IL-10 Dampens an IL-17-Mediated Periodontitis-Associated Inflammatory Network.

Emerging evidence suggests comprehensive immune profiling represents a highly promising, yet insufficiently tapped approach to identify potentially prognostic signatures for periodontitis. In this report, we agnostically identified a periodontitis-associated inflammatory expression network with multiple biomarkers identified within gingival crevicular fluid samples from study participants by applying principal component analysis. We identified an IL-17-dominated trait that is associated with periodontal disease and is inversely modified by the level of IL-10. IL-10 mitigated chemokine CXCL5 and CXCL1 expressions in IL-17-stimulated peripheral blood monocytic cells and peripheral blood monocytic cell-derived macrophages. Il10-deficient mice presented more bone loss, which was associated with more Il17 and IL-17-mediated chemokine and cytokine expression at the transcriptional levels in comparison with control wild-type mice in both the Porphyromonas gingivalis-induced experimental murine periodontitis and ligature-induced alveolar bone-loss models. The dampening effect of IL-10 on the excessive signaling of IL-17 appeared to be mediated by innate immune cells populations rather than by gingival epithelial cells, which are the major cell target for IL-17 signaling. Additionally, elevated IL-17 response in Il10-deficient mice specifically elicited an M1-skewing macrophage phenotype in the gingiva that was associated with the advanced bone loss in the ligature model. In summary, IL-17 dominated an inflammatory network characteristic of periodontitis, and IL-10 dampens this excessive IL-17-mediated periodontitis trait.

Impaired function of epithelial plakophilin-2 is associated with periodontal disease.

BACKGROUND AND OBJECTIVES: Plakophilin-2 (PKP2) is an intracellular desmosomal anchoring protein that has been implicated in a genome-wide association study, in which genetic variants of PKP2 are associated with Porphyromonas gingivalis (P.gingivalis) -dominant periodontal dysbiosis. In this study, we compared the ex vivo PKP2 expression in periodontitis gingival biopsies to periodontitis-free subjects and assessed the in vitro role of PKP2 in gingival epithelial barrier function and the mechanism by which P.gingivalis modulates PKP2 expression. MATERIAL AND METHODS: Using reverse transcription quantitative real-time PCR (RT-qPCR), we determined PKP2 mRNA expression levels in gingival biopsies collected from 11 periodontally healthy, 10 experimental gingivitis, and 10 chronic periodontitis subjects. PKP2 protein expression in gingival biopsies was detected by immunohistochemistry. We then challenged primary gingival epithelial cells with bacteria including P.gingivalis, Campylobacter rectus, and various Toll-like receptor agonists. Western blot and immunofluorescence staining were used to detect protein expression. Inhibitors blocking proteases pathways were tested for P.gingivalis-mediated PKP2 protein degradations. We also knocked down endogenous epithelial PKP2 using lentiviral short-hairpin RNA (shRNA) and evaluated cell proliferation, spreading, and barrier function. RESULTS: Periodontitis gingival biopsies had approximately twofold less PKP2 mRNA than did healthy controls (p < .05). PKP2 protein was predominantly expressed in gingival epithelium. In primary gingival epithelial cells, P.gingivalis challenge increased PKP2 mRNA levels, while protein expression decreased, which suggests that P.gingivalis has a protein degradation mechanism. Cysteine proteases inhibitors greatly attenuated P.gingivalis-mediated PKP2 protein degradation. Epithelial cells with deficient PKP2 exhibited inhibited cell proliferation and spreading and failed to form monolayers. Finally, P.gingivalis impaired gingival epithelial barrier function. CONCLUSIONS: PKP2 appears to be critical in maintaining gingival epithelial barrier function and is susceptible to degradation by cysteine proteases produced by P.gingivalis. Our findings have identified a mechanism by which P.gingivalis impairs epithelial barrier function by promoting PKP2 degradation.

Periodontal inflammation: Integrating genes and dysbiosis.

