Periodontitis and cardiometabolic disorders: The role of lipopolysaccharide and endotoxemia.

Lipopolysaccharide is a virulence factor of gram-negative bacteria with a crucial importance to the bacterial surface integrity. From the host's perspective, lipopolysaccharide plays a role in both local and systemic inflammation, activates both innate and adaptive immunity, and can trigger inflammation either directly (as a microbe-associated molecular pattern) or indirectly (by inducing the generation of nonmicrobial, danger-associated molecular patterns). Translocation of lipopolysaccharide into the circulation causes endotoxemia, which is typically measured as the biological activity of lipopolysaccharide to induce coagulation of an aqueous extract of blood cells of the assay. Apparently healthy subjects have a low circulating lipopolysaccharide activity, since it is neutralized and cleared rapidly. However, chronic endotoxemia is involved in the pathogenesis of many inflammation-driven conditions, especially cardiometabolic disorders. These include atherosclerotic cardiovascular diseases, obesity, liver diseases, diabetes, and metabolic syndrome, where endotoxemia has been recognized as a risk factor. The main source of endotoxemia is thought to be the gut microbiota. However, the oral dysbiosis in periodontitis, which is typically enriched with gram-negative bacterial species, may also contribute to endotoxemia. As endotoxemia is associated with an increased risk of cardiometabolic disorders, lipopolysaccharide could be considered as a molecular link between periodontal microbiota and cardiometabolic diseases.

Subgingival lipid A profile and endotoxin activity in periodontal health and disease.

OBJECTIVES: Regulation of lipopolysaccharide (LPS) chemical composition, particularly its lipid A domain, is an important, naturally occurring mechanism that drives bacteria-host immune system interactions into either a symbiotic or pathogenic relationship. Members of the subgingival oral microbiota can critically modulate host immuno-inflammatory responses by synthesizing different LPS isoforms. The objectives of this study were to analyze subgingival lipid A profiles and endotoxin activities in periodontal health and disease and to evaluate the use of the recombinant factor C assay as a new, lipid A-based biosensor for personalized, point-of-care periodontal therapy. MATERIALS AND METHODS: Subgingival plaque samples were collected from healthy individuals and chronic periodontitis patients before and after periodontal therapy. Chemical composition of subgingival lipid A moieties was determined by ESI-Mass Spectrometry. Endotoxin activity of subgingival LPS extracts was assessed using the recombinant factor C assay, and their inflammatory potential was examined in THP-1-derived macrophages by measuring TNF-α and IL-8 production. RESULTS: Characteristic lipid A molecular signatures, corresponding to over-acylated, bi-phosphorylated lipid A isoforms, were observed in diseased samples. Healthy and post-treatment samples were characterized by lower m/z peaks, related to under-acylated, hypo-phosphorylated lipid A structures. Endotoxin activity levels and inflammatory potentials of subgingival LPS extracts from periodontitis patients were significantly higher compared to healthy and post-treatment samples. CONCLUSIONS: This is the first study to consider structure-function-clinical implications of different lipid A isoforms present in the subgingival niche and sheds new light on molecular pathogenic mechanisms of subgingival biofilm communities. CLINICAL RELEVANCE: Subgingival endotoxin activity (determined by lipid A chemical composition) could be a reliable, bacterially derived biomarker and a risk assessment tool for personalized periodontal care.

The benefits of integrating dental and dental therapy and hygiene students in undergraduate curricula.

PURPOSE: The purpose of the current study was to evaluate the impact of integrating the teaching of Bachelor of Dental Surgery (BDS) and Bachelor of Dental Therapy and Hygiene (BScDTH) students in enquiry-based learning (EBL) sessions, using performance on multiple related integrated dental science (IDS) multiple-choice question assessments. METHOD: IDS assessments are sat twice in the first stages of both the BDS and BScDTH programmes. IDS scores from integrated and non-integrated cohorts were collated and compared across test occasions (first or second assessment of the stage) and programmes (BDS and BScDTH). RESULTS: The results revealed that IDS scores were, overall, significantly higher for students in integrated (M = 63.46, SD = 13.06) than non-integrated EBL groups (M = 60.75, SD = 13.67; F(1,207)  = 4.277, P = 0.040, < ! [ C D A T A [ η p 2 ] ] >  = 0.020). Although this effect was not statistically significant when each programme was considered separately, the effect of integration on both programmes was nevertheless positive, with a more pronounced improvement for BScDTH (+7.88) than BDS (+0.63) students. CONCLUSIONS: Integrating students from different programmes for the teaching of core dental knowledge in team environments improves student performance in subsequent dental science assessments-and more so for BScDTH than BDS students. The fact that both groups benefit from integration should go some way towards reassuring institutions that are considering integration but are cautious of threats to "established" programmes.

