples to identify genetic risk variants of periodontitis.

Vaccination with recombinant RgpA peptide protects against Porphyromonas gingivalis-induced bone loss.

OBJECTIVE: Following Porphyromonas gingivalis infection in mice, the efficacy of vaccination by recombinant and native RgpA in modulating the early local anti-inflammatory and immune responses and periodontal bone loss were examined. MATERIAL AND METHODS: Using the subcutaneous chamber model, exudates were analyzed for cytokines after treatment with native RgpA and adjuvant (test), or adjuvant and saline alone (controls). Mice were also immunized with recombinant RgpA after being orally infected with P. gingivalis. After 6 wk, serum was examined for anti-P. gingivalis IgG1 and IgG2a titers and for alveolar bone resorption. RESULTS: Immunization with native RgpA shifted the immune response toward an anti-inflammatory response as demonstrated by decreased proinflammatory cytokine IL-1ß production and greater anti-inflammatory cytokine IL-4 in chamber exudates. Systemically, immunization with recombinant RgpA peptide prevented alveolar bone loss by 50%, similar to immunization with heat-killed whole bacteria. Furthermore, recombinant RgpA shifted the humoral response toward high IgG1 and low IgG2a titers, representing an in vivo anti-inflammatory response. CONCLUSIONS: The present study demonstrates the potential of RgpA to shift the early local immune response toward an anti-inflammatory response while vaccination with recRgpA protected against P. gingivalis-induced periodontitis.

Host susceptibility to periodontitis: mapping murine genomic regions.

Host susceptibility to periodontal infection is controlled by genetic factors. As a step toward identifying and cloning these factors, we generated an A/J x BALB/cJ F2 mouse resource population. A genome-wide search for Quantitative Trait Loci (QTL) associated with periodontitis was performed. We aimed to quantify the phenotypic response of the progenies to periodontitis by microCT analysis, to perform a genome-wide search for QTL associated with periodontitis, and, finally, to suggest candidate genes for periodontitis. We were able to produce 408 F2 mice. All mice were co-infected with Porphyromonas gingivalis and Fusobacterium nucleatum bacteria. Six weeks following infection, alveolar bone loss was quantified by computerized tomography (microCT) technology. We found normal distribution of the phenotype, with 2 highly significant QTL on chromosomes 5 and 3. A third significant QTL was found on chromosome 1. Candidate genes were suggested, such as Toll-like receptors (TLR) 1 and 6, chemokines, and bone-remodeling genes (enamelin, ameloblastin, and amelotin). This report shows that periodontitis in mice is a polygenic trait with highly significant mapped QTL.

Evaluation of plant and fungal extracts for their potential antigingivitis and anticaries activity.

The link between diet and health has lead to the promotion of functional foods which can enhance health. In this study, the oral health benefits of a number of food homogenates and high molecular mass and low molecular mass fractions were investigated. A comprehensive range of assays were performed to assess the action of these foods on the development of gingivitis and caries using bacterial species associated with these diseases. Both antigingivitis and anticaries effects were investigated by assays examining the prevention of biofilm formation and coaggregation, disruption of preexisting biofilms, and the foods' antibacterial effects. Assays investigating interactions with gingival epithelial cells and cytokine production were carried out to assess the foods' anti- gingivitis properties. Anti-caries properties such as interactions with hydroxyapatite, disruption of signal transduction, and the inhibition of acid production were investigated. The mushroom and chicory homogenates and low molecular mass fractions show promise as anti-caries and anti-gingivitis agents, and further testing and clinical trials will need to be performed to evaluate their true effectiveness in humans.

Involvement of autoimmunity in the pathogenesis of aggressive periodontitis.

The aim of this study was to investigate the involvement of autoimmune reactions to native and post-translationally modified extracellular matrix components in the pathogenesis of periodontitis. Sera from individuals with aggressive periodontitis (AgP, n = 25), chronic periodontitis (CP, n = 14), and gingivitis (G, n = 18) were tested for the presence of autoantibodies against: (a) native collagen type I (CI) and collagen type III (CIII); (b) CI and CIII post-translationally modified by reactive oxygen species (ROS) of the type present during inflammation; and (c) citrullinated filaggrin-derived peptides (CCP). Autoantibodies to native and ROS-modified CI and CIII as well as autoantibodies to CCP were observed exclusively in patients with AgP and not in those with CP or G. In conclusion, autoimmune reactions to native and post-translationally modified self-antigens may play a role specifically in the pathogenesis of AgP.

IL-10 gene transfer attenuates P. gingivalis-induced inflammation.

