Specific host metabolite and gut microbiome alterations are associated with bone loss during spaceflight.

Understanding the axis of the human microbiome and physiological homeostasis is an essential task in managing deep-space-travel-associated health risks. The NASA-led Rodent Research 5 mission enabled an ancillary investigation of the gut microbiome, varying exposure to microgravity (flight) relative to ground controls in the context of previously shown bone mineral density (BMD) loss that was observed in these flight groups. We demonstrate elevated abundance of Lactobacillus murinus and Dorea sp. during microgravity exposure relative to ground control through whole-genome sequencing and 16S rRNA analyses. Specific functionally assigned gene clusters of L. murinus and Dorea sp. capable of producing metabolites, lactic acid, leucine/isoleucine, and glutathione are enriched. These metabolites are elevated in the microgravity-exposed host serum as shown by liquid chromatography-tandem mass spectrometry (LC-MS/MS) metabolomic analysis. Along with BMD loss, ELISA reveals increases in osteocalcin and reductions in tartrate-resistant acid phosphatase 5b signifying additional loss of bone homeostasis in flight.

Resolvin D1 modulates periodontal ligament fibroblast function.

BACKGROUND: The resolution of inflammation is an active process mediated by specialized lipid mediators called lipoxins and resolvins. Periodontal ligament fibroblasts (PDLFs) play a significant role in periodontal regeneration. The purpose of the current study was to determine the impact of resolvin D1 (RvD1) on human PDLF cell wound healing and proliferation, receptor expression (G-protein-coupled receptor 32 [GPR32] and formyl peptide receptor 2 [ALX/FPR2]), and cytokine expression and release. METHODS: PDLFs were stimulated with interleukin-1ß (IL-1ß) (500 pg/ml) with and without RvD1 (100 nM). RvD1 receptor expression was determined by quantitative real-time polymerase chain reaction (qPCR), immunofluorescence microscopy, and fluorescence-activated cell sorting. Wound closure was measured by a scratch assay, and proliferation was determined by bromodeoxyuridine incorporation. Interleukin-6 (IL-6), interleukin-8 (IL-8), monocyte chemoattractant protein-1, cyclooxygenase-2, matrix metalloproteinases-1, -2, and -3 (MMP-1, -2, and -3), tissue inhibitors of metalloproteinases-1 and -2 (TIMP-1 and -2), prostaglandin E2, and osteoprotegerin (OPG) gene expression and production were measured using qPCR and Western blotting, multiplex immunoassay, and enzyme-linked immunosorbent assay. RESULTS: PDLF expressed GPR32 and ALX/FPR2. RvD1 reversed IL-1ß-induced inhibition of wound healing and proliferation of PDLF. IL-1ß also induced the production of proinflammatory cytokines and MMPs. This effect was reversed by RvD1. RvD1 reversed IL-1ß-induced inhibition of TIMP-1, TIMP-2, and OPG. CONCLUSION: The data suggested that RvD1 has a pro-wound healing, proliferative, and anti-inflammatory impact on the PDLF that favors periodontal regeneration.

An isolated population reveals greater genetic structuring of the Australian dingo.

The Australian dingo is a recent anthropogenic addition to the Australian fauna, which spread rapidly across the continent and has since widely interbred with modern dogs. Genetic studies of dingoes have given rise to speculation about their entry to the continent and subsequent biogeographic effects, but few studies of their contemporary population structure have been conducted. Here we investigated the dingo ancestry and population structure of free-living dogs in western Victoria and contrasted it with a wider southern Australian sample. We wished to determine whether their geographic isolation was mirrored in genetic isolation. To address this question, we analysed 34 microsatellite markers using Bayesian clustering and discriminant analysis of principal components, and summarised genetic diversity at the population and individual level. The broader southern Australia sample (n = 1138) comprised mostly hybrid animals, with 30% considered pure dingoes. All western Victorian individuals (n = 59) appeared to be hybrids with high dingo ancestry. The population showed no evidence of admixture with other populations and low genetic diversity on all measures tested. Based upon our characterisation of this unusual mainland population, we advise against assuming homogeneity of dingoes across the continent.

