Subgingival microbial profiles in pre- and postmenopausal women: Associations with serum estradiol levels.

BACKGROUND: Subgingival dental plaque is an ecosystem playing a key role in supporting both oral health and systemic health. Menopause-related changes have the potential to disrupt its balance, which is crucial to postmenopausal well-being. Our study explored how circulating estradiol levels correlate with subgingival microbial composition using checkerboard DNA-DNA hybridization in premenopausal and postmenopausal women. We also demonstrated that combining this method with 16S ribosomal RNA (rRNA) sequencing insights remains valuable for examining subgingival ecology. METHODS: We assessed 40 bacterial species in 77 premenopausal and 81 postmenopausal women using checkerboard DNA-DNA hybridization and measured serum estradiol with enzyme-linked immunosorbent assay (ELISA). Women were categorized by subgingival dysbiosis severity using a modified Subgingival Microbial Dysbiosis Index (mSMDI). Six women from each normobiotic and dysbiotic subgroup across premenopausal and postmenopausal women underwent 16S rRNA sequencing analysis. RESULTS: DNA checkerboard analysis revealed that most observed variability in individual bacterial proportions is associated with periodontitis. Two species, Leptotrichia buccalis and Streptococcus constellatus, exhibited differences related to estradiol levels within the premenopausal group (p = 0.055 and p = 0.009, respectively). 16S rRNA sequencing confirmed the mSMDI's validity in categorizing normobiotic and dysbiotic states. Menopausal status was not associated with a dysbiotic shift in the subgingival microbiome despite significantly more attachment loss in postmenopausal compared to premenopausal women. CONCLUSIONS: Our results indicate that decreased estradiol levels or increased attachment loss during menopause are not associated with changes in species abundance or dysbiotic shifts in women. The mSMDI may be a useful tool for classifying subgingival ecology based on its normobiotic or dysbiotic inclination.

Arginine-specific gingipains (RgpA/RgpB) knockdown modulates neutrophil machinery.

Background: Gingipains are important virulence factors present in Porphyromonas gingivalis. Arginine-specific gingipains (RgpA and RgpB) are critically associated with increased proteolytic activity and immune system dysfunction, including neutrophilic activity. In this study, we assessed the impact of gingipains (RgpA and RgpB) on neutrophil function. Methods: Peripheral blood samples were obtained; neutrophils were isolated and incubated with P. gingivalis A7436, W50, and the double RgpA/RgpB double knockout mutant E8 at MOI 20 for 2 hours. Neutrophil viability was assessed by Sytox staining. Phagocytic capacity and apoptosis were measured by flow cytometry. Superoxide release was measured by superoxide dismutase and cytochrome c reduction assay. Gene expression of TLR2, p47-phox, p67-phox, and P2 × 7was measured by qPCR. Inflammatory cytokine and chemokine production was measured by IL-1ß, IL-8, RANTES, and TNF-α in cell supernatants. Results: Neutrophil TLR2 gene expression was reduced in the absence of RgpA/RgpB (p < 0.05), while superoxide production was not significantly impacted. RgpA/RgpB-/- significantly impaired neutrophil phagocytic function (p < 0.05) and increased TNF-α production when compared with the wild-type control (p < 0.05). Neutrophil apoptosis was not altered when exposed to RgpA/RgpB-/- E8 (p > 0.05). Conclusion: These data suggest that arginine-specific gingipains (RgpA/RgpB) can modulate neutrophil responses against P. gingivalis infection.

P. gingivalis-derived arginine-specific gingipains impaired the phagocytic and apoptotic function in neutrophils.

Oral microbial dysbiosis in patients with oral cavity cancers.

OBJECTIVES: The pathogenesis of oral cavity cancers is complex. We tested the hypothesis that oral microbiota dysbiosis is associated with oral cavity cancer. MATERIALS AND METHODS: Patients with primary oral cavity cancer who met the inclusion and exclusion criteria were included in the study. Matching healthy individuals were recruited as controls. Data on socio-demographic and behavioral factors, self-reported periodontal measures and habits, and current dental status were collected using a structured questionnaire and periodontal chartings. In addition to self-reported oral health measures, each participant received a standard and detailed clinical examination. DNA was extracted from saliva samples from patients and healthy controls. Next-generation sequencing was performed by targeting V3-V4 gene regions of the 16 S rRNA with subsequent bioinformatic analyses. RESULTS: Patients with oral cavity cancers had a lower quality of oral health than healthy controls. Proteobacteria, Aggregatibacter, Haemophilus, and Neisseria decreased, while Firmicutes, Bacteroidetes, Actinobacteria, Lactobacillus, Gemella, and Fusobacteria increased in oral cancer patients. At the species level, C. durum, L. umeaens, N. subflava, A. massiliensis, and V. dispar were significantly lower, while G. haemolysans was significantly increased (p < 0.05). Major periodontopathogens associated with periodontal disease (P. gingivalis and F.nucleatum) increased 6.5- and 2.8-fold, respectively. CONCLUSION: These data suggested that patients with oral cancer had worse oral health conditions and a distinct oral microbiome composition that is affected by personal daily habits and may be associated with the pathogenicity of the disease and interspecies interactions. CLINICAL RELEVANCE: This paper demonstrates the link between oral bacteria and oral cancers, identifying mechanistic interactions between species of oral microbiome.