IL-10 is an anti-inflammatory cytokine secreted by stimulated Th2 lymphocytes that can down-regulate inflammatory responses to bacterial challenge. We hypothesized that local delivery of IL-10 using gene-transfer will down-regulate inflammatory responses. We examined the effect of IL-10 plasmid injection on the local cytokine response. Two weeks after the implantation of chambers, either IL-10 plasmid or vector was injected into the mice. Four days later, they were challenged with an intra-chamber injection of P. gingivalis. The intra-chamber levels of IL-10, IFNgamma, TNFalpha, and IL-1beta were evaluated after 2 and 24 hrs. The results showed that local IL-10 gene delivery elevated the levels of IL-10 at both time periods. It attenuated the levels of IFNgamma (656 +/- 154 to 218 +/- 144 pg/mL) and TNFalpha (23 +/- 2.0 to 12.5 +/- 2.9 ng/mL) at 2 hrs, and of IL-1beta (21.5 +/- 5.7 to 12.4 +/- 3.0 ng/mL) at 24 hrs. The results suggest the possibility of modulating the local inflammatory response to P. gingivalis by direct IL-10 gene transfer.

[Hi-tech trends in future dentistry--part A: computerized technologies].

Growing knowledge and information in the field of dentistry has had a great impact on dental education, management and practice. A prominent change is the digital revolution, which presents new computed technologies and accessible means of communication for sharing written and visual information. Biotechnology, yet another innovated field, will change dentistry even further. This article presents dental innovations forwarded by Israeli Hi-Tech companies. An overview of some advances in technology in the field of dentistry is presented. Part of the described technologies is in R&D phase, part is under preliminary clinical evaluation, and a few have undergone short-term clinical studies. Part A of the article discusses the application of computerized imaging systems to educational purposes, diagnostics and treatment; part B presents other diagnostic and therapeutic technologies.

[Hi-tech trends in future dentistry--Part B: diagnostic and therapeutic technologies].

The technology advance and the growing amount of knowledge had a great impact on dental practice for the last decade. The prominent change began with digital revolution presenting new computed technologies and accessible communicational means for sharing literal and imaging information. Following toward the coming biotechnological revolution, dentistry will be even further changed. This article presents dental innovations of Israeli Hi-Tech companies, sorted into two groups. Part A of the article discussed the area of computed imaging systems for educational purposes, diagnostic and treatment. While part B presents other diagnostic or therapeutic technologies. However, because some of the described technologies are still on their R&D (Research and Development) phases, they are not commercialized yet in the market.

Cranberry juice constituents affect influenza virus adhesion and infectivity.

Cranberry juice contains high molecular weight materials (NDM) that inhibit bacterial adhesion to host cells as well as the co-aggregation of many oral bacteria. Because of its broad-spectrum activity, we investigated NDM's potential for inhibiting influenza virus adhesion to cells, and subsequent infectivity. Hemagglutination (HA) of red blood cells (RBC) caused by representatives of both influenza virus A subtypes (H1N1)and H3N2) and the B type was inhibited by NDM at concentrations of 125 microg/ml or lower, which is at least 20-fold lower than that usually found in cranberry juice. A dose-response effect of NDM on HA was demonstrated. The infectivity of the A and B types was significantly reduced by preincubation with NDM (250 microg/ml), as reflected by the lack of cytopathic effect on Madine-Darby canine kidney (MDCK) cells and the lack of HA activity in the media of infected cells. The effect of NDM was also tested after A or B type viruses were allowed to adsorb to and penetrate the cells. Various levels of reduction in virus tissue culture infective dose TCID50 were observed. The effect was most pronounced when NDM was added several times to the infected MDCK cells. Our cumulative findings indicate that the inhibitory effect of NDM on influenza virus adhesion and infectivity may have a therapeutic potential.

Interferon-gamma deficiency attenuates local P. gingivalis-induced inflammation.

Infection with the periodontal pathogen Porphyromonas gingivalis causes a strong local inflammatory reaction. Using IFNgamma-deficient mice, we tested the hypothesis that the absence of IFNgamma would result in a reduction of the local pro-inflammatory response to P. gingivalis. Cytokine secretion by macrophages from IFNgamma(-/-) animals was significantly attenuated. Addition of IFNgamma restored cytokine secretion. In vivo injection of P. gingivalis into subcutaneous chambers increased the intra-chamber leukocyte counts and TNFalpha and IL-1beta levels. This increase was significantly lower in the IFNgamma(-/-) mice. Local reconstitution of IFNgamma(-/-) mice at the site of inflammation with the IFNgamma gene increased the levels of TNFalpha and decreased the IL-10 levels. Anti-P. gingivalis IgG1 levels, a marker of Th2 response, were higher in immunized IFNgamma(-/-) than in IFNgamma(+/+) mice. The results suggest that lack of IFNgamma reduced the amplitude of the local pro-inflammatory response without decreasing the humoral protective response. The higher IgG1/IgG2a ratio observed supports the possibility of a Th2-dominant response in IFNgamma-deficient animals.

Immunization to Porphyromonas gingivalis enhances the local pro-inflammatory response to subcutaneous bacterial challenge.