Resolvin E1 Reduces Tumor Growth in a Xenograft Model of Lung Cancer.

Inflammation plays a significant role in carcinogenesis and tumor growth. The current study was designed to test the hypothesis that resolvin E1 (RvE1) and overexpression of the receptor for RvE1 (ERV1) will prevent and/or reverse tumor generation in a gain-of-function mouse model of tumor seeding with lung cancer cells. To measure the impact of enhanced resolution of inflammation on cancer pathogenesis, ERV1-overexpressing transgenic (TG) and wild-type FVB mice were given an injection of 1 × 106 LA-P0297 cells subcutaneously and were treated with RvE1 (100 ng; intraperitoneally) or placebo. To assess the impact of RvE1 as an adjunct to chemotherapy, ERV1-TG and wild-type FVB mice were treated with cisplatin or cisplatin + RvE1. RvE1 significantly prevented tumor growth and reduced tumor size, cyclooxygenase-2, NF-κB, and proinflammatory cytokines in TG animals as compared to wild-type animals. A significant decrease in Ki-67, vascular endothelial growth factor, angiopoietin (Ang)-1, and Ang-2 was also observed in TG animals as compared to wild-type animals. Tumor-associated neutrophils and macrophages were significantly reduced by RvE1 in transgenics (P < 0.001). RvE1 administration with cisplatin led to a significant reduction of tumor volume and reduced cyclooxygenase-2, NF-κB, vascular endothelial growth factor-A, Ang-1, and Ang-2. These data suggest that RvE1 prevents inflammation and vascularization, reduces tumor seeding and tumor size, and, when used as an adjunct to chemotherapy, enhances tumor reduction at significantly lower doses of cisplatin.

Active shedding of Neospora caninum detected in Australian wild canids in a nonexperimental context.

Infection with Neospora caninum parasites is a leading cause of reproduction losses in cattle worldwide. In Australia, this loss is estimated to total AU$110 million every year. However, despite this considerable economic impact, the transmission cycle and the host(s) responsible for the sylvatic transmission of the parasite remain to be defined. Dingoes (Canis familiaris) have been suggested to be a wildlife host of N. caninum in Australia, but this is yet to be proven in a nonexperimental setting. This study aimed to determine the prevalence of natural N. caninum shedding in Australian wild dogs (defined as dingoes, dingo-domestic dog hybrids and feral dogs) by performing molecular analysis of faecal samples collected in wild dog populations in south-east Australia. Molecular analysis allowed host species identification and dingo purity testing, while genetic analysis of Coccidia and Neospora conserved genes allowed for parasite identification. Among the 115 samples collected and determined to belong to dingoes, dingo-domestic dog hybrids and foxes, Coccidian parasites were detected in 41 samples and N. caninum was identified in one sample of canine origin from South East Australia (Mansfield). Across all samples collected in Mansfield only 15 individuals were successfully identified by genotype. Thereby our study determined that 6.7% (1/15, 95% confidence intervals 1.2-29.9) of wild dogs were actively shedding N. caninum oocysts at this site. Further, only four individuals were identified at a second site (Swift Creek), and none were positive. This study conclusively confirms the role of wild dogs in the horizontal transmission of N. caninum parasites in Australia.

Longitudinal assessment of salivary vitamin D binding protein during orthodontic tooth movement.

