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.

Human amylin (hA) is a small protein cosecreted with insulin from pancreatic islet b-cells upon stimulation by glucose or other chemical signals [1,2]. Wild-type hA, also known as insulinoma amyloid peptide, insulinoma amyloid polypeptide (IAPP) or islet amyloid polypeptide [3], demonstrates a strong in vitro tendency to aggregate into fibrils [4,5], which is dependent on specific residues in different regions of the molecule, notably, the amyloidogenic region of amino acids 20-29 [6]. It is the main protein in the amyloid aggregates that are frequently present in the islets of human subjects with type 2 diabetes mellitus (T2DM) [7,8], as well as in diabetic cats and primates [9]. It has been reported that aggregated hA might contribute to the loss of insulin-producing pancreatic b-cells cells in T2DM [10,11]. The following lines of evidence have associated hA with the pancreatic pathology of T2DM: (a) amyloid is often concentrated near areas of islet b-cell degeneration in humans or primates with T2DM [10,[12][13][14]; (b) synthetic hA is toxic to pancreatic b-cells in vitro [11,[15][16][17]; (c) spontaneous loss of b-cells has been reported in a line of hA-transgenic mice [18]; and (d) toxicity of hA towards cultured cells correlates with its ability to form fibrils -for example, rat amylin does not form fibrils and nor does it evoke b-cell apoptosis in vitro [11,17,19]. Human amylin is a small fibrillogenic protein that is the major constituent of pancreatic islet amyloid, which occurs in most subjects with type 2 diabetes. There is evidence that it can elicit in vitro apoptosis in islet b-cells, but the physical properties that underpin its cytotoxicity have not been clearly elucidated. Here we employed electron microscopy, thioflavin T fluorescence and CD spectroscopy to analyze amylin preparations whose cytotoxic potential was established by live-dead assay in cultured b-cells. Highly toxic amylin contained few preformed fibrils and initially showed little b-sheet content, but underwent marked time-dependent aggregation and b-conformer formation following dissolution. By contrast, low-toxicity amylin contained abundant preformed fibrils, and demonstrated high initial b-sheet content but little propensity to aggregate further once dissolved. Thus, mature amylin fibrils are not toxic to b-cells, and aggregates of fibrils such as occur in pancreatic islet amyloid in vivo are unlikely to contribute to b-cell loss. Rather, the toxic molecular species is likely to comprise soluble oligomers with significant b-sheet content. Attempts to find ways of protecting b-cells from amylin-mediated death might profitably focus on preventing the conformational change from random coil to b-sheet.Abbreviations AM, acetomethoxyl ester; EthD, ethidium homodimer; hA, human amylin; HFIP, hexafluoroisopropanol; IAPP, insulinoma amyloid polypeptide; JNK1, Jun NH 2 -terminal kinase 1; KRB, Krebs-Ringer bicarbonate buffer; RINm5F, rat insulinoma m5F; T2DM, type 2 diabetes mellitus; TEM, transmission electron microscopy; ThT, thioflav...

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-1β) 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-1β 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.