Biofilm bacteria co-evolve and reach a symbiosis with the host on the gingival surface. The disruption of the homeostatic relationship between plaque bacteria and the host can initiate and promote periodontal disease progression. Recent advances in sequencing technologies allow researchers to profile disease-associated microbial communities and quantify microbial metabolic activities and host transcriptional responses. In addition to confirming the findings from previous studies, new putative pathogens and novel genes that have not previously been associated with periodontitis, emerge. For example, multiple studies have reported that Synergistetes bacteria are associated with periodontitis. Genes involved in epithelial barrier defense were downregulated in periodontitis, while excessive expression of interleukin-17 was associated with a hyperinflammatory response in periodontitis and with a unique microbial community. Bioinformatics-enabled gene ontology pathway analyses provide a panoramic view of the bacterial and host activities as they shift from periodontal health to disease. Additionally, host innate factors, such as genetic variants identified by either a candidate-gene approach or genome-wide association analyses, have an impact on subgingival bacterial colonization. Transgenic mice carrying candidate genetic variants, or with the deletion of candidate genes mimicking the deleterious loss-of-function variant effect, provide experimental evidence validating the biologic relevance of the novel markers associated with the microbial phenotype identified through a statistical approach. Further refinement in bioinformatics, data management approaches, or statistical tools, are required to gain insight into host-microbe interactions by harmonizing the multidimensional "big" data at the genomic, transcriptional, and proteomic levels.

Role of inflammasomes in the pathogenesis of periodontal disease and therapeutics.

Inflammasomes are a group of multimolecular intracellular complexes assembled around several innate immune proteins. Recognition of a diverse range of microbial, stress and damage signals by inflammasomes results in direct activation of caspase-1, which subsequently induces the only known form of secretion of active interleukin-1ß and interleukin-18. Although the importance of interleukin-1ß in the periodontium is not questioned, the impact of inflammasomes in periodontal disease and its potential for therapeutics in periodontology is still in its very early stages. Increasing evidence in preclinical models and human data strongly implicate the involvement of inflammasomes in a number of inflammatory, autoinflammatory and autoimmune disorders. Here we review: (a) the currently known inflammasome functions, (b) clinical/preclinical data supporting inflammasome involvement in the context of periodontal and comorbid diseases and (c) potential therapies targeting inflammasomes. To clarify further the inflammasome involvement in periodontitis, we present analyses of data from a large clinical study (n = 5809) that measured the gingival crevicular fluid-interleukin-1ß and grouped the participants based on current periodontal disease classifications. We review data on 4910 European-Americans that correlate 16 polymorphisms in the interleukin-1B region with high gingival crevicular fluid-interleukin-1ß levels. We show that inflammasome components are increased in diseased periodontal tissues and that the caspase-1 inhibitor, VX-765, inhibits ~50% of alveolar bone loss in experimental periodontitis. The literature review further supports that although patients clinically present with the same phenotype, the disease that develops probably has different underlying biological pathways. The current data indicate that inflammasomes have a role in periodontal disease pathogenesis. Understanding the contribution of different inflammasomes to disease development and distinct patient susceptibility will probably translate into improved, personalized therapies.

Genome-wide association study of biologically informed periodontal complex traits offers novel insights into the genetic basis of periodontal disease.

Genome-wide association studies (GWAS) of chronic periodontitis (CP) defined by clinical criteria alone have had modest success to-date. Here, we refine the CP phenotype by supplementing clinical data with biological intermediates of microbial burden (levels of eight periodontal pathogens) and local inflammatory response (gingival crevicular fluid IL-1ß) and derive periodontal complex traits (PCTs) via principal component analysis. PCTs were carried forward to GWAS (∼2.5 million markers) to identify PCT-associated loci among 975 European American adult participants of the Dental ARIC study. We sought to validate these findings for CP in the larger ARIC cohort (n = 821 participants with severe CP, 2031-moderate CP, 1914-healthy/mild disease) and an independent German sample including 717 aggressive periodontitis cases and 4210 controls. We identified six PCTs with distinct microbial community/IL-1ß structures, although with overlapping clinical presentations. PCT1 was characterized by a uniformly high pathogen load, whereas PCT3 and PCT5 were dominated by Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis, respectively. We detected genome-wide significant signals for PCT1 (CLEC19A, TRA, GGTA2P, TM9SF2, IFI16, RBMS3), PCT4 (HPVC1) and PCT5 (SLC15A4, PKP2, SNRPN). Overall, the highlighted loci included genes associated with immune response and epithelial barrier function. With the exception of associations of BEGAIN with severe and UBE3D with moderate CP, no other loci were associated with CP in ARIC or aggressive periodontitis in the German sample. Although not associated with current clinically determined periodontal disease taxonomies, upon replication and mechanistic validation these candidate loci may highlight dysbiotic microbial community structures and altered inflammatory/immune responses underlying biological sub-types of CP.