Neutrophil response to Porphyromonas gingivalis is modulated by low-level laser application.

OBJECTIVES: Neutrophil response is critical in inflammatory regulation and immune response to bacterial infections. During periodontal disease, pathogenic bacteria lead to exaggerated neutrophil responses. We hypothesized that low-level laser application (LLLT), therapeutic strategy for dampening inflammatory processes, will regulate neutrophil activity in response to periodontopathogens. MATERIALS AND METHODS: The impact of LLLT on neutrophil responses was measured by light delivered at wavelength of 850 nm. The direct effect of LLLT on P. gingivalis A7436 was determined by flow cytometry using LIVE/DEADTM Cell Vitality kit. The phagocytosis of P. gingivalis A7436 by human neutrophils was measured using flow cytometry. Superoxide generation was measured by cytochrome-C-reduction in the presence of N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP; 1 mM). Cytokine release by neutrophils was measured by multiplex immunoassay. RESULTS: The phagocytosis of P. gingivalis by primary human neutrophils was significantly reduced in response to LLLT (p < 0.05). While LLLT led to increased superoxide production in neutrophils that were not challenged by P. gingivalis, it dampened the increased superoxide and IL-6 release by the neutrophils in response to P. gingivalis. LLLT did not directly affect the viability of P. gingivalis. CONCLUSION: These results suggested that LLLT can provide therapeutic strategy in periodontal disease, regulating the neutrophil response to P. gingivalis.

Oral and gut microbial profiling in periodontitis and Parkinson's disease.

Objectives: We tested the hypothesis that Parkinson's disease (PA) alters the periodontitis-associated oral microbiome. Method: Patients with periodontitis with Parkinson's disease (PA+P) and without PA (P) and systemically and periodontally healthy individuals (HC) were enrolled. Clinical, periodontal and neurological parameters were recorded. The severity of PA motor functions was measured. Unstimulated saliva samples and stool samples were collected. Next-generation sequencing of 16S ribosomal RNA (V1-V3 regions) was performed. Results: PA patients had mild-to-moderate motor dysfunction and comparable plaque scores as those without, indicating that oral hygiene was efficient in the PA+P group. In saliva, there were statistically significant differences in beta diversity between HC and PA+P (p = 0.001), HC and P (p = 0.001), and P and PA+P (p = 0.028). The microbial profiles of saliva and fecal samples were distinct. Mycoplasma faucium, Tannerella forsythia, Parvimonas micra, and Saccharibacteria (TM7) were increased in P; Prevotella pallens, Prevotella melaninogenica, Neisseria multispecies were more abundant in PA+P group, Ruthenibacterium lactatiformans, Dialister succinatiphilus, Butyrivibrio crossotus and Alloprevotella tannerae were detected in fecal samples in P groups compared to healthy controls. Conclusions: No significant differences were detected between Parkinson's and non-Parkinson's gut microbiomes, suggesting that Parkinson's disease modifies the oral microbiome in periodontitis subjects independent of the gut microbiome.

Genetic and environmental impact on mandibular growth in mono- and dizygotic twins during adolescence: A retrospective cohort study.

INTRODUCTION: This study aimed to discover the genetic and environmental factors that contribute to the mandibular development of untreated monozygotic and dizygotic twins. MATERIAL AND METHODS: The sample, taken from the Forsyth Moorrees Twin Study, included 52 untreated monozygotic twins (36 male, 16 female) and 46 untreated dizygotic twins (23 male, 23 female). At the ages of 12 and 17, lateral cephalograms were collected and traced to assess total mandibular length, mandibular ramus length, mandibular corpus length, gonial angle, SNB, and bony chin prominence. The genetic and environmental components of variation were assessed using multilevel mixed-effects structural equation modelling. RESULTS: At 12 years of age, high additive genetic influences were observed for total mandibular length (74%), gonial angle (76%), SNB (41%), and bony chin prominence (64%), whereas strong dominant genetic components were observed for corpus length (72%), and mandibular ramus length was under unique environment influence (54%). At 17 years of age, only total mandibular length (45%), ramus length (53%), gonial angle (76%), and bony chin prominence (68%) were under strong additive genetic control, while the remainder were under strong dominant genetic control. CONCLUSIONS: Although monozygotic and dizygotic twins share at least a portion of their DNA, additive, dominant, or environmental components were discovered during adolescence. Nonetheless, by the age of 17, the majority of the mandibular traits are under either additive or dominant genetic impact.