Effect of constant versus variable small-group facilitators on students' basic science knowledge in an enquiry-based dental curriculum.

INTRODUCTION: The role of small-group facilitators is of pivotal importance for the success of curricula based on active learning. Disorganised tutorial processes and superficial study of the problem have been identified as main hindering factors for students' learning. The aim of this study was to evaluate the influence of consistency of facilitation on students' performance in knowledge-based basic science assessments in a hybrid, enquiry-based (EBL) undergraduate dental curriculum. MATERIALS AND METHODS: This was a retrospective study of 519 first- and second-year undergraduate dental students, enrolled at Peninsula Dental School between 2013 and 2018. Twice in each academic year, students sat a 60-item single-best-answer, multiple-choice examination. Percentage and Z-scores were compared between students whose EBL groups had the same facilitator throughout the academic year, and those whose EBL group was facilitated by different members of staff. All EBL facilitators were dentally qualified but with different levels of expertise in basic dental sciences, prior EBL facilitation, involvement in the curriculum design and university affiliation. RESULTS: No statistically significant difference was observed in the percentage or Z-scores of students whose EBL sessions were supported by consistent or variable facilitators in any of the 18 MCQ tests. Z-scores of first-year students were more variable than for second-year students. In addition, pairwise comparisons revealed no statistically significant differences in students' Z-scores between any of the permanent facilitators' groups. CONCLUSIONS: The results of our study may influence the design and delivery of enquiry-based curricula as well as human resources management by shifting the focus from maintaining facilitator consistency to ensuring comparable training and approaches across facilitators.

Eradication of gastric Helicobacter pylori ameliorates halitosis and tongue coating.

BACKGROUND: The influence of gastric Helicobacter pylori infection on the development of oral pathoses remains unclear. The aim of this study is to examine the influence of gastric H. pylori infection on occurrence of halitosis and coated tongue. MATERIALS AND METHODS: Ninety-eight patients with dyspepsia were included in the study and their salivary samples and gastric biopsies were analyzed for the presence of H. pylori by Nested-PCR. Halitosis and coated tongue were assessed at the initial examination and 3 months after systemic eradication therapy against H. pylori. RESULTS: Gastric biopsies of 66 patients were positive for H. pylori. Only one saliva sample was H. pylori positive. At initial examination, halitosis was observed in 20 patients (30.3%) out of 66 who had gastric H. pylori infection and in only 3 patients (9.4%) out of 32 without H. pylori infection (p = 0.0236). Coated tongue was diagnosed in 18 (27.2%) patients with the infection compared to only 2 (6.25%) patients negative for gastric H. pylori (p = 0.0164). Patients with gastric infection were treated with the triple eradication therapy (Amoxicillin, Clarythromycin, Pantoprazol) and their gastric biopsies and oral status were examined 3 months later. Halitosis was significantly more prevalent in the group of patients with persistent H. pylori infection (42.1%) compared to only 6.4% of patients in the group where infection was successfully eradicated (p = 0.0012). Coated tongue was diagnosed in 47.4% of patients where H. pylori was still present after eradication therapy and in only 6.4% where eradication succeeded (p = 0.0003). CONCLUSION: Our findings suggest that eradication of gastric H. pylori significantly alleviates halitosis and coated tongue, the two oral conditions that may be considered as extragastric manifestations of this common chronic bacterial infection.

Altered Toll-like receptor 2-mediated endotoxin tolerance is related to diminished interferon beta production.