The pathogenic mechanism of diabetes varies with the degree of overexpression and oligomerization of human amylin in the pancreatic islet ß cells.

The aggregation of human amylin (hA) to form cytotoxic structures has been closely associated with the causation of type 2 diabetes. We sought to advance understanding of how altered expression and aggregation of hA might link ß-cell degeneration with diabetes onset and progression, by comparing phenotypes between homozygous and hemizygous hA-transgenic mice. The homozygous mice displayed elevated islet hA that correlated positively with measures of oligomer formation (r=0.91; P<0.0001). They also developed hyperinsulinemia with transient insulin resistance during the prediabetes stage and then underwent rapid ß-cell loss, culminating in severe juvenile-onset diabetes. The prediabetes stage was prolonged in the hemizygous mice, wherein ß-cell dysfunction and extensive oligomer formation occurred in adulthood at a much later stage, when hA levels were lower (r=-0.60; P<0.0001). This is the first report to show that hA-evoked diabetes is associated with age, insulin resistance, progressive islet dysfunction, and ß-cell apoptosis, which interact variably to cause the different diabetes syndromes. The various levels of hA elevation cause different extents of oligomer formation in the disease stages, thus eliciting early- or adult-onset diabetes syndromes, reminiscent of type 1 and 2 diabetes, respectively. Thus, the hA-evoked diabetes phenotypes differ substantively according to degree of amylin overproduction. These findings are relevant to the understanding of the pathogenesis and the development of experimental therapeutics for diabetes.

IL-1 receptor antagonist (IL-1RA) suppresses a hyper-IL-17 response-mediated bone loss in a murine experimental periodontitis.

OBJECTIVE: We aimed to assess the role of interleukin - 1 receptor antagonist (IL-1RA) in a ligature-induced periodontal (LIP) model and the mechanism of IL-1RA in regulating the IL-17-mediated periodontal bone loss. DESIGN: Periodontal bone loss was induced through the LIP model in WT and Il1ra-/- mice and measured by micro(µ) CT. Transcription of upstream IL-17 production signals and downstream targets in the ligated gingiva was compared by the real-time quantitative PCR (RT-qPCR) between WT and Il1ra-/- mice. Single-cell suspensions were prepared in gingiva and cervical lymph nodes and were analyzed by fluorescence-activated cell sorting to quantify IL-17+ cells and IL-17-secreting subpopulations. We locally delivered an anti-IL-17 neutralizing antibody to the ligated gingiva and compared the bone loss with the isotype control antibody-treated Il1ra-/- mice. RESULTS: Il1ra-/- mice manifested significantly more bone loss than that of WT mice in the LIP model. Il17 and IL-17-associated transcripts (Il1b, Il6, Il23, Tgfb), Inos, Mrc1, Mmp13, and Rank were upregulated in the gingiva of Il1ra-/- mice in comparison to WT mice. Significantly more IL-17+ immune cells (CD45+IL17+) are present in the gingiva of Il1ra-/- mice with the majority of being TCR γδ T cells (CD45+IL-17+CD3+TCR γδ+) than WT mice. The anti-IL-17 neutralizing antibody treatment attenuated the alveolar bone loss in the LIP model. CONCLUSION: IL-1RA plays a protective role in the murine LIP model by suppressing an expansion of the IL-17+ cells and preventing a hyper-IL-17 response in the gingiva.

Plasma treatment effects on destruction and recovery of Porphyromonas gingivalis biofilms.