Modulating the sEH/EETs Axis Restrains Specialized Proresolving Mediator Impairment and Regulates T Cell Imbalance in Experimental Periodontitis.

Epoxyeicosatrienoic acids (EETs) and other epoxy fatty acids are short-acting lipids involved in resolution of inflammation. Their short half-life, due to its metabolism by soluble epoxide hydrolase (sEH), limits their effects. Specialized proresolving mediators (SPMs) are endogenous regulatory lipids insufficiently synthesized in uncontrolled and chronic inflammation. Using an experimental periodontitis model, we pharmacologically inhibited sEH, examining its impact on T cell activation and systemic SPM production. In humans, we analyzed sEH in the gingival tissue of periodontitis patients. Mice were treated with sEH inhibitor (sEHi) and/or EETs before ligature placement and treated for 14 d. Bone parameters were assessed by microcomputed tomography and methylene blue staining. Blood plasma metabololipidomics were carried out to quantify SPM levels. We also determined T cell activation by reverse transcription-quantitative PCR and flow cytometry in cervical lymph nodes. Human gingival samples were collected to analyze sEH using ELISA and electrophoresis. Data reveal that pharmacological sEHi abrogated bone resorption and preserved bone architecture. Metabololipidomics revealed that sEHi enhances lipoxin A4, lipoxin B4, resolvin E2, and resolvin D6. An increased percentage of regulatory T cells over Th17 was noted in sEHi-treated mice. Lastly, inflamed human gingival tissues presented higher levels and expression of sEH than did healthy gingivae, being positively correlated with periodontitis severity. Our findings indicate that sEHi preserves bone architecture and stimulates SPM production, associated with regulatory actions on T cells favoring resolution of inflammation. Because sEH is enhanced in human gingivae from patients with periodontitis and connected with disease severity, inhibition may prove to be an attractive target for managing osteolytic inflammatory diseases.

Wnt signaling in periodontitis.

OBJECTIVE: This study aimed to evaluate the Wnt/ß-catenin signaling pathway activity in gingival samples obtained from patients with periodontitis. MATERIALS AND METHODS: Fifteen patients with stage III grade B (SIIIGB) and eleven with stage III grade C (SIIIGC) periodontitis were included and compared to 15 control subjects. ß-Catenin, Wnt 3a, Wnt 5a, and Wnt 10b expressions were evaluated by Q-PCR. Topographic localization of tissue ß-catenin, Wnt 5a, and Wnt 10b was measured by immunohistochemical analysis. TNF-α was used to assess the inflammatory state of the tissues, while Runx2 was used as a mediator of active destruction. RESULTS: Wnt 3a, Wnt 5a, and Wnt 10b were significantly higher in gingival tissues in both grades of stage 3 periodontitis compared to the control group (p < 0.05). ß-Catenin showed intranuclear staining in connective tissue in periodontitis, while it was confined to intracytoplasmic staining in epithelial tissue and the cell walls in the control group. Wnt5a protein expression was elevated in periodontitis, with the most intense staining observed in the connective tissue of SIIIGC samples. Wnt10b showed the highest density in the connective tissue of patients with periodontitis. CONCLUSIONS: Our findings suggested that periodontal inflammation disrupts the Wnt/ß-catenin signaling pathway. CLINICAL RELEVANCE: Periodontitis disrupts Wnt signaling in periodontal tissues in parallel with tissue inflammation and changes in morphology. This change in Wnt-related signaling pathways that regulate tissue homeostasis in the immunoinflammatory response may shed light on host-induced tissue destruction in the pathogenesis of the periodontal disease.

Periodontal Disease, Local and Systemic Inflammation in Puerto Ricans with Type 2 Diabetes Mellitus.