BACKGROUND: Vitamin D is critical for bone physiology. In this study, we quantified Vitamin D Binding Protein (VitDBP) levels in saliva as a measure of Vitamin D during orthodontic tooth movement. METHODS: In this longitudinal study, saliva samples were collected from 73 orthodontic patients for 4 timepoints for the first six months of orthodontic treatment, along with dental casts at the beginning and the end of the study period. The saliva was measured for VitDBP as a biological marker for bone apposition and clinical tooth movement. We used the absolute change in Little's Irregularity Index as a quantitative measure for alignment. In addition, we measured the levels of alkaline phosphatase (ALP) in saliva as a marker of bone turnover. RESULTS: Both low (< 2.75 ng/ml) and high (> 6.48 ng/ml) VitDBP levels were associated with reduced tooth movement. Significant (p < 0.05) seasonal changes in VitDBP using a two-season year model were found with lower levels observed in the summer (Apr-Sept) than in the winter (Oct-Mar). CONCLUSIONS: Clinically significant orthodontic tooth movement is associated with an optimal range of VitDBP in saliva. Normal levels of VitDBP correlated with more orthodontic tooth movement, suggesting a "normal" range of salivary content of VitDBP. Given the strong trend that is independent of the confounding factors (ex. age, race or gender), the predictive value or salivary VitDBP for tooth movement should be studied in larger cohorts in future studies.

Resolvin E1 Regulates Th17 Function and T Cell Activation.

Resolvin E1 (RvE1) is a specialized pro-resolving lipid mediator derived from eicosapentaenoic acid and plays a critical role in resolving inflammation and tissue homeostasis. Th17 cells are a distinct group of T helper (Th) cells with tissue-destructive functions in autoimmune and chronic inflammatory diseases via the secretion of IL-17. Dendritic cell (DC)-mediated antigen presentation regulates the Th17-induced progression of inflammation and tissue destruction. In this study, we hypothesized that the RvE1 would restore homeostatic balance and inflammation by targeting the Th17 function. We designed three experiments to investigate the impact of RvE1 on different phases of Th17 response and the potential role of DCs: First CD4+ T cells were induced by IL-6/TGFß to measure the effect of RvE1 on Th17 differentiation in an inflammatory milieu. Second, we measured the impact of RvE1 on DC-stimulated Th17 differentiation in a co-culture model. Third, we measured the effect of RvE1 on DC maturation. RvE1 blocked the CD25, CCR6 and IL-17 expression; IL-17, IL-21, IL-10, and IL-2 production, suggesting inhibition of T cell activation, Th17 stimulation and chemoattraction. RvE1 also suppressed the activation of DCs by limiting their pro-inflammatory cytokine production. Our findings collectively demonstrated that the RvE1 targeted the Th17 activation and the DC function as a potential mechanism for inflammatory resolution and acquired immune response.

Periodontal inflammation and distinct inflammatory profiles in saliva and gingival crevicular fluid compared with serum and joints in rheumatoid arthritis patients.

BACKGROUND: The association of periodontitis and Porphyromonas gingivalis (Pg) with rheumatoid arthritis (RA) is incompletely understood. To gain further insights, we evaluated periodontal status, oral, serum and joint inflammatory profiles, and Pg biomarkers in RA patients. METHODS: In this cross-sectional study, we evaluated 33 patients with predominantly untreated new-onset RA, 20 healthy individuals (HIs), and 20 non-RA chronic periodontitis patients. Thirteen mediators (IFN-γ, IL-10, IL-17A, IL-6, IL-8, CXCL10, TNF-α, CXCL13, IL-23, MMP-1, MMP-3, MMP-8, MMP-9) were measured in serum, synovial fluid, saliva and gingival crevicular fluid (GCF) by multiplex immunoassay. Serum Pg IgG antibodies and subgingival Pg DNA were determined. RESULTS: Most RA patients (91%) received routine dental care; only one currently smoked. Ten (30.3%) had periodontal health, 13 (39.4%) had gingivitis, and 10 (30.3%) had periodontitis. Th1 and innate immune responses predominated in serum. Many mediators were concentrated in joints, particularly IL-6, IL-8, and CXCL10. However, salivary and GCF profiles were more restricted, emphasizing neutrophilic inflammation (IL-8, MMP-8) and MMP-9. Compared with HI, most RA patients, regardless of periodontal status, had significantly elevated oral fluid levels of these mediators, with suppression of GCF IL-10, a pattern similar to non-RA periodontitis patients. Pg antibodies or DNA however were primarily associated with clinical periodontitis. CONCLUSIONS: Despite routine dental care, RA patients often had inflammation in oral fluids, but inflammatory profiles differed from serum and joints. Neutrophilic inflammatory profiles in oral fluids, regardless of periodontal status, suggests that gingival tissues are a common, and often unrecognized, site of extra-articular inflammation in RA.