The objective of this study was to investigate the treatment effects of non-thermal atmospheric gas plasmas (NTAP) on destruction and the recovery (or re-colonization) of Porphyromonas gingivalis (P. gingivalis) in biofilms. P. gingivalis is a well-known keystone periodontal pathogen strongly associated with periodontal diseases, especially periodontitis. P. gingivalis biofilms were formed on stainless steel coupons and treated for 1, 2, and 5 minutes by NTAP of pure argon gas and argon+oxygen gas mixture. MTT assay, colony forming unit (CFU) counting assay and confocal laser scanning microscopy (CLSM) were used to assess the destruction efficiency. In addition, the plasma treated biofilms were re-cultured in the medium supplemented with antibiotics and oxidative stress sources to determine the synergy of the NTAP with other antimicrobial agents. The results showed the plasma treatment could result in 2.7 log unit reduction in bacterial load. The recovered biofilm CFU with NTAP treatment combined with sub minimal inhibition concentration of amoxicillin was 0.33 log units less than the biofilm treated with amoxicillin alone. The recovered biofilm CFU in NTAP groups was about 2.0 log units less than that in the untreated controls under H2O2 treatment. There was approximately 1.0 log unit reduction of biofilm CFU in plasma treated biofilm compared with untreated control under paraquat treatment. The plasma treated biofilms exhibited less resistance to amoxicillin and greater susceptibility to hydrogen peroxide (H2O2) and paraquat, suggesting that NTAP may enhance biofilm susceptibility to host defense. These in vitro findings suggested that NTAP could be a novel and effective treatment method of oral biofilms that cause periodontal diseases.

Periodontal disease, undiagnosed diabetes, and body mass index: Implications for diabetes screening by dentists.

BACKGROUND: Periodontal disease and diabetes are widespread comorbid conditions that are detrimental to oral and overall health. Dentists' performing chairside screenings for undiagnosed diabetes mellitus (UDM) can be beneficial to both patients and providers. The authors determined UDM rates in a population-based study and whether UDM and periodontal disease were independently associated. METHODS: Data from 7,343 participants in the Atherosclerosis Risk in Communities study visit 4 were used to determine rates of UDM by periodontal status, edentulism, and body mass index. The authors used a χ2 test or analysis of variance, along with a 2-stage logistic regression model, to determine relationships with UDM. UDM was defined as no self-reported diabetes and blood glucose levels (fasting glucose ≥ 126 milligrams/deciliter or nonfasting glucose > 200 mg/dL). Periodontal disease was defined using the Periodontal Profile Classes system adapted to stages and the Centers for Disease Control and Prevention and American Academy of Periodontology index. RESULTS: UDM rates overall were 5.6%. The highest rates occurred in patients who were obese and edentulous (12.6%) and obese and had severe periodontal disease (12.2%). Significant associations were found for UDM and severe periodontal disease (Periodontal Profile Classes system stage IV) (odds ratio, 1.78; 95% confidence interval, 1.10 to 2.88). Edentulism was significantly associated with UDM in the Periodontal Profile Classes system model (odds ratio, 1.87; 95% confidence interval, 1.27 to 2.75) and Centers for Disease Control and Prevention and American Academy of Periodontology index (odds ratio, 1.70; 95% confidence interval, 1.08 to 2.67). Hyperglycemia was found in participants of all body mass index categories. CONCLUSIONS: UDM is significantly associated with obesity, edentulism, and periodontitis. These characteristics could help dentists identify patients at higher risk of developing DM. Patients without these characteristics still have UDM, so dentists performing chairside diabetes screening for all patients would yield additional benefit. PRACTICAL IMPLICATIONS: Dental offices are a major point of contact within the US health care system. Diabetes screening in this setting can provide important health information with direct relevance to patient care.

Periodontitis and Risk of Diabetes in the Atherosclerosis Risk In Communities (ARIC) Study: A BMI-Modified Association.

PURPOSE: To determine whether periodontal disease is positively associated with incident diabetes across the continuum of body mass levels (BMI) and test the hypothesis that the periodontal risk for incident diabetes is modified by BMI. METHODS: We included 5569 diabetes-free participants from Visit 4 (1996-1998) of the Atherosclerosis Risk in Communities study and followed them until 2018. Periodontal disease status was classified by periodontal profile class (PPC)-Stages , and incident diabetes was based on participant report of physician diagnosis. We estimated the hazard ratios (HR) for diabetes using a competing risk model for each PPC-Stage. We assessed multiplicative interactions between periodontal disease and BMI (as a continuous variable) on risk of diabetes. RESULTS: During a median time of 19.4 years of follow-up, 1348 incident diabetes cases and 1529 deaths occurred. Compared to the "Health/Incidental Disease" stage, participants with PPC "Severe Periodontal Disease" or "Severe Tooth Loss" stage and lower BMI had elevated risk for diabetes adjusting for demographic, smoking, education, and biological variables when accounting for death as a competing risk with HRs of 1.76 (95% CI 1.10-2.80) and 2.11 (95% CI 1.46-3.04), respectively. The interaction between PPC-Stages and BMI was significant (P = 0.01). No significant associations of PPC-Stages with incident diabetes were present when BMI was above 31 kg/m2. CONCLUSION: Periodontal disease was associated with incident diabetes, especially in nonobese participants. Dentists should be aware that periodontal disease is associated with incident diabetes but the association may be modified for patient's at higher BMI levels.