Periodontal disease (PD) is prevalent in type 2 diabetic condition (T2DM). OBJECTIVES: We assessed the associations between serum or gingival crevicular fluid (GCF) endothelial and inflammatory mediators and chronic PD among T2DM Hispanic adults. METHODS: We enrolled 248 Puerto Rican residents with T2DM aged 40-65 years. The exposures included serum inflammatory mediators (IL-1b, IL-6, IL-10, and TNF-α), endothelial adhesion molecules, RANKL levels, and the GCF content of these analytes from a subset of 158 samples. The outcomes included the percent of sites with a probing pocket depth (PPD) ≥ 4 mm and clinical attachment loss ≥ 4 mm. Adjusted logistic regression models were fit to the categorized outcomes. RESULTS: Increased serum IL-10 (Adj. OR: 1.10, p = 0.04), sICAM-1 (Adj. OR: 1.01; p = 0.06), and elevated serum IL-1ß (Adj. OR: 1.93; p = 0.06) were statistically significant or close to being significantly associated with a percent of sites with PPD ≥ 4 mm. An increase in GCF IL-1α (Adj. OR: 1.16; p < 0.01) and IL-1ß (Adj: 2.40; p = 0.02) was associated with periodontal parameters. CONCLUSIONS: Our findings suggested that oral and systemic endothelial and inflammatory mediators are associated with periodontal clinical parameters among Hispanic adults with T2DM.

Porphyromonas gingivalis enhances the senescence-induced increase of 5-alpha reductase in gingival fibroblasts.

OBJECTIVES: Aging is characterized by chronic inflammatory activity. Senescent cells increase with chronic inflammation and age-related pathologies, including periodontal disease. As a critical regulator of tissue inflammaging, we hypothesized that 5α reductase (5αR) is associated with periodontal disease and bacteria-induced senescence in gingival fibroblasts. MATERIALS AND METHODS: We recruited 36 patients with periodontitis, measured 5αR immunohistochemically before and after periodontal treatment, and compared the expression of 5αR in gingival biopsies from 12 healthy individuals. We then tested the impact of Porphyromonas gingivalis on gingival fibroblasts treated with or without D-galactose-induced cell senescence. We treated primary gingival fibroblasts with D-galactose-supplemented media (0 µM, 50 µM, 100 µM, 1 mM, 10 mM, 50 mM) to induce senescence. The expression of type 1 and type 2 5αR was analyzed with real-time PCR and immunocytochemistry. The levels of IL-6, IL-8, TNF-α, and MCP-1 in fibroblast cultures were evaluated by multiplex immunoassay. RESULTS: In gingival biopsies from patients with periodontal disease, the expression of 5αR was significantly higher than in samples from individuals without periodontal disease (p < 0.001). Periodontal treatment significantly reduced the expression of 5αR in gingival tissues (p < 0.001) to levels comparable in healthy individuals. Gingival fibroblasts exposed to D-galactose-supplemented media had a dose-dependent and significant increase in 5αR expression (p < 0.001). P. gingivalis caused statistically higher type 1 and type 2 5αR expression in gingival fibroblast cells. This effect was exacerbated by the lower doses of D-galactose (p = 0.037). Cells infected with P. gingivalis produced significantly higher levels of IL-6, IL-8, TNF-α, and MCP-1 (p < 0.05) regardless of the D-galactose exposure. CONCLUSION: The results suggested that 5αR plays a role in periodontal disease and mediates the senescence-induced response to P. gingivalis in gingival fibroblasts. CLINICAL RELEVANCE: Periodontal diseases and aging can increase the production of 5-alpha reductase in the gingival tissue.

Parkinson's disease alters the composition of subgingival microbiome.

