Periodontal diseases are highly prevalent chronic diseases, and severe periodontitis creates functional and esthetic problems and decreases self-esteem for a large percentage of the older population worldwide. In many cases of periodontitis, there is no distinct tell-tale pain that motivates a patient to seek treatment, rather the signs become clinically detectable late, and typically when the disease has progressed to a problematic level for the life of the dentition. Early periodontal screening and diagnostics tools will provide early recognition of periodontal diseases and facilitate timely management of the disease to reduce tooth loss. To this goal, gingival crevicular fluid is easily sampled, can be repeatedly and non-invasively collected, and can be tested for potential biomarkers. Moreover, the site specificity of periodontal diseases enhances the usefulness of gingival crevicular fluid sampled from specific sites as a biofluid for diagnosis and longitudinal monitoring of periodontal diseases. The present review aimed to provide up-to-date information on potential diagnostic biomarkers with utility that can be assayed from gingival crevicular fluid samples, focusing on what is new and useful and providing only general historic background textually and in a tabulated format.

The aim of this narrative review is to relate the contribution of European researchers to the complex topic of the host immune system in periodontal disease, focusing on acquired immunity. Other chapters in this volume will address the genetics and autoantibody responses and other forms of immunity to periodontal disease. While the contribution of European authors is the focus, global literature is included in this descriptive narrative for contextual clarity, albeit many with European co-authors. The topic is relatively intense and is thus broken down into sections outlined below, tackled as descriptive narratives to enhance understanding. Any attempt at a systematic or scoping review was quickly abandoned given the descriptive nature and marked variation of approach in almost all publications. Even the most uniform area of this acquired periodontal immunology literature, antibody responses to putative pathogens in periodontal diseases, falls short of common structures and common primary outcome variables one would need and expect in clinical studies, where randomized controlled clinical trials (RCTs) abound. Addressing 'the host's role' in immunity immediately requires a discussion of host susceptibility, which necessitates consideration of genetic studies (covered elsewhere in the volume and superficially covered here).

Chronic inflammatory periodontal disease and its related condition, peri-implant disease, are highly prevalent globally and require accurate and speedy diagnosis. The focus of this volume dedicated to diagnostics is to cover modern enhancements in accuracy, simplicity and speed. An international assortment of experts has been tasked with reviewing defined areas of current best practice as well innovation in the field of periodontitis and peri-implantitis diagnostics. Periodontitis and peri-implantitis are irreversible, chronic, cumulative conditions propagated by bacteria and host factors, which involve soft and hard tissue changes, and these changes are measured in the diagnostic process. Clinically relevant modifications to the healthy state are detected using clinical, radiological and laboratory or point of care testing, and these testing approaches are critically reviewed at length in this state-of-the-art resume of periodontal diagnostics.

Liquid biopsy is a noninvasive diagnostic technique used for monitoring cancer utilizing specific genetic biomarkers present in bodily fluids, such as blood, saliva, or urine. These analyses employ multiple biomolecular sources including circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), and exosomes (that contain DNA fragments) to detect genetic biomarkers that can predict, disclose, and/or monitor cancers. Levels of these biomarkers can inform on the presence of cancer, its genetic characteristics, and its potential treatment response and also provide predictive genetic predisposition information for specific cancers including oral squamous cell carcinomas (OSCC). Liquid biopsies can aid cancer management as they offer real-time dynamic information on the response to say chemotherapy or radiotherapy and recurrence following surgical excision. Unlike traditional tissue biopsies, which are invasive with a degree of morbidity and require specific tumor location sampling, liquid biopsies are noninvasive and can be repeated frequently. For oral squamous cell carcinoma, on which this review focuses, liquid biopsy of blood or saliva can be valuable in predicting susceptibility, providing early detection, and monitoring the disease's progression and response to therapy. This review gives a general narrative overview of the technology, its current medical usage, and advantages and disadvantages compared with current techniques and discusses a range of current potential biomarkers for disclosing OSCC and predicting its risk. Oral squamous cell carcinoma is all too often detected in the late stages. In future, liquid biopsy may provide an effective screening process such that cancers including OSCC will be detected in the early stages rather than later when prognosis is poor and morbidity and debilitation are greater. In this screening process, periodontists and hygienists have a critical role in that they are adept in examining mucosa, they see patients with shared risk factors for periodontitis and OSCC, namely smoking and poor oral hygiene, and they see patients frequently such that OSCC examinations should be a routine part of the recall visit. With this additional screening manpower, oral medicine and oral surgery colleagues will detect OSCC earlier and this coupled with new techniques such as liquid biopsy may greatly decrease global morbidity in OSCC.