Use of amnion-derived cellular cytokine solution for the treatment of gingivitis: A 2-week safety, dose-ranging, proof-of-principle randomized trial.

BACKGROUND: A 6-week Phase I clinical trial was performed to primarily evaluate the safety and secondarily determine the preliminary efficacy of a novel biological solution, ST266, comprised of a mixture of cytokines, growth factors, nucleic acids, and lipids secreted by cultured amnion-derived multipotent progenitor cells on gingival inflammation. METHODS: Fifty-four adults with gingivitis/periodontitis were randomly assigned to 1X ST266 or diluted 0.3X ST266 or saline topically applied on facial/lingual gingiva (20 µL/tooth). Safety was assessed through oral soft/hard tissue exam, adverse events, and routine laboratory tests. Efficacy was assessed by modified gingival index (MGI), bleeding on probing, plaque index, probing depth (PD), and clinical attachment level (CAL). Assessments were performed on day 0, 8, 12, and 42. ST266 and saline applied daily starting at day 0 through day 12 except weekend days. Plasma was analyzed for safety and proinflammatory cytokines, interleukin (IL)-1ß, IL-6, tumor necrosis factor-alpha, and interferon gamma. Gingival crevicular fluid (GCF) was analyzed for the same cytokines. Subgingival plaque was primarily analyzed by checkerboard DNA-DNA hybridization. Comparisons with saline were modeled through a generalized estimating equations method adjusting for baseline. RESULTS: No safety concern was found related to ST266. Statistically significant reduction in MGI was noted at day 42 by 1X ST266 compared with saline (P = 0.044). PD and CAL were reduced by both doses of ST266 at day 42 (P <0.01) and by 1X ST266 at day 12 (P <0.05). GCF IL-1ß and IL-6 levels were reduced by both doses of ST266 at day 12 (P <0.05, P <0.01, respectively). IL-6 was also significantly reduced in plasma of both ST266 groups (P <0.05). Significant reductions in red complex bacteria were detected in both ST266 doses. CONCLUSIONS: In this "first in human oral cavity" study, topical ST266 was safe and effective in reducing gingival inflammation in 6 weeks. Longitudinal studies with large sample sizes are warranted to assess the therapeutic value of this novel host modulatory compound in the treatment of periodontal diseases.

TLR2 and TLR4 Differentially Regulate the Osteogenic Capacity of Human Periodontal Ligament Fibroblasts.

AIMS: To test that the osteogenic capacity of periodontal ligament (PDL) fibroblasts can be mediated by TLR2 and TLR4 activation. MATERIALS AND METHODS: Human PDL fibroblasts were cultured in osteogenic medium and treated with TLR2 and TLR4 agonists (Pam3CSK4 and monophosphoryl Lipid A (MPLA), respectively). Cell proliferation was measured by MTT and BrdU incorporation. Osteogenic differentiation was measured by alkaline phosphatase (ALP) activity. Nodule formation was measured for osteoblast function. The expression of markers of potential signaling pathways (RUNX2, OCN, BSP and Osterix) was evaluated by quantitative PCR. RESULTS: PDL fibroblasts grew at the same rate during the first 5 days in response to both Pam3CSK5 and MPLA. On day 7, cells cultured in the presence of Pam3CSK4 had a significantly higher rate of DNA replication, while cells in MPLA group had a significantly lower DNA replication rate (one-third) compared to the control (p less than 0.05). Pam3CSK4 induced significantly higher ALP activity and larger calcified nodules. TLR4 activation significantly reduced the expression of RUNX2 and osterix and enhanced OCN. Neither TLR2 nor TLR4 affected BSP expression. CONCLUSIONS: These data suggest that the activation of TLR2 and TLR4 differentially and perhaps antagonistically modulate osteogenesis by human PDL fibroblasts and have a direct role of TLR-mediated PDL function during periodontal regeneration as a potential target for therapeutics.