Aim: The current study aimed to test the hypothesis that Parkinson's disease exacerbates periodontitis by altering its microbiome. Materials and Methods: Clinical periodontal parameters were recorded. Subgingival samples from healthy controls, periodontitis patients (PD), and Parkinson's patients with periodontitis (PA+PD) were analyzed using the checkerboard DNA-DNA hybridization technique for targeting 40 bacterial species typically associated with periodontal disease and health. Next-generation sequencing (NGS) of the 16S ribosomal RNA gene (V1-V3 regions) was performed to analyze the microbiome comprehensively. Results: Parkinson's patients had mild-to-moderate motor dysfunctions. Bleeding on probing was significantly increased in the PA+PD group compared to PD (p < 0.05). With checkerboard analysis, PA was associated with increased Treponema socranskii (p = 0.0062), Peptostreptococcaceae_[G-6] [Eubacterium]_nodatum (p = 0.0439), Parvimona micra (p < 0.0001), Prevotella melaninogenica (p = 0.0002), Lachnoanaerobaculum saburreum (p < 0.0001), and Streptococcus anginosus (p = 0.0020). Streptococcus intermedia (p = 0.0042), P.nodatum (p = 0.0022), P. micra (p = 0.0002), Treponema denticola (p = 0.0045), L.saburreum (p = 0.0267), P.melaninogenica (p = 0.0017), Campylobacter rectus (p = 0.0020), and T.socranskii (p = 0.0002) were higher; Aggregatibacter actinomycetemcomitans (p = 0.0072) was lower in deep pockets in the PA+PD compared to PD. Schaalia odontolytica (p = 0.0351) and A.actinomycetemcomitans (p = 0.002) were lower; C.rectus (p = 0.0002), P. micra (p = 0065), Streptococcus constellatus (p = 0.0151), T.denticola (p = 0.0141), P.melaninogenica (p = 0.0057), and T.socranskii (p = 0.0316) were higher in shallow pockets in the PA+PD. Diversity decreased in PD (p = 0.001) and PA+PD (p = 0.026) compared to control, with minimal differences in alpha and beta diversities among PD and PA+PD based on NGS results. Conclusion: These data demonstrated that Parkinson's disease modifies PD-associated subgingival microbiome.

Association between androgenetic alopecia and periodontitis.

AIM: Androgenic alopecia (AGA) is men's most common form of hair loss. It is affected by changes in the expression and activity of 5αR and the metabolism of testosterone and DHT. There is an association between AGA and systemic inflammatory diseases. We hypothesized that there is an association between AGA and periodontal disease, as inflamed gingiva and periodontal fibroblasts have been shown to express more 5αR. Thus, this study aimed to evaluate the relationship between periodontal disease and AGA and the potential effect of aging on this association. MATERIALS AND METHODS: Out of a cohort of 1088 individuals, 385 white males aged 25-65 with similar socioeconomic levels and without systemic disease were included. Periodontitis was defined using NHANES data. AGA was evaluated using the Norwood-Hamilton scale. The relationship between AGA, periodontal disease severity, and age was assessed. RESULTS: There was a correlation between age and baldness (r = .421, p < .001). There was a significant correlation between AGA and periodontal disease in younger patients aged 25-34 and 35-44. (p < .042 and p < .036, respectively). There was no significant correlation between AGA and periodontal disease in the 45-54 and 55-65 age groups (p > .05). CONCLUSION: There may be a relationship between periodontal disease and AGA in the 25-44 age range, suggesting that this association starts at an early age in adulthood.

Parkinson's disease is positively associated with periodontal inflammation.

BACKGROUND: Parkinson's disease (PA) affects 1% of the global population above 60 years. PA pathogenesis involves severe neuroinflammation that impacts systemic and local inflammatory changes. We tested the hypothesis that PA is associated with periodontal tissue inflammation promoting a greater systemic inflammatory burden. METHODS: We recruited 60 patients with Stage III, Grade B periodontitis (P) with and without PA (n = 20 for each). We also included systemically and periodontally healthy individuals as controls (n = 20). Clinical periodontal parameters were recorded. Serum, saliva, and gingival crevicular fluid (GCF) samples were collected to measure the inflammatory and neurodegenerative targets (YKL-40, fractalkine, S100B, alpha-synuclein, tau, vascular cell adhesion protein-1 (VCAM-1), brain-derived neurotrophic factor (BDNF), neurofilament light chain (NfL). RESULTS: Parkinson's patients in this study had mild to moderate motor dysfunctions, which did not prevent them from performing optimal oral hygiene control. Periodontal parameters and GCF volume were significantly higher in the P and P+PA groups than in the control group. PA was associated with significantly increased bleeding on probing (BOP) compared to P-alone (p < 0.05), while other clinical parameters were similar between P and P+PA groups. In saliva and serum, YKL-40 levels were higher in the P+PA group than in P and C groups (p < 0.001). GCF NfL levels from shallow sites were significantly higher in the P+PA group compared to the C group (p = 0.0462). GCF S100B levels from deep sites were higher in the P+PA group than in healthy individuals (p = 0.0194). CONCLUSION: The data suggested that PA is highly associated with increased periodontal inflammatory burden-bleeding upon probing and inflammatory markers-in parallel with PA-related neuroinflammation.

Differential effects of resolvin D1 and resolvin E1 on cementoblast function.