DNA methylation controls several inflammatory genes affecting bone homeostasis. Hitherto, inhibition of DNA methylation in vivo in the context of periodontitis and osteoclastogenesis has not been attempted. Ligature-induced periodontitis in C57BL/6J mice was induced by placing ligature for five days with Decitabine (5-aza-2′-deoxycytidine) (1 mg/kg/day) or vehicle treatment. We evaluated bone resorption, osteoclast differentiation by tartrate-resistant acid phosphatase (TRAP) and mRNA expression of anti-inflammatory molecules using cluster differentiation 14 positive (CD14+) monocytes from human peripheral blood. Our data showed that decitabine inhibited bone loss and osteoclast differentiation experimental periodontitis, and suppressed osteoclast CD14+ human monocytes; and conversely, that it increased bone mineralization in osteoblastic cell line MC3T3-E1 in a concentration-dependent manner. In addition to increasing IL10 (interleukin-10), TGFB (transforming growth factor beta-1) in CD14+ monocytes, decitabine upregulated KLF2 (Krüppel-like factor-2) expression. Overexpression of KLF2 protein enhanced the transcription of IL10 and TGFB. On the contrary, site-directed mutagenesis of KLF2 binding site in IL10 and TFGB abrogated luciferase activity in HEK293T cells. Decitabine reduces bone loss in a mouse model of periodontitis by inhibiting osteoclastogenesis through the upregulation of anti-inflammatory cytokines via KLF2 dependent mechanisms. DNA methyltransferase inhibitors merit further investigation as a possible novel therapy for periodontitis.

The role of the adaptor molecule MyD88 is thought to be independent of Toll-like receptor 3 (TLR3) signaling. In this report, we demonstrate a previously unknown role of MyD88 in TLR3 signaling in inducing endogenous ligands of TLR2 to elicit innate immune responses. Of the various TLR ligands examined, the TLR3-specific ligand polyinosinic:polycytidylic acid (poly I:C), significantly induced TNF production and the upregulation of other TLR transcripts, in particular, TLR2. Accordingly, TLR3 stimulation also led to a significant upregulation of endogenous TLR2 ligands mainly, HMGB1 and Hsp60. By contrast, the silencing of TLR3 significantly downregulated MyD88 and TLR2 gene expression and pro-inflammatory IL1β, TNF, and IL8 secretion. The silencing of MyD88 similarly led to the downregulation of TLR2, IL1β, TNF and IL8, thus suggesting MyD88 to somehow act downstream of TLR3. Corroborating in vitro data, Myd88−/− knockout mice downregulated TNF, CXCL1; and phospho-p65 and phospho-IRF3 nuclear localization, upon poly I:C treatment in a mouse model of skin infection. Taken together, we identified a previously unknown role for MyD88 in the TLR3 signaling pathway, underlying the importance of TLRs and adapter protein interplay in modulating endogenous TLR ligands culminating in pro-inflammatory cytokine regulation.

In aggressive periodontitis, the dysbiotic microbial community in the subgingival crevice, which is abundant in Aggregatibacter actinomycetemcomitans, interacts with extra- and intracellular receptors of host cells, leading to exacerbated inflammation and subsequent tissue destruction. Our goal was to understand the innate immune interactions of A. actinomycetemcomitans with macrophages and human gingival epithelial cells (HGECs) on the signaling cascade involved in inflammasome and inflammatory responses. U937 macrophages and HGECs were co-cultured with A. actinomycetemcomitans strain Y4 and key signaling pathways were analyzed using real-time PCR, Western blotting and cytokine production by ELISA. A. actinomycetemcomitans infection upregulated the transcription of TLR2, TLR4, NOD2 and NLRP3 in U937 macrophages, but not in HGECs. Transcription of IL-1β and IL-18 was upregulated in macrophages and HGECs after 1 h interaction with A. actinomycetemcomitans, but positive regulation persisted only in macrophages, resulting in the presence of IL-1β in macrophage supernatant. Immunoblot data revealed that A. actinomycetemcomitans induced the phosphorylation of AKT and ERK1/2, possibly leading to activation of the NF-κB pathway in macrophages. On the other hand, HGEC signaling induced by A. actinomycetemcomitans was distinct, since AKT and 4EBP1 were phosphorylated after stimulation with A. actinomycetemcomitans, whereas ERK1/2 was not. Furthermore, A. actinomycetemcomitans was able to induce the cleavage of caspase-1 in U937 macrophages in an NRLP3-dependent pathway. Differences in host cell responses, such as those seen between HGECs and macrophages, suggested that survival of A. actinomycetemcomitans in periodontal tissues may be favored by its ability to differentially activate host cells.