Biomimetic polydopamine-laced hydroxyapatite collagen material orients osteoclast behavior to an anti-resorptive pattern without compromising osteoclasts' coupling to osteoblasts.

Polydopamine-assisted modification for bone substitute materials has recently shown great application potential in bone tissue engineering due to its excellent biocompatibility and adhesive properties. A scaffold material's impact on osteoclasts is equally as important as its impact on osteoblasts when considering tissue engineering for bone defect repair, as healthy bone regeneration requires an orchestrated coupling between osteoclasts and osteoblasts. How polydopamine-functionalized bone substitute materials modulate the activity of osteoblast lineage cells has been extensively investigated, but much less is known about their impact on osteoclasts. Moreover, most of the polydopamine-functionalized materials would need to additionally load a biomolecule to exert the modulation on osteoclast activity. Herein, we demonstrated that our biomimetic polydopamine-laced hydroxyapatite collagen (PDHC) scaffold material, which does not need to load additional bioactive agent, is sufficiently able to modulate osteoclast activity in vitro. First, PDHC showed an anti-resorptive potential, characterized by decreased osteoclast differentiation and resorption capacity and changes in osteoclasts' transcriptome profile. Next, cAMP response element-binding protein (CREB) activity was found to mediate PDHC's anti-osteoclastogenic effect. Finally, although PDHC altered clastokines expression pattern of osteoclasts, as revealed by transcriptomic and secretomic analysis, osteoclasts' coupling to osteoblasts was not compromised by PDHC. Collectively, this study demonstrated the PDHC material orients osteoclast behavior to an anti-resorptive pattern without compromising osteoclasts' coupling to osteoblasts. Such a feature is favorable for the net increase of bone mass, which endows the PDHC material with great application potential in preclinical/clinical bone defect repair.

Interferon-gamma promoter hypomethylation and increased expression in chronic periodontitis.

AIM: The goal of this investigation was to determine whether epigenetic modifications in the IFNG promoter are associated with an increase of IFNG transcription in different stages of periodontal diseases. MATERIALS AND METHODS: DNA was extracted from gingival biopsy samples collected from 47 total sites from 47 different subjects: 23 periodontally healthy sites, 12 experimentally induced gingivitis sites and 12 chronic periodontitis sites. Levels of DNA methylation within the IFNG promoter containing six CpG dinucleotides were determined using pyrosequencing technology. Interferon gamma mRNA expression was analysed by quantitative polymerase chain reactions using isolated RNA from part of the biological samples mentioned above. RESULTS: The methylation level of all six analysed CpG sites within the IFNG promoter region in the periodontitis biopsies {52% [interquartile range, IQR (43.8%, 63%)]} was significantly lower than periodontally healthy samples {62% [IQR (51.3%, 74%)], p=0.007} and gingivitis biopsies {63% [IQR (55%, 74%)], p=0.02}. The transcriptional level of IFNG in periodontitis biopsies was 1.96-fold and significantly higher than tissues with periodontal health (p=0.04). Although the mRNA level from experimental gingivitis samples exhibited an 8.5-fold increase as compared with periodontally healthy samples, no significant methylation difference was observed in experimental gingivitis sample. CONCLUSIONS: A hypomethylation profile within IFNG promoter region is related to an increase of IFNG transcription present in the chronic periodontitis biopsies, while such an increase of IFNG in experimentally induced gingivitis seems independent of promoter methylation alteration.

Three-Dimensional Volumetric Changes After Socket Augmentation with Deproteinized Bovine Bone and Collagen Matrix.