Microglial response to experimental periodontitis in a murine model of Alzheimer's disease.

Periodontal disease (PD) has been suggested to be a risk factor for Alzheimer's disease (AD). We tested the impact of ligature-induced PD on 5xFAD mice and WT littermates. At baseline, 5xFAD mice presented significant alveolar bone loss compared to WT mice. After the induction of PD, both WT and 5xFAD mice experienced alveolar bone loss. PD increased the level of Iba1-immunostained microglia in WT mice. In 5xFAD mice, PD increased the level of insoluble Aß42. The increased level in Iba1 immunostaining that parallels the accumulation of Aß in 5xFAD mice was not affected by PD except for a decrease in the dentate gyrus. Analysis of double-label fluorescent images showed a decline in Iba1 in the proximity of Aß plaques in 5xFAD mice with PD compared to those without PD suggesting a PD-induced decrease in plaque-associated microglia (PAM). PD reduced IL-6, MCP-1, GM-CSF, and IFN-γ in brains of WT mice and reduced IL-10 in 5xFAD mice. The data demonstrated that PD increases neuroinflammation in WT mice and disrupts the neuroinflammatory response in 5xFAD mice and suggest that microglia is central to the association between PD and AD.

Spatially-explicit model for assessing wild dog control strategies in Western Australia.

Large predators can significantly impact livestock industries. In Australia, wild dogs (Canis lupus familiaris, Canis lupus dingo, and hybrids) cause economic losses of more than AUD$40M annually. Landscape-scale exclusion fencing coupled with lethal techniques is a widely practiced control method. In Western Australia, the State Barrier Fence encompasses approximately 260,000km2 of predominantly agricultural land, but its effectiveness in preventing wild dogs from entering the agricultural region is difficult to evaluate. We conducted a management strategy evaluation (MSE) based on spatially-explicit population models to forecast the effects of upgrades to the Western Australian State Barrier Fence and several control scenarios varying in intensity and spatial extent on wild dog populations in southwest Western Australia. The model results indicate that populations of wild dogs on both sides of the State Barrier Fence are self-sustaining and current control practices are not sufficient to effectively reduce their abundance in the agricultural region. Only when a combination of control techniques is applied on a large scale, intensively and continuously are wild dog numbers effectively controlled. This study identifies the requirement for addressing extant populations of predators within fenced areas to meet the objective of preventing wild dog expansion. This objective is only achieved when control is applied to the whole area where wild dogs are currently present within the fence plus an additional buffer of ~20 km. Our modelling focused on the use of baiting, trapping and shooting; however, we acknowledge that additional tools may also be applied. Finally, we recommend that a cost-benefit analysis be performed to evaluate the economic viability of an integrated control strategy.

Hypoxia-induced endothelial cell responses - possible roles during periodontal disease.