Induction of endotoxin tolerance leads to a reduced inflammatory response after repeated challenge by LPS and is important for resolution of inflammation and prevention of tissue damage. Enterobacterial LPS is recognized by the TLR4 signaling complex, whereas LPS of some non-enterobacterial organisms is capable of signaling independently of TLR4 utilizing TLR2-mediated signal transduction instead. In this study we report that Porphyromonas gingivalis LPS, a TLR2 agonist, fails to induce a fully endotoxin tolerant state in a human monocytic cell line (THP-1) and mouse bone marrow-derived macrophages. In contrast to significantly decreased production of human IL-8 and TNF-α and, in mice, keratinocyte-derived cytokine (KC), macrophage inflammatory protein-2 (MIP-2), and TNF-α after repeated challenge with Escherichia coli LPS, cells repeatedly exposed to P. gingivalis LPS responded by producing less TNF-α but sustained elevated secretion of IL-8, KC, and MIP-2. Furthermore, in endotoxin-tolerant cells, production of IL-8 is controlled at the signaling level and correlates well with NF-κB activation, whereas TNF-α expression is blocked at the gene transcription level. Interferon ß plays an important role in attenuation of chemokine expression in endotoxin-tolerized cells as shown in interferon regulatory factor-3 knock-out mice. In addition, human gingival fibroblasts, commonly known not to display LPS tolerance, were found to be tolerant to repeated challenge by LPS if pretreated with interferon ß. The data suggest that the inability of the LPS-TLR2 complex to induce full endotoxin tolerance in monocytes/macrophages is related to diminished production of interferon ß and may partly explain the involvement of these LPS isoforms in the pathogenesis of chronic inflammatory diseases.

Evaluating clinical utility of subgingival and salivary endotoxin activity levels as periodontal biomarkers.

Objectives: The use of periodontal biomarkers for identification and monitoring of unique patient populations could foster better stratification of at-risk groups, increase access to treatment for those most in need, facilitate preventive measures and improve personalised care plans. The aim of this study was to examine the diagnostic and prognostic utility of oral lipopolysaccharides as bacterially-derived periodontal biomarkers. Methods: Periodontal parameters were recorded, and saliva and subgingival plaque samples were collected at the beginning of the study from periodontally healthy volunteers and periodontitis patients, and three months after completion of conventional periodontal treatment in the periodontitis group. Endotoxin activity in the samples was measured using the recombinant factor C assay. Associations between clinical periodontal parameters and subgingival and salivary endotoxin activities were analysed using a multivariate regression model, while the ROC curve was applied to estimate the sensitivity, specificity and c-statistics for salivary and subgingival endotoxin activities as diagnostic biomarkers for periodontitis. Results: Significant correlations were found between subgingival endotoxin activities, probing pocket depth and periodontal diagnosis, which were independent from patients' age, gender and smoking status. In addition, subgingival endotoxin levels had high specificity and sensitivity in detecting periodontal health and disease (0.91 and 0.85 respectively). Salivary endotoxin activity was positively associated with periodontal diagnosis, mean probing pocket depth, percentages of sites over 4 mm and full mouth bleeding score. However, it was inferior in discriminating patients with stable periodontium from those with periodontitis (sensitivity = 0.69, specificity = 0.61) compared to subgingival endotoxin activity. Conclusions: Subgingival endotoxin activity has good diagnostic and prognostic values as a site-specific periodontal biomarker and is not influenced by the patient's age, gender or smoking status. In contrast, salivary endotoxin activity, as a patient-level biomarker, is dependent on patient's age, has poorer diagnostic and prognostic capability, but shows good correlations with disease susceptibility and both its extent and severity.

Impaired immune tolerance to Porphyromonas gingivalis lipopolysaccharide promotes neutrophil migration and decreased apoptosis.