AIM The purpose of this study was to determine inflammatory and epigenetic features following induction of oral and gut dysbiosis in experimental periodontitis in order to examine the interplay between oral and systemic infection. MATERIALS AND METHODS Periodontitis was induced in 6- to 8-week-old C57BL/6 mice by (a) Ligature placement (Lig group) (oral challenge); (b) P. gingivalis gavage (Pg group) (systemic challenge); and (c) the combination of the two models oral and systemic challenge (Pg + Lig). The duration of the experiment was 60 days, and the animals were then sacrificed for analyses. Alveolar bone loss was assessed, and a multiplex immunoassay was performed. Maxillae and gut tissues were immunostained for DNMT3b (de novo methylation marker), B and T lymphocyte attenuator (BTLA) and IL-18R1 (inflammation markers). RESULTS Pg and Pg + Lig groups exhibited higher bone loss when compared to Sham. BAFF, VEGF, RANKL, RANTES and IP-10 were significantly higher with Pg gavage. Likewise, DNMT3b was overexpressed in both gut and maxilla after the Pg administration. The same pattern was observed for BTLA and IL-18R1 in gut tissues. CONCLUSIONS The systemic microbial challenge either alone or in combination with local challenge leads to distinct patterns of inflammatory and epigenetic features when compared to simply locally induced experimental periodontitis.

Polymorphonuclear neutrophils (PMNs) are indispensable in fighting infectious microbes by adopting various antimicrobial strategies including phagocytosis and neutrophil extracellular traps (NETs). Although the role and importance of PMNs in periodontal disease are well established, the specific molecular mechanisms involved in NET formation are yet to be characterized. In the present study, we sought to determine the role of periodontal pathogen on NET formation by utilizing Fusobacterium nucleatum. Our data demonstrates that F. nucleatum activates neutrophils and induces robust NETosis in a time-dependent manner via the upregulation of the Nucleotide oligomerization domain 1 (NOD1) and NOD2 receptors. Furthermore, CRISPR/Cas9 knockout of HL-60 cells and the use of ligands/inhibitors confirmed the involvement of NOD1 and NOD2 receptors in F. nucleatum-mediated NET formation. When treated with NOD1 and NOD2 inhibitors, we observed a significant downregulation of peptidylarginine deiminase 4 (PAD4) activity. In addition, neutrophils showed a significant increase and decrease of myeloperoxidase (MPO) and neutrophil elastase (NE) when treated with NOD1/NOD2 ligands and inhibitors, respectively. Taken together, CRISPR/Cas9 knockout of NOD1/NOD2 HL-60 cells and inhibitors of NOD signaling confirmed the role of NLRs in F. nucleatum-mediated NETosis. Our data demonstrates an important pathway linking NOD1 and NOD2 to NETosis by F. nucleatum, a prominent microbe in periodontal biofilms. This is the first study to elucidate the role of NOD-like receptors in NETosis and their downstream signaling network.