Background and objective Inflammatory periodontal pockets are known to be hypoxic. Hypoxia influences vascular response to periodontal inflammation, including angiogenesis, which is critical for oxygen and nutrient delivery to periodontal tissues and granulation tissue formation. Our previous work suggests that periodontal bacteria may actively contribute to pocket hypoxia. Herein, we test the hypothesis that Fusobacterium nucleatum actively induces low oxygen tension, which modulates angiogenesis and endothelial cell activity. HUVEC cells were incubated in 1.5% oxygen for (Folkman & Shing, 1992)48 hours. Cell proliferation was measured by MTT; surface expression of CD31, CD34 and VEGF receptors (VEGFR1, VEGFR2) were analyzed by FACS. mRNA expression of HIF isoforms, iNOS, eNOS, COX-2, and VEGF was measured by quantitative PCR. Supernatants were analyzed for the release of IL-1α, TNF-α, and VEGF by ELISA or multiplex immunoassays and nitric oxide was measured by colorimetric assay. F. nucleatum actively depleted oxygen. Hypoxia resulted in a significant increase of HIF isoforms. iNOS was increased while nitric oxide was unchanged. VEGF release was increased at 4 hours followed by an increase in VEGFR1 at 12 hours, but not VEGFR2. CD31 expression was reduced and CD34 was increased after 48 hours (p < 0.05). IL-1α and TNF-α release were decreased at 4 hours (p < 0.05), but both increased by 24 hours; TNF-α increased at 24 h. The data highlight the role of hypoxia in endothelial cell inflammatory changes. F. nucleatum, considered a bridging species in the development of periodontopathic biofilms induces hypoxia in the periodontium leading to angiogenic changes in periodontal disease pathogenesis.

Combined administration of resolvin E1 and lipoxin A4 resolves inflammation in a murine model of Alzheimer's disease.

Dysfunction in the resolution of inflammation may play a key role in Alzheimer's disease (AD). In this study, we found that the levels of specialized pro-resolving lipid mediators (SPMs) in the hippocampus of 5xFAD mice are significantly lower than in non-transgenic littermates. We, therefore, tested the hypothesis that treatment with resolvin E1 (RvE1) and lipoxin A4 (LXA4) alone or in combination will reverse the neuroinflammatory process and decrease Aß pathology. 5xFAD mice were treated intraperitoneally starting at 1month of age with RvE1 or LXA4 alone or in combination at a dose of 1.5 µg/kg, 3 times a week until 3months of age. We found that treatment with RvE1 or LXA4 alone or in combination increased the concentration of RvE1, LXA4, and RvD2 in the hippocampus as measured by ELISA. Combination treatment of RvE1 and LXA4 had a more potent effect on the activation of microglia and astrocytes than either treatment alone, measured by immunohistochemistry with Iba1 and GFAP antibodies, respectively. The concentrations of Aß40 and Aß42 were measured by ELISA and the percentage of Aß plaques were analyzed by immunohistochemistry. All treatments single and in combination, decreased the measures of Aß pathology and restored the homeostasis reversing the inflammatory process for inflammatory cytokines and chemokines (GM-CSF, IFN-γ, IL-1ß, IL-6, IL-10, TNF-α, MCP-1, MIP-1α, MIP-1ß, and RANTES) as measured by multiplex immunoassay. Overall, the study showed that the levels of SPMs in the hippocampus of 5xFAD mice were significantly lower than in wild-type mice; that treatment with RvE1 and LXA4 restored the level of these compounds, reversed the inflammatory process, and decreased the neuroinflammation associated with Aß pathology in 5xFAD mice.

Spontaneous PMN apoptosis in type 2 diabetes and the impact of periodontitis.

The purpose of this study was to test the hypothesis that peripheral blood neutrophils (PMN) exhibit delayed spontaneous apoptosis in individuals with diabetes mellitus type 2 (T2DM) and that the delay is exacerbated further among people who coexpress chronic periodontitis (CP). Seventy-three individuals were enrolled, including those with T2DM (n = 16), CP (n = 15), T2DM + CP (n = 21), and healthy volunteers (n = 21). PMN apoptosis was determined by flow cytometry using TUNEL and Annexin V assays. The activity of caspase-3, -8, and -9 was measured by colorimetric assay. PMN surface death receptor quantification was performed by flow cytometry staining with fluorescence-conjugated anti-CD120a (TNFR1) and anti-CD95 [Fas receptor (FasR)] antibody. Analysis of inflammatory markers in serum samples was performed using multiplexed sandwich immunoassays. In healthy volunteers and individuals with T2DM, CP, and T2DM + CP, spontaneous PMN apoptosis observed at 12 h reached 85.3 ± 3.1, 67.3 ± 3.9, 62.9 ± 3.5 and 62.5 ± 5.4%, respectively (P < 0.05). Caspase-3 activity was significantly reduced in individuals with T2DM and T2DM + CP (P < 0.05) when compared with healthy volunteers. Caspase-8 activity was also significantly decreased in CP and T2DM + CP (P < 0.05), associated with reduced cell-surface FasR, TNFRs, and Fas ligand (FasL) serum levels. Glucose alone was not observed to impact PMN apoptosis; simultaneous incubation with the receptor for advanced glycation endproducts (RAGE) agonist S100B induced significant PMN apoptosis (P < 0.05). These data support the premise that the inhibition of PMN apoptosis in individuals with T2DM occurs through an advanced glycation endproducts/RAGE ligand/receptor-mediated interaction.