Periodontitis, a chronic inflammatory disease of the tissues supporting the teeth, is characterized by an exaggerated host immune and inflammatory response to periopathogenic bacteria. Toll-like receptor activation, cytokine network induction, and accumulation of neutrophils at the site of inflammation are important in the host defense against infection. At the same time, induction of immune tolerance and the clearance of neutrophils from the site of infection are essential in the control of the immune response, resolution of inflammation, and prevention of tissue destruction. Using a human monocytic cell line, we demonstrate that Porphyromonas gingivalis lipopolysaccharide (LPS), which is a major etiological factor in periodontal disease, induces only partial immune tolerance, with continued high production of interleukin-8 (IL-8) but diminished secretion of tumor necrosis factor alpha (TNF-α) after repeated challenge. This cytokine response has functional consequences for other immune cells involved in the response to infection. Primary human neutrophils incubated with P. gingivalis LPS-treated naïve monocyte supernatant displayed a high migration index and increased apoptosis. In contrast, neutrophils treated with P. gingivalis LPS-tolerized monocyte supernatant showed a high migration index but significantly decreased apoptosis. Overall, these findings suggest that induction of an imbalanced immune tolerance in monocytes by P. gingivalis LPS, which favors continued secretion of IL-8 but decreased TNF-α production, may be associated with enhanced migration of neutrophils to the site of infection but also with decreased apoptosis and may play a role in the chronic inflammatory state seen in periodontal disease.

Comparative analysis of total salivary lipopolysaccharide chemical and biological properties with periodontal status.

OBJECTIVE: Clinical manifestations of Gram-negative bacteria mediated diseases can be influenced by how the host senses their major microbe-associated molecular pattern, the cell wall lipopolysaccharide (LPS). Keystone periodontal pathogens can produce a heterogeneous population of LPS molecules, with strikingly different host-microbiome interactions and immune outcomes. DESIGN: Structure-function correlations of salivary LPS extracts in patients with periodontitis before and after periodontal treatment and healthy volunteers were analysed by comparing its lipid A and carbohydrate chain chemical structure and evaluating its endotoxin activity and inflammatory potential. RESULTS: Salivary LPS extracts from periodontitis patients were characterised by high m/z lipid A mass-spectrometry peaks, corresponding to over-acylated and phosphorylated lipid A ions and by a combination of rough and smooth LPS carbohydrate moieties. In contrast, gingival health was defined by the predominance of low m/z lipid A peaks, consistent with under-acylated and hypo-phosphorylated lipid A molecular signatures, with long and intermediate carbohydrate chains as determined by silver staining. Total, diseased salivary LPS extracts were stronger inducers of the recombinant factor C assay and triggered significantly higher levels of TNF-α, IL-8 and IP-10 production in THP-1 cells, compared to almost immunosilent healthy samples. Interestingly, salivary LPS architecture, endotoxin activity, and inflammatory potential were well conserved after periodontal therapy and showed similarities to diseased samples. CONCLUSIONS: This study sheds new light on molecular pathogenic mechanisms of oral dysbiotic communities and indicates that the regulation of LPS chemical structure is an important mechanism that drives oral bacteria-host immune system interactions into either a symbiotic or pathogenic relationship.

Sialylation of Porphyromonas gingivalis LPS and its effect on bacterial-host interactions.

Porphyromonas gingivalis produces different LPS isoforms with significant structural variations of their lipid A and O-antigen moieties that can affect its pro-inflammatory and bone-resorbing potential. We show here, for the first time, that P. gingivalis LPS isolated from W83 strain is highly sialylated and possesses significantly reduced inflammatory potential compared with less sialylated ATCC 33277 strain LPS. Nevertheless, the reduction in the endotoxin activity is not mediated by the presence of sialic acid LPS moieties as the sialic acid-free LPS produced by the mutant W83 strain exhibits a similar inflammatory potential to the wild type strain. Furthermore, our findings suggest that the interaction between the sialic acid LPS moieties and the inhibitory CD33 receptor is prevented by endogenously expressed sialic acid on the surface of THP-1 cells that cannot be out-competed by sialic acid containing P. gingivalis LPS. The present study also highlights the importance of endogenous sialic acid as a 'self-associated molecular pattern' and CD33 receptors in modulation of innate immune response as human gingival fibroblasts, which do not express CD33 receptors, and desialylated THP-1 cells have both been found to have much higher spontaneous IL-8 production than naïve THP-1 cells.