The oral cavity is home for a plethora of bacteria and viruses. Epithelial barriers encounter these micro-organisms and recognize them via pathogen recognition receptors (PRRs) that instigate antibacterial and antiviral responses. We and others have shown that human gingival epithelial cells (HGECs) express PRRs to defend invading pathogens. Among these PRRs, TLR2, TLR3 and TLR4 are highly expressed in HGECs and appear to be important based on our previous findings. IFN-β is one of the major type 1 interferons induced to defend viral attack. In this report, we sought to dissect TLR3 and TLR4 mediated secretion of IFN-β in HGECs. We stimulated HGECs with ultrapure LPS (TLR4 ligand) and Poly I:C (TLR3 ligand) for 24 h and supernatant was used to determine IFN-β secretion. We show that cells treated with Poly I:C induced IFN-β secretion but not cells treated with LPS. In addition, silencing of TLR3 prior to Poly I:C stimulation significantly downregulated IFN-β secretion. On the contrary, overexpression of MD2 and TLR4 in HGECs restored IFN-β secretion. Upon further evaluation, we found that TLR3 stimulation but not TLR4 induced the phosphorylation of interferon regulatory factor 3 (IRF3), which is critical for IFN-β secretion. We conclude that IFN-β secretion is through TLR3 and not via TLR4 in HGECs.

The purpose is to provide current knowledge and recent development and understanding of periodontal disease dysbiosis in the perspective of epigenetic changes. Epigenetic changes, where environmental factors modify the gene expression network without changing the DNA sequence, may influence inflammatory diseases such as chronic periodontitis. These chemical modifications of DNA and histone proteins cause epigenetic changes that alter cellular function and host defenses. Findings suggest that the methylation of cytosine residues on DNA particularly at CpG Islands is commonly associated with gene silencing, and covalent modifications on histones are associated with chromatin structural integrity and function that play crucial roles in gene expression. In periodontal immune dysbiosis, aberrant DNA methylation and/or histone modifications could potentially play a role in disease state. We broadly discuss epigenetic modifications related to immune regulation and comprehensively discuss recent developments in the dynamics of epigenetic changes pertaining to chronic inflammatory periodontal disease.

MicroRNAs are small functional RNAs that modulate various biological processes in cells by interfering with gene translation. We have previously demonstrated that certain miRNAs play a crucial role in the innate immune responses of human oral epithelial cells to Porphyromonas gingivalis. While addressing the mechanisms of P. gingivalis induced apoptosis in these cells, we discovered that certain miRNAs are upregulated upon stimulation with live bacteria. These upregulated miRNAs include hsa-miR-584, hsa-miR-572, hsa-miR-210, hsa-miR-492, hsa-miR-623 and hsa-miR-663. Further analysis revealed an unexpected role for hsa-miR-663 (miR-663). To further evaluate miR-663 function, we overexpressed miR-663 in epithelial cells which resulted in cellular apoptosis. The bioinformatics analysis of the miR-663 target prediction, revealed a strong binding affinity to a 3′ UTR region of Apoptosis Antagonizing Transcription Factor (AATF) mRNA. To demonstrate the binding of miR-663 to AATF mRNA, the putative miR-663 target site within the 3′-UTR region of AATF was cloned in luciferase vector and transfected to HEK293T cells. Luminescence data showed the downregulation of luciferase activity in cells that had the full length target region of the putative binding site, confirming that AATF is one of the targets for miR-663. This prompted us to further evaluate its role in a cancer cell line (MCF-7) to determine miR-663s’ apoptotic function. The overexpression of miR-663 led to a significant increase in apoptosis of MCF-7 cells. Taken together, miR-663 may function as an ‘apoptomiR’ by inhibiting the anti-apoptotic gene AATF to induce apoptosis. These findings could have therapeutic implications for epithelial cell targeting in cancer therapy.

Polymorphonuclear neutrophils (PMNs) are indispensable in fighting infectious microbes by adopting various antimicrobial strategies including phagocytosis and neutrophil extracellular traps (NETs). Although the role and importance of PMNs in periodontal disease are well established, the specific molecular mechanisms involved in NET formation are yet to be characterized. In the present study, we sought to determine the role of periodontal pathogen on NET formation by utilizing Fusobacterium nucleatum. Our data demonstrates that F. nucleatum activates neutrophils and induces robust NETosis in a time-dependent manner via the upregulation of the Nucleotide oligomerization domain 1 (NOD1) and NOD2 receptors. Furthermore, CRISPR/Cas9 knockout of HL-60 cells and the use of ligands/inhibitors confirmed the involvement of NOD1 and NOD2 receptors in F. nucleatum-mediated NET formation. When treated with NOD1 and NOD2 inhibitors, we observed a significant downregulation of peptidylarginine deiminase 4 (PAD4) activity. In addition, neutrophils showed a significant increase and decrease of myeloperoxidase (MPO) and neutrophil elastase (NE) when treated with NOD1/NOD2 ligands and inhibitors, respectively. Taken together, CRISPR/Cas9 knockout of NOD1/NOD2 HL-60 cells and inhibitors of NOD signaling confirmed the role of NLRs in F. nucleatum-mediated NETosis. Our data demonstrates an important pathway linking NOD1 and NOD2 to NETosis by F. nucleatum, a prominent microbe in periodontal biofilms. This is the first study to elucidate the role of NOD-like receptors in NETosis and their downstream signaling network.