Collaborative Cross Mouse Population for Studying Alveolar Bone Changes and Impaired Glucose Tolerance Comorbidity After High-Fat Diet Consumption.

BACKGROUND: High-fat diet (HFD), body weight (BW) gain, and impaired glucose tolerance development are associated with alveolar bone loss (ABL) in susceptible individuals. This report explores the Collaborative Cross (CC) mouse population for studying the impact of genetic background on comorbidity of alveolar bone change and glucose tolerance after HFD consumption. METHODS: Seventy-eight mice from 19 different CC lines were maintained on rodent chow diet for 8 weeks and were subsequently transferred to an HFD (42% fat) for an additional 12 weeks. BW changes were assessed, and glucose tolerance was measured using an intraperitoneal glucose tolerance test (IPGTT). Six cytokines/chemokines were quantified by multiplex immunoassay, alveolar bone volume was quantified by microcomputed tomography, and the ABL phenotype was calculated relative to a control group (143 mice maintained on standard chow diet for 20 weeks). RESULTS: The glucose tolerance response after HFD significantly varied among CC lines (P <0.01), with a significant effect of sex (P <0.01). Alveolar bone changes significantly varied among CC lines (P <0.01). Overall, there was no significant correlation between alveolar bone volume changes and increased BW or glucose tolerance response. However, individual CC lines were identified that showed type 2 diabetes mellitus (t2DM) development and significant alveolar bone volume change (P <0.05), whereas others showed t2DM development, regardless of periodontitis. Interleukin-6 significantly correlated with alveolar bone changes (P <0.05), whereas adipsin showed a negative correlation with IPGTT area under the curve values (P <0.05). CONCLUSION: The present results demonstrate the power of CC mice for studying the genetic background impact between comorbidity of t2DM and bone loss.

Strain-Specific Impact of Fusobacterium nucleatum on Neutrophil Function.