PURPOSE: Socket augmentation decreases the magnitude of alveolar ridge resorption, but the literature is limited in respect to quantifying soft tissue remodeling. The aim of this study was to determine the volumetric and linear dimensional changes at the buccal surface for both hard and soft tissues after socket augmentation treated with a xenogeneic collagen matrix in combination with bone grafting. MATERIALS AND METHODS: Twenty-four individuals indicated for tooth extraction were enrolled in this investigation. Each participant was randomly assigned to one of two groups: (1) deproteinized bovine bone + collagen plug, or (2) deproteinized bovine bone + xenogeneic collagen matrix. A cone beam computed tomography scan was taken prior to extraction and at 6 months postextraction. Intraoral scanning images were taken at baseline, 3 months, and 6 months postextraction. Hard and soft tissue analyses were performed to compare linear ridge remodeling and volumetric changes by noncontact reverse-engineering software. RESULTS: Both groups showed bone and soft tissue remodeling. For hard tissue remodeling, there was no significant difference between the collagen plug and collagen matrix groups. For soft tissue remodeling, the collagen matrix group showed a reduced soft tissue loss compared with the collagen plug group. The volumetric analysis demonstrated that the mean buccal soft tissue volume loss for the collagen matrix group was 68.6 mm3 compared with 87.6 mm3 found in the collagen plug group (P = .009) over a 6-month period. CONCLUSION: This clinical investigation provides early evidence of using the total tissue volume to compare soft and hard tissue remodeling after socket augmentation. The results of this study demonstrated that the use of a xenogeneic collagen matrix reduced the buccal soft tissue loss after tooth extraction, but additional studies are necessary to evaluate the clinical significance of soft tissue augmentation after tooth extraction.

Evaluation of a Curved Design Rubber Bristle Interdental Cleaner on Patients with Gingivitis.

Purpose: The purpose of this clinical study was to evaluate the effectiveness of a curved rubber bristle interdental cleaner, as compared to dental floss, in the reduction of gingivitis and plaque.Methods: Gingival Index (GI), Bleeding on Probing (BOP), Periodontal Probing Depth (PPD) and Modified QH Plaque Index (MQH-PI) parameters were evaluated in an examiner-masked, parallel group, controlled clinical study. A total of 50 participants with gingivitis (no site with PPD >4 mm, BOP ≥10% but ≤50%) met the eligibility criteria. Participants were randomly assigned to either the curved rubber bristle interdental cleaner (cRBIC) group or the ADA-accepted dental floss (Floss) group. Participants used the devices for four weeks. Parameters were obtained at 2 and 4 weeks. Participants scored their level of product familiarity, satisfaction and motivation for interdental cleaning.Results: There were no statistically significant differences between the two groups in changes from baseline to 2 or 4 weeks in GI, BOP%, and MQH-PI. However, cRBIC group showed greater reduction of PPD at 4 weeks from baseline, compared with Floss group (p<0.05). The cRBIC group showed overall better compliance level than Floss group. The mean score of "ease of use" of the cRBIC group was significantly greater than that of Floss group. However, Floss group showed higher levels of "satisfaction" than cRBIC group. Motivation for interdental cleaning was higher in cRBIC.Conclusion: The cRBIC was similar to Floss in clinical effectiveness; however, PPD reduction at 4 weeks was greater with the cRBIC. Ease of use of cRBIC may have affected the participants' motivation for interdental cleaning, resulting in better compliance.

MicroRNA Expression Profiles in External Cervical Resorption.