BACKGROUND, AIMS: Human and animal studies have suggested that immunization to P. gingivalis might be beneficial for controlling periodontitis, by the induction of protective antibody response. The present study was designed to examine the effect of immunization on the local cellular, cytokine and antibody response to P. gingivalis in mice. METHODS: Subcutaneous chambers were implanted in 3 groups of mice. 2 groups were then immunized with P. gingivalis in either incomplete Freund's (IFA) or an Alum-based adjuvant. The 3rd group served as the control. At baseline, all mice were challenged with an intra-chamber injection of P. gingivalis. Chamber exudates were sampled at baseline, 1 and 7 days post-challenge, following by determination of leukocyte counts and the cytokines TNF-alpha, IFNgamma (pro-inflammatory) and IL-10 (anti-inflammatory). IgG levels to P. gingivalis were analyzed in both the exudates and serum. RESULTS: Leukocyte accumulation increased in the chambers over the study period and was more marked in the immunized groups. P. gingivalis challenge induced the expression of the tested cytokines in all groups. Levels of IFNgamma showed a significantly greater increase in the immunized groups on day 1 post-challenge. By day 7, the levels in the controls had reached those of the immunized groups. IL-10 levels were significantly higher in the control group compared to the immunized groups on day 1 and by day 7 they were reduced significantly in all groups to barely detectable levels. While there were no significant differences in TNF-alpha levels between IFA and control groups, they were significantly higher in the Alum group on day 0 and 7. Both immunization protocols induced anti-P. gingivalis IgG. The Alum group achieved the highest antibody levels, which were due to the increased expression of IgG1, a marker of a Th2-response. CONCLUSIONS: The results suggest that immunization to P. gingivalis results in enhanced expression of pro-inflammatory, tissue-destructive cytokines in the inflammatory site. The nature of the adjuvant used for immunization allows manipulation of the T-cell response, and alum was more effective in reducing the inflammatory response than IFA.

Repeat bacterial challenge in a subcutaneous chamber model results in augmented tumour necrosis factor-alpha and interferon-gamma response, and suppression of interleukin-10.

The present study compared the effect of a single or a repeat challenge with the Gram-negative pathogen Porphyromonas gingivalis on the local inflammatory response within subcutaneous chamber model in mice. Subcutaneous chambers were implanted 2 weeks prior to the final challenge. The repeat-challenge (REP) group received two intrachamber bacterial injections 14 days apart, while the single-injection group (SIN) received only a single bacterial challenge. Injection of saline was used as the control. The cellular contents of the chamber exudates were used for differential cell counts, and the supernatants were analysed for tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), and interleukin (IL)-10 levels. Immunoglobulin G1 (IgG1) and IgG2a levels to P. gingivalis in the exudates were also determined. The results showed that the leucocyte counts increased significantly post-challenge, and the REP group showed the highest number of lymphocytes and neutrophils. Both P. gingivalis-challenged groups exhibited significant increase in TNF-alpha and IL-10 levels at day 1 post-challenge. TNF-alpha levels in the chamber exudate were threefold higher in the REP group compared with the SIN group on day 1 post-challenge (P < 0.05). In contrast, IL-10 levels were significantly lower in the REP group 1 day post-challenge compared with the SIN group. The REP group had significantly higher levels of IFN-gamma at baseline, and this difference remained significant 1 day post-challenge. Analysis of antibody levels to P. gingivalis showed that while the control and the SIN groups had no anti-P. gingivalis IgG in the chamber exudate during the 7-day study period, the REP group showed high anti-P. gingivalis IgG levels. In addition, the titres of IgG2a were fivefold higher than the IgG1 titres. The results showed that a repeat local challenge with P. gingivalis augmented the proinflammatory cytokines TNF-alpha and IFN-gamma, while inhibiting the accumulation of the anti-inflammatory cytokine IL-10. This shift towards a T helper 1 (Th1)-dominant response was reflected in the relatively high anti-P. gingivalis IgG2a titres in the local inflammatory environment 7 days post-challenge.

The effect of stress on the inflammatory response to Porphyromonas gingivalis in a mouse subcutaneous chamber model.

BACKGROUND: The impact of emotional stress on the outcome of infectious diseases was studied in animal models and humans, but data related to the effect of stress on periodontal infection are limited. Using the subcutaneous chamber model in mice, the present study was carried out to investigate the effect of stress on the host response to Porphyromonas gingivalis. METHODS: Mice with subcutaneous chambers (2 per animal) were divided into 4 treatment groups: cold-stress; isolation-stress; corticosterone (CS)-injected; and controls. On the third day of stress conditions, heat-killed P. gingivalis were injected into the chambers. The chambers were sampled 1 and 5 days later and analyzed for leukocyte number, tumor necrosis factor (TNF)-alpha levels, and interferon (IFN)-gamma levels. RESULTS: Injection of P. gingivalis induced the migration of leukocytes into the chambers and increased the intrachamber levels of IFN-gamma and TNF-alpha. There were no significant differences in cell number and IFN-gamma levels between the different treatment groups, but the levels of TNF-alpha were significantly lower in the isolation-stress and cold-stress groups compared to control animals. CS-injected animals were not different from controls. In addition, the levels of TNF-alpha in the stressed animals were lower on the fifth day post-injection than on the first day, but not in the CS and control group. CONCLUSIONS: The results suggest that the levels of TNF-alpha induced by P. gingivalis in the infection site are downregulated in stressed animals, and CS is not the sole mediator responsible. The stress-induced reduction in TNF-alpha levels might have an impact on the pathogenesis of periodontal disease in humans experiencing emotional stress.