Periodontal diseases comprise a wide range of inflammatory conditions that affect the supporting structures of the teeth (the gingiva, bone and periodontal ligament), which could lead to tooth loss and contribute to systemic inflammation. Chronic periodontitis predominantly affects adults, but aggressive periodontitis may occasionally occur in children. Periodontal disease initiation and propagation is through a dysbiosis of the commensal oral microbiota (dental plaque), which then interacts with the immune defences of the host, leading to inflammation and disease. This pathophysiological situation persists through bouts of activity and quiescence, until the affected tooth is extracted or the microbial biofilm is therapeutically removed and the inflammation subsides. The severity of the periodontal disease depends on environmental and host risk factors, both modifiable (for example, smoking) and non-modifiable (for example, genetic susceptibility). Prevention is achieved with daily self-performed oral hygiene and professional removal of the microbial biofilm on a quarterly or bi-annual basis. New treatment modalities that are actively explored include antimicrobial therapy, host modulation therapy, laser therapy and tissue engineering for tissue repair and regeneration.

Periodontal diseases comprise a wide range of inflammatory conditions that affect the supporting structures of the teeth the gingiva, bone and periodontal ligament), which could lead to tooth loss and contribute to systemic inflammation. Chronic periodontitis predominantly affects adults, but aggressive periodontitis may occasionally occur in children. Periodontal disease initiation and propagation is through a dysbiosis of the commensal oral microbiota (dental plaque), which then interacts with the immune defences of the host, leading to inflammation and disease. This pathophysiological situation persists through bouts of activity and quiescence, until the affected tooth is extracted or the microbial biofilm is therapeutically removed and the inflammation subsides. The severity of the periodontal disease depends on environmental and host risk factors, both modifiable (for example, smoking) and non-modifiable (for example, genetic susceptibility). Prevention is achieved with daily self-performed oral hygiene and professional removal of the microbial biofilm on a quarterly or bi-annual basis. New treatment modalities that are actively explored include antimicrobial therapy, host modulation therapy, laser therapy and tissue engineering for tissue repair and regeneration.

BACKGROUND AND OBJECTIVE:Adiponectin is produced by adipose cells and is considered an anti-inflammatory molecule. In contrast, C-reactive protein (CRP) has been identified as a hallmark of systemic inflammation and used as a risk marker of cardiovascular disease (CVD). Of interest was the relationship of these two biomarkers to oral health and CVD risk.MATERIAL AND METHODS:This investigation examined these two molecules in serum and unstimulated whole saliva of patients within 48 h of an acute myocardial infarction (AMI) compared to control subjects. We hypothesized a differential response in these biomolecules resulting from the heart attack that would be affected by both the body mass index and oral health characteristics of the individuals.RESULTS:Significantly lower adiponectin levels were observed in the serum of patients with AMI. Serum adiponectin in both groups and salivary adiponectin in patients with AMI decreased with increasing body mass index. Oral health was significantly worse in patients with AMI, and both serum and salivary adiponectin were elevated with better oral health in control subjects. Serum CRP levels were increased in patients with AMI regardless of their oral health, and both serum and salivary CRP were significantly elevated in S-T wave elevated patients with MI.CONCLUSIONS:These initial data provide evidence relating obesity and oral health to salivary and serum analyte levels that occur in association with cardiac events. Relationships have been described between CVD risk and periodontal disease. Additionally, various systemic inflammatory biomarkers appear to reflect both the CVD risk and the extent/severity of periodontitis. Our findings indicated that oral health and obesity contribute to altering levels of these salivary and serum analytes in association with cardiac events. The potential that serum and/or salivary biomarkers could aid in evaluating CVD risk requires knowledge regarding how the oral health of the individual would impact the effectiveness of these biological measures.