INTRODUCTION: External cervical resorption (ECR) has been challenging for its diagnosis, prevention, and treatment. Its etiology and pathogenesis are largely unknown. This study characterized microRNA (miRNA) expression patterns of human tissues from ECR lesions and identified potential messenger RNA targets and pathways. METHODS: Granulomatous tissues from ECR (n = 5) and their adjacent nonaffected asymptomatic gingival connective tissues (n = 5) were collected. Similarly, chronic periodontitis (CP) and control samples were collected (n = 3). Quantitative reverse transcription polymerase chain reaction array analysis compared the expression profiles of 88 miRNAs between diseases. Differentially expressed miRNAs were identified using the Student t test. Bioinformatics for messenger RNA (miRWalk) and KEGG pathway analyses were performed to identify predicted target genes and biological/cellular functions and signaling pathways. RESULTS: Three miRNAs (miR-20a-5p, miR-210-3p, and miR-99a-4p) were significantly down-regulated and 1 miRNA (miR-122-5p) was significantly up-regulated in ECR (P < .05). One up-regulated and 1 down-regulated miRNA reached the significance threshold in CP. A comparison of miRNA expression in ECR and CP identified 3 differentially expressed miRNAs, indicating differences in disease pathobiology. Inflammation-associated Wnt, PI3K-Akt, mitogen-activated protein kinases signaling, and bone formation-associated transforming growth factor beta pathways were identified and predicted to be modulated by differentially expressed miRNAs in both ECR and CP. Biological processes unique to each disease entity were identified, such as T- and B-cell receptor signaling pathways, osteoclast differentiation, and extracellular matrix-receptor interaction for CP. Glycosaminoglycan biosynthesis, mineral absorption, and insulin signaling pathways for ECR were identified. CONCLUSIONS: This proof-of-principle in vivo study indicated that ECR has both common and unique miRNA expression profiles in comparison with CP, which are predicted to target genes regulating inflammation, immunity, and metabolism of mineralized tissues.

Calretinin expression in the differential diagnosis of human ameloblastoma and keratocystic odontogenic tumor.

Ameloblastoma is a benign, locally aggressive epithelial odontogenic tumor that has the potential to become malignant and produce metastasis to distant sites such as lungs and kidneys. The histologic presentation can be, in some instances, mistaken for keratocystic odontogenic tumor (KCOT) (formerly known as odontogenic keratocyst). The expression of calretinin [calbindin2 (CALB2)] was investigated on both ameloblastoma and KCOT. Nineteen cases of ameloblastoma and 17 cases of KCOT were stained with calretinin antiserum 18-0211 (Zymed, San Francisco, CA). All cases (100%) of ameloblastoma showed positive calretinin staining, restricted to the neoplastic epithelial component and none (0%) of the 17 KCOTs showed positive calretinin staining. Gene expression profiling of ameloblastomas showed CALB2 expressed in the basal cell layer of columnar cells resembling preameloblasts, in all 5 of the ameloblastomas evaluated. Taken together, the results of this study strongly support calretinin as a useful immunohistochemical marker for ameloblastoma and malignant ameloblastoma and it can also be used in the differential diagnosis of KCOT.

An experimental murine model to study periodontitis.

Periodontal disease (PD) is a common dental disease associated with the interaction between dysbiotic oral microbiota and host immunity. It is a prevalent disease, resulting in loss of gingival tissue, periodontal ligament, cementum and alveolar bone. PD is a major form of tooth loss in the adult population. Experimental animal models have enabled the study of PD pathogenesis and are used to test new therapeutic approaches for treating the disease. The ligature-induced periodontitis model has several advantages as compared with other models, including rapid disease induction, predictable bone loss and the capacity to study periodontal tissue and alveolar bone regeneration because the model is established within the periodontal apparatus. Although mice are the most convenient and versatile animal models used in research, ligature-induced periodontitis has been more frequently used in large animals. This is mostly due to the technical challenges involved in consistently placing ligatures around murine teeth. To reduce the technical challenge associated with the traditional ligature model, we previously developed a simplified method to easily install a bacterially retentive ligature between two molars for inducing periodontitis. In this protocol, we provide detailed instructions for placement of the ligature and demonstrate how the model can be used to evaluate gingival tissue inflammation and alveolar bone loss over a period of 18 d after ligature placement. This model can also be used on germ-free mice to investigate the role of human oral bacteria in periodontitis in vivo. In conclusion, this protocol enables the mechanistic study of the pathogenesis of periodontitis in vivo.

GWAS for Interleukin-1ß levels in gingival crevicular fluid identifies IL37 variants in periodontal inflammation.