BACKGROUND: Resolvins are endogenous mediators of the resolution of inflammation. They are derived from omega-3 polyunsaturated fatty acid precursors. Resolvin D1 (RvD1) and Resolvin E1 (RvE1) are the best-characterized members for actively promoting periodontal regeneration in experimental animal models. Here, we evaluated the efficacy of RvD1 and RvE1 on cementoblasts, the key cells involved in dental cementum regeneration and the attachment of the tooth to the alveolar bone. METHODS: Immortalized mouse cementoblasts (OCCM-30) were treated with different concentrations (0.1-1000 ng/mL) of RvD1 and RvE1. Cell proliferation was measured using an electrical impedance-based real-time cell analyzer. Mineralization was evaluated with von Kossa staining. The mRNA expression of mineralized tissue-associated markers of bone sialoprotein (BSP), Type I collagen (COL I), osteocalcin (OCN), osteopontin (OPN), runt-related transcription factor 2 (RunX2), alkaline phosphatase (ALP), osteoprotegerin (OPG), receptor activator of nuclear factor kappa B (NF-κB) (RANK), receptor activator of NF-κB ligand (RANKL), and extracellular matrix-degrading enzymes [matrix metalloproteinase (MMP)-1, MMP-2, MMP-3, MMP-9, and their tissue inhibitors (TIMP-1, TIMP-2)], RvE1 receptor (ChemR23) and RvD1 receptor (ALX/PFR2), cytokines (tumor necrosis factor-alpha {TNF-α}, interleukin {IL}-1ß, IL-6, IL-8, IL-10, IL-17), oxidative stress enzymes [superoxide dismutase (SOD), glutathione peroxidase (GPX), and cyclooxygenase-2 (Cox-2)] were analyzed using quantitative polymerase chain reaction (qPCR). RESULTS: Both RvD1 and RvE1 (10-100 ng/mL) significantly increased the proliferation of cementoblasts and mineralized nodules at all concentrations (p < 0.05). RvE1 increased BSP, RunX2, and ALP compared with the RvD1 dose and time-dependently, while RvD1 and RvE1 differentially regulated COL-I. RvE1 increased OPG mRNA expression, whereas RANK-RANKL mRNA expression decreased by RvE1. MMP-2, MMP-3, MMP-9, TIMP-1, and TIMP-2 expressions were reduced by RvE1 compared with RvD1. Treatment of cementoblasts with RvD1 and RvE1 differentially affected cytokine and oxidative stress enzymes while significantly increasing their receptor expressions (ChemR23 and ALX/PFR2). CONCLUSIONS: RvD1 and RvE1 regulate proliferation, mineralization, and gene expression in cementoblasts using similar pathways while differentially affecting tissue degradation, suggesting a targeted therapeutic approach for cementum turnover during periodontal regeneration.

Anti-inflammatory and pro-regenerative effects of a monoterpene glycoside on experimental periodontitis in a rat model of diabetes.

OBJECTIVE: Paeoniflorin (Pae) is a monoterpene glycoside with immune-regulatory effects. Several studies have already demonstrated the impact of Pae on periodontitis, but its effect on diabetic periodontitis is unclear. In this study, our aim was to test the hypothesis that Pae had a strong anti-inflammatory effect that prevented bone loss in diabetic periodontitis. METHODS: Thirty male Wistar albino rats were randomly divided into control (healthy, n = 10), periodontitis (PD) + diabetes (DM; n = 10), and PD + DM + Pae (n = 10) groups. Ligature-induced periodontitis was created by placing 4-0 silk ligatures around the lower first molars on both sides of the mandibulae. Experimental DM was created via an injection of 50 mg/kg and streptozotocin (STZ). Hyperglycemia was confirmed by the blood glucose levels of rats (>300 mg/dL). The bone mineral density (BMD), trabecular number, trabecular thickness, and bone loss were measured by micro-CT. The expression levels of IL-1ß, IL-6, and TNF-α were measured in tissue homogenates by ELISA. RESULTS: The PD + DM + Pae group had significantly less alveolar crest resorption when compared to the PD + DM group. There was also a significant difference between the PD + DM + Pae group compared to PD + DM group in trabecular thickness, BMD, and the number of trabeculae. Pae application led to a statistically significant decrease in IL-1ß, IL-6, and TNF-α levels in diabetic periodontitis. CONCLUSION: Systemic application of Pae suppressed inflammation caused by PD and DM, leading to reduced bone loss and enhanced bone quality.

Fusobacterium nucleatum dissemination by neutrophils.