Background Tumor necrosis factor-alpha (TNF-α) plays a central role in the molecular pathogenesis of periodontal disease. However, the epigenetic regulation attributable to microbial and inflammatory signals at the biofilm gingival interface are poorly understood. In this study, we investigated the DNA methylation alteration within the TNFA promoter in human gingival biopsies from different stages of periodontal disease, and explored the regulatory mechanism of TNFA transcription by DNA methylation. Methods Gingival biopsies were harvested from 17 chronic periodontitis patients and 18 subjects with periodontal health. Another 11 subjects participated in an experimentally induced gingivitis study, and gingival biopsies were collected at the baseline, induction, and resolution phase. To confirm that TNFA promoter methylation modulated TNFA transcription we treated THP.1 cells with a DNA methyltransferase inhibitor, 5-aza-2-deoxycytidine and used a RAW 294.7 cell line transfected with a TNFA promoter-specific luciferase reporter system with or without methlyaiton, Results In gingival biopsies from subjects with severe chronic periodontitis two individual CpG sites within the TNFA promoter (at -163bp and -161bp) displayed increased methylation in periodontitis samples as compared to gingival health (16.1±5.1% vs. 11.0±4.6%, p=0.02, 19.8±4.1% vs. 15.4±3.6%, p=0.04, respectively). The methylation level at -163bp was inversely associated with the transcription level of TNFA (p=0.018). However, no significant difference in the TNFA promoter methylation pattern was observed in samples biopsied during the induction or resolution phase of experimentally induced gingivitis, which represented a reversible periodontal lesion. THP.1 cells treated with 5-aza-2-deoxycytidine demonstrated a time-dependent increase in TNFA messenger level. We also found that the luciferase activity decreased 2.6 fold in a construct containing an in vitro methylated TNFA promoter as compared to the unmethylated insert (p=0.03). Conclusion Although the biopsy samples represented a mixed cell population, the change in promoter methylation status in chronic periodontal disease suggested that DNA methylation may be an important regulatory mechanism in controlling TNFA transcriptional expression in disease.

Fibrillogenic human amylin elicits pancreatic ␤-cell apoptosis that may contribute to development of type-2 diabetes. Here, we demonstrated that activation of a caspase cascade is necessary for induction of apoptosis by fibrillogenic amylin variants in two pancreatic ␤-cell lines. Human amylin, as well as truncated 8 -37 human amylin, evoked sequential activation of caspases-8 and -3, and apoptosis, whereas non-␤-sheet forming and nonfibrillogenic homologs, such as [ 25,28,29 triprolyl]human amylin, did not, implying that the ␤-sheet conformer is required for human amylin-induced caspase activation. Significant inhibition of apoptosis was evoked by a selective caspase-1 inhibitor, indicating that caspase-1 is also essential for activation of the caspase cascade. Furthermore, we showed that specific jnk1 antisense oligonucleotides, which suppress phospho-JNK1 expression, effectively decreased human amylin-induced activation of c-Jun. Studies of the interplay between the caspase cascade and the JNK pathway showed that both apoptosis and caspase-3 activation were suppressed by treatment with a JNK inhibitor and by transfection of antisense jnk1 oligonucleotides or antisense-c-jun, whereas a selective inhibitor of caspases-1 and -3 prevented apoptosis but not c-Jun activation. Thus, the JNK1 activation preceded activation of caspases-1 and -3. However, selective JNK inhibition had no effect on caspase-8 activation, and selective caspase-8 inhibition only partially suppressed apoptosis and c-Jun activation, indicating that caspase-8 may partially act upstream of the JNK pathway. Our studies demonstrate a functional interaction of a caspase cascade and JNK1. Fibrillogenic amylin can evoke a JNK1-mediated apoptotic pathway, which is partially dependent and partially independent of caspase-8, and in which caspase-3 acts as a common downstream effector.Pancreatic islet amyloid commonly occurs in T2DM 1 (type-2 diabetes mellitus), where it may contribute to islet ␤-cell dysfunction and impairment of insulin secretion (1-4). Amylin (known also as islet amyloid polypeptide) is the main protein in this type of amyloid (4). It is synthesized by islet ␤-cells, and its co-secretion with insulin increases rapidly following nutrient stimulation (5, 6). There is substantive evidence that those amylin molecules that aggregate in vivo, such as human amylin, are cytotoxic to islet ␤-cells, whereas the non-aggregating variants are not (2, 7-11). Thus, aggregation of human amylin could cause progressive in vivo damage to ␤-cells. Recent studies have indicated that there are structure-function relationships in the amylin molecule that link aggregation and fibril formation to the associated cytotoxicity (12-15). Human amylin, when present in physiological solutions in vitro, undergoes progressive time-dependent physical changes that include: the adoption of increasing amounts of ␤-sheet conformation (16), formation of oligomers of progressively increasing size (17), and aggregation into polymorphic fibrillar assemblies (17,18). By contra...

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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.

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‐1β and interleukin‐18. Although the importance of interleukin‐1β 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‐1β 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‐1β 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.