There is no agnostic GWAS evidence for the genetic control of IL-1ß expression in periodontal disease. Here we report a GWAS for "high" gingival crevicular fluid IL-1ß expression among 4910 European-American adults and identify association signals in the IL37 locus. rs3811046 at this locus (p = 3.3 × 10-22) is associated with severe chronic periodontitis (OR = 1.50; 95% CI = 1.12-2.00), 10-year incident tooth loss (≥3 teeth: RR = 1.33; 95% CI = 1.09-1.62) and aggressive periodontitis (OR = 1.12; 95% CI = 1.01-1.26) in an independent sample of 4927 German/Dutch adults. The minor allele at rs3811046 is associated with increased expression of IL-1ß in periodontal tissue. In RAW macrophages, PBMCs and transgenic mice, the IL37 variant increases expression of IL-1ß and IL-6, inducing more severe periodontal disease, while IL-37 protein production is impaired and shows reduced cleavage by caspase-1. A second variant in the IL37 locus (rs2708943, p = 4.2 × 10-7) associates with attenuated IL37 mRNA expression. Overall, we demonstrate that IL37 variants modulate the inflammatory cascade in periodontal disease.

Common Polymorphisms in IFI16 and AIM2 Genes Are Associated With Periodontal Disease.

BACKGROUND: The single nucleotide polymorphism (SNP) context of a previously identified periodontitis-associated locus is investigated, and its association with microbial, biologic, and periodontal disease clinical parameters is examined. METHODS: A 200-kb spanning region of 1q12 previously highlighted in a genome-wide association scan among 4,766 European American individuals (SNP rs1633266) was annotated. Two haplotype blocks were selected. Association of these polymorphisms with data on microbial plaque composition, gingival crevicular fluid (GCF)-interleukin (IL)-1ß levels, and clinical parameters of periodontal disease were examined. Descriptive analysis of IFI16 and AIM2 protein expression in gingival tissues from healthy individuals (n = 2) and individuals with chronic periodontitis (n = 2) was done via immunohistochemistry. RESULTS: The highlighted locus is a 100-kb region containing the interferon γ-inducible protein 16 (IFI16) and absent in melanoma 2 (AIM2) genes. Two haplotype blocks, rs6940 and rs1057028, were significantly associated with increased extent bleeding on probing and levels of microorganisms Porphyromonas gingivalis, Tannerella forsythia, and Campylobacter rectus (P ≤0.05). Haplotype block rs1057028 was also significantly associated with pathogens Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans, increased GCF-IL-1ß levels, and extent of probing depth ≥4 mm (P ≤0.05). Prevalence of severe periodontitis (biofilm-gingival interface P3 classification) was positively associated with haplotype block rs1057028. Similar trends were observed for haplotype block rs1057028. IFI16 and AIM2 protein expression was observed in multiple cell types of gingival tissues, including inflammatory cells. CONCLUSION: This study found IFI16 and AIM2 SNPs associated with higher levels of periodontal microorganisms and an increased percentage of periodontal disease clinical parameters, suggesting the need for functional studies and additional fine-mapping of variants in the 1q12-locus.

The Novel ASIC2 Locus is Associated with Severe Gingival Inflammation.

An increasing body of evidence suggests a significant genetic regulation of inflammatory response mechanisms; however, little is known regarding the genetic determinants of severe gingival inflammation (GI). We conducted a genome-wide association study of severe GI among 4077 European American adults, participants in the Dental Atherosclerosis Risk In Communities cohort. The severe GI trait was defined dichotomously using the 90th percentile of gingival index ≥2 extent score. Genotyping was performed with the Affymetrix 6.0 array platform and an imputed set of 2.5 million markers, based on HapMap Phase II CEU build 36, was interrogated. Genetic models were based on logistic regression and controlled for ancestry (10 principal components), sex, age, and examination center. One locus on chromosome 17 met genome-wide statistical significance criteria-lead single nucleotide polymorphism (SNP): rs11652874 [minor allele frequency=0.06, intronic to ASIC2 (acid sensing ionic channel-2, formerly named ACCN1); odds ratio=2.1, 95% confidence interval=1.6-2.7, p=3.9×10-8]. This association persisted among subjects with severe periodontitis and was robust to adjustment for microbial plaque index. Moreover, the minor [G] allele was associated with higher levels of severe GI in stratified analyses among subsets of participants with high load of either "red" or "orange" complex pathogens, although this association was not statistically significant. While these results will require replication in independent samples and confirmation by mechanistic studies, this locus appears as a promising candidate for severe gingival inflammation. Our findings suggest that genetic variation in ASIC2 is significantly associated with severe gingival inflammation and the association is plaque-independent.