Recent studies uncovered that Fusobacterium nucleatum (Fn), a common, opportunistic bacterium in the oral cavity, is associated with a growing number of systemic diseases, ranging from colon cancer to Alzheimer's disease. However, the pathological mechanisms responsible for this association are still poorly understood. Here, we leverage recent technological advances to study the interactions between Fn and neutrophils. We show that Fn survives within human neutrophils after phagocytosis. Using in vitro microfluidic devices, we determine that human neutrophils can protect and transport Fn over large distances. Moreover, we validate these observations in vivo by showing that neutrophils disseminate Fn using a zebrafish model. Our data support the emerging hypothesis that bacterial dissemination by neutrophils is a mechanistic link between oral and systemic diseases. Furthermore, our results may ultimately lead to therapeutic approaches that target specific host-bacteria interactions, including the dissemination process.

Microglial cell response to experimental periodontal disease.

OBJECTIVES: Microglial activation is critical for modulating the neuroinflammatory process and the pathological progression of neurodegenerative diseases, such as Alzheimer's disease (AD). Microglia are involved in forming barriers around extracellular neuritic plaques and the phagocytosis of ß-amyloid peptide (Aß). In this study, we tested the hypothesis that periodontal disease (PD) as a source of infection alters inflammatory activation and Aß phagocytosis by the microglial cells. METHODS: Experimental PD was induced using ligatures in C57BL/6 mice for 1, 10, 20, and 30 days to assess the progression of PD. Animals without ligatures were used as controls. Maxillary bone loss and local periodontal tissue inflammation associated with the development of PD were confirmed by morphometric bone analysis and cytokine expression, respectively. The frequency and the total number of activated microglia (CD45+ CD11b+ MHCII+) in the brain were analyzed by flow cytometry. Mouse microglial cells (1 × 105) were incubated with heat-inactivated bacterial biofilm isolated from the ligatures retrieved from the teeth or with Klebsiella variicola, a relevant PD-associated bacteria in mice. Expression of pro-inflammatory cytokines, toll-like receptors (TLR), and receptors for phagocytosis was measured by quantitative PCR. The phagocytic capacity of microglia to uptake ß-amyloid was analyzed by flow cytometry. RESULTS: Ligature placement caused progressive periodontal disease and bone resorption that was already significant on day 1 post-ligation (p < 0.05) and continued to increase until day 30 (p < 0.0001). The severity of periodontal disease increased the frequency of activated microglia in the brains on day 30 by 36%. In parallel, heat-inactivated PD-associated total bacteria and Klebsiella variicola increased the expression of TNFα, IL-1ß, IL-6, TLR2, and TLR9 in microglial cells (1.6-, 83-, 3.2-, 1.5-, 1.5-fold, respectively p < 0.01). Incubation of microglia with Klebsiella variicola increased the Aß-phagocytosis by 394% and the expression of the phagocytic receptor MSR1 by 33-fold compared to the non-activated cells (p < 0.0001). CONCLUSIONS: We showed that inducing PD in mice results in microglia activation in vivo and that PD-associated bacteria directly promote a pro-inflammatory and phagocytic phenotype in microglia. These results support a direct role of PD-associated pathogens in neuroinflammation.

The role of the host-Neutrophil biology.

Neutrophilic polymorphonuclear leukocytes (neutrophils) are myeloid cells packed with lysosomal granules (hence also called granulocytes) that contain a formidable antimicrobial arsenal. They are terminally differentiated cells that play a critical role in acute and chronic inflammation, as well as in the resolution of inflammation and wound healing. Neutrophils express a dense array of surface receptors for multiple ligands, ranging from integrins to support their egress from bone marrow into the circulation and from the circulation into tissues, to cytokine/chemokine receptors that drive their navigation to the site of infection or tissue damage and also prime them for a second stimulus, to pattern recognition receptors and immunoglobulin receptors to facilitate the destruction and removal of infective agents or debridement of damaged tissues. When afferent neutrophil signals are proportionate and coordinated they will phagocytose opsonized and unopsonized bacteria, activating the nicotinamide adenine dinucleotide phosphate oxidase (respiratory burst) to generate reactive oxygen species, which augment the proteolytic destruction of microbes secured within the phagosome. A highly orchestrated process of apoptosis follows, forming membrane-bound substructures that are removed by macrophages. Neutrophils are capable of various other forms of programmed cell death, such as NETosis and pyroptotic cell death, as well as nonprogrammed cell death by necrosis. In recent years, research has revealed that neutrophils are capable of far more subtle cell-cell interactions than previously thought possible. This includes synthesis of various inflammatory mediators and also myeloid cell training within bone marrow, where epigenetic and metabolic signals associated with returning neutrophils that undergo reverse egress from tissues into the vasculature and back to bone marrow program a hyperreactive subset of neutrophils during myelopoiesis that are capable of hypersensitive reactions to microbial aggressors. These characteristics are evident in various neutrophil subsets/subpopulations, creating broad heterogeneity in the behavior and biological repertoire of these seemingly schizophrenic immune cells. Moreover, neutrophils are critical effector cells of adaptive and innate immunity, binding to opsonized bacteria and destroying them by extracellular and intracellular methods. The former creates substantial collateral host tissue damage, as they are less specific than T-cytotoxic cell-killing mechanisms, and in conditions such as peri-implantitis, where plasma cells and neutrophils dominate the immune infiltrate, bone and tissue destruction are rapid and appear relentless. Finally, the role of neutrophils as conduits for periodontal-systemic disease connections and for oxidative damage to act as a causal link between the two has only recently been realized. In this chapter, we attempt to expand on these issues, emphasizing the contributions of European scientists throughout a detailed appraisal of the benefits and side effects of neutrophilic inflammation and immune function.