BACKGROUND: Neutrophil function is critical for initiation and progression of infecto-inflammatory diseases. Key quorum-sensing plaque bacteria, such as Fusobacterium nucleatum, act as bridging species between early and late colonizer pathogens, such as Porphyromonas gingivalis, as the biofilm ages and periodontal inflammation increases. This study is designed to determine impact of different F. nucleatum strains on neutrophil function. METHODS: Cells of human promyelocytic leukemia cell line-60 were differentiated into neutrophil-like cells and cultured with F. nucleatum strains of subspecies (ssp.) nucleatum ATCC 25586, ssp. polymorphum ATCC 10953, and ssp. vincentii ATCC 49256. Neutrophil phagocytosis of F. nucleatum strains and neutrophil apoptosis were analyzed by flow cytometry. Superoxide generation was measured by cytochrome C reduction in the presence and absence of N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) (1 µM) stimulation. Proinflammatory cytokine release was determined after 2, 6, and 24 hours of culture in the presence/absence of different F. nucleatum strains. Expression of Toll-like receptor (TLR)2, TLR4, and nuclear factor (NF)-kappa B mRNA levels were analyzed using real-time quantitative polymerase chain reaction. Each experiment was repeated at least three times in triplicate. Data were analyzed using analysis of variance followed by post hoc Bonferroni correction. RESULTS: All strains of F. nucleatum significantly increased phagocytic capacity of neutrophils. Neutrophil phagocytosis of F. nucleatum ssp. polymorphum was significantly greater than that of F. nucleatum ssp. vincentii and ssp. nucleatum (P <0.001). F. nucleatum ssp. nucleatum and ssp. polymorphum significantly blocked fMLP-induced superoxide generation (P <0.001). Although F. nucleatum vincentii also reduced superoxide generation (25%), the impact was not as strong as that of ssp. nucleatum (83%) and ssp. polymorphum (100%). All F. nucleatum strains stimulated significant increase in neutrophil apoptosis compared with control (P <0.001) and significantly increased expression of NF-κB mRNA in neutrophils (P <0.05). Levels of interleukin-8 and tumor necrosis factor-α produced by neutrophils were significantly increased in all F. nucleatum groups compared with control (P <0.001). CONCLUSIONS: These findings suggest that different strains of F. nucleatum impact neutrophil function in different ways. Two of three subspecies blocked neutrophil superoxide generation in response to a secondary stimulus, preventing oxidative killing by neutrophils. The direct role of bridging species in pathogenesis of periodontitis may be greater than previously suspected in which they create a favorable environment for pathogenic transition of the dental ecosystem.

Endothelial Cell Response to Fusobacterium nucleatum.

Vascular response is an essential aspect of an effective immune response to periodontal disease pathogens, as new blood vessel formation contributes to wound healing and inflammation. Gaining a greater understanding of the factors that affect vascular response may then contribute to future breakthroughs in dental medicine. In this study, we have characterized the endothelial cell response to the common bacterium Fusobacterium nucleatum, an important bridging species that facilitates the activity of late colonizers of the dental biofilm. Endothelial cells were infected with Fusobacterium nucleatum (strain 25586) for periods of 4, 12, 24, or 48 h. Cell proliferation and tube formation were analyzed, and expression of adhesion molecules (CD31 and CD34) and vascular endothelial growth factor (VEGF) receptors 1 and 2 was measured by fluorescence-activated cell sorter (FACS) analysis. Data indicate that F. nucleatum impaired endothelial cell proliferation and tube formation. The findings suggest that the modified endothelial cell response acts as a mechanism promoting the pathogenic progression of periodontal diseases and may potentially suggest the involvement of periodontopathogens in systemic diseases associated with periodontal inflammation.

Unhealthy Phenotype as Indicated by Salivary Biomarkers: Glucose, Insulin, VEGF-A, and IL-12p70 in Obese Kuwaiti Adolescents.

OBJECTIVE: Here, we investigated the relationships between obesity and the salivary concentrations of insulin, glucose, and 20 metabolic biomarkers in Kuwaiti adolescents. Previously, we have shown that certain salivary metabolic markers can act as surrogates for blood concentrations. METHODS: Salivary samples of whole saliva were collected from 8,317 adolescents. Salivary glucose concentration was measured by a high-sensitivity glucose oxidase method implemented on a robotic chemical analyzer. The concentration of salivary insulin and 20 other metabolic biomarkers was assayed in 744 randomly selected saliva samples by multiplexed bead-based immunoassay. RESULTS: Obesity was seen in 26.5% of the adolescents. Salivary insulin predicting hyperinsulinemia occurred in 4.3% of normal-weight adolescents, 8.3% of overweight adolescents, and 25.7% of obese adolescents (p < 0.0001). Salivary glucose predicting hyperglycemia was found in only 3% of obese children and was not predictive (p = 0.89). Elevated salivary glucose and insulin occurring together was associated with elevated vascular endothelial growth factor and reduced salivary interleukin-12. CONCLUSION: Considering the surrogate nature of salivary insulin and glucose, this study suggests that elevated insulin may be a dominant sign of metabolic disease in adolescent populations. It also appears that a proangiogenic environment may accompany elevated glucose in obese adolescents.