Impact of fixed orthodontic appliances on blood count and high-sensitivity C-reactive protein levels: A prospective cohort study.

INTRODUCTION: The aim was to elucidate the magnitude of alterations in systemic blood counts in healthy patients during the first 14 days after fixed orthodontic appliance placement. METHODS: This prospective cohort study consecutively included 35 White Caucasian patients starting orthodontic treatment with fixed appliances. The mean age was 24.48 ± 6.68 years. All patients were physically and periodontally healthy. Blood samples were collected at 3 time points: (1) baseline (exactly before the placement of appliances), (2) 5 days after bonding, and (3) 14 days after baseline. Whole blood and erythrocyte sedimentation rates were analyzed in automated hematology and erythrocyte sedimentation rate analyzer. Serum high-sensitivity C-reactive protein levels were measured by the nephelometric method. Standardized sample handling and patient preparation procedures were adopted to reduce preanalytical variability. RESULTS: A total of 105 samples were analyzed. All clinical and orthodontic procedures were performed without complications or side effects during the study period. All laboratory procedures were performed per protocol. Significantly lower white blood cell counts were detected 5 days after bracket bonding, compared with baseline (P <0.05). Hemoglobin levels were lower at 14 days than baseline (P <0.05). No other significant shifts or alteration patterns were observed over time. CONCLUSIONS: Orthodontic fixed appliances led to a limited and transient change in white blood cell counts and hemoglobin levels during the first days after bracket placement. The fluctuation of high-sensitivity C-reactive protein levels was not significant, demonstrating a lack of association between systemic inflammation and orthodontic treatment.

Soluble epoxide hydrolase inhibition enhances production of specialized pro-resolving lipid mediator and promotes macrophage plasticity.

BACKGROUND AND PURPOSE: Epoxyeicosatrienoic acids (EETs) and other epoxy fatty acids (EpFA) are lipid mediators that are rapidly inactivated by soluble epoxide hydrolase (sEH). Uncontrolled and chronic inflammatory disorders fail to sufficiently activate endogenous regulatory pathways, including the production of specialized pro-resolving mediators (SPMs). Here, we addressed the relationship between SPMs and the EET/sEH axis and explored the effects of sEH inhibition on resolving macrophage phenotype. EXPERIMENTAL APPROACH: Mice were treated with a sEH inhibitor, EETs, or sEH inhibitor + EETs (combination) before ligature placement to induce experimental periodontitis. Using RT-qPCR, gingival samples were used to examine SPM receptors and osteolytic and inflammatory biomarkers. Maxillary alveolar bone loss was quantified by micro-CT and methylene blue staining. SPM levels were analysed by salivary metabolo-lipidomics. Gingival macrophage phenotype plasticity was determined by RT-qPCR and flow cytometry. Effects of sEH inhibition on macrophage polarization and SPM production were assessed with bone marrow-derived macrophages (BMDMs). KEY RESULTS: Pharmacological inhibition of sEH suppressed bone resorption and the inflammatory cytokine storm in experimental periodontitis. Lipidomic analysis revealed that sEH inhibition augmented levels of LXA4, RvE1, RvE2, and 4-HDoHE, concomitant with up-regulation of LTB4R1, CMKLR1/ChemR23, and ALX/FPR2 SPM receptors. Notably, there is an impact on gingival macrophage plasticity was affected suggesting an inflammation resolving phenotype with sEH inhibition. In BMDMs, sEH inhibition reduced inflammatory macrophage activation, and resolving macrophages were triggered to produce SPMs. CONCLUSION AND IMPLICATIONS: Pharmacological sEH inhibition increased SPM synthesis associated with resolving macrophages, suggesting a potential target to control osteolytic inflammatory disorders.