From manual periodontal probing to digital 3-D imaging to endoscopic capillaroscopy: Recent advances in periodontal disease diagnosis.

Deepened periodontal pockets exert a significant pathological burden on the host and its immune system, particularly in a patient with generalized moderate to severe periodontitis. This burden is extensive and longitudinal, occurring over decades of disease development. Considerable diagnostic and prognostic successes in this regard have come from efforts to measure the depths of the pockets and their contents, including level of inflammatory mediators, cellular exudates and microbes; however, the current standard of care for measuring these pockets, periodontal probing, is an analog technology in a digital age. Measurements obtained by probing are variable, operator dependent and influenced by site-specific factors. Despite these limitations, manual probing is still the standard of care for periodontal diagnostics globally. However, it is becoming increasingly clear that this technology needs to be updated to be compatible with the digital technologies currently being used to image other orofacial structures, such as maxillary sinuses, alveolar bone, nerve foramina and endodontic canals in 3 dimensions. This review aims to summarize the existing technology, as well as new imaging strategies that could be utilized for accurate evaluation of periodontal pocket dimensions.

Application of radiopaque micro-particle fillers for 3-D imaging of periodontal pocket analogues using cone beam CT.

BACKGROUND: Periodontitis is an infectious/inflammatory disease most often diagnosed by deepening of the gingival sulcus, which leads to periodontal pockets (PPs) conventional manual periodontal probing does not provide detailed information on the three-dimensional (3-D) nature of PPs. OBJECTIVES: To determine whether accurate 3-D analyses of the depths and volumes of calibrated PP analogues (PPAs) can be obtained by conventional cone beam computed tomography (CBCT) coupled with novel radiopaque micro-particle fillers (described in the companion paper) injected into the PPAs. METHODS: Two PPA models were employed: (1) a human skull model with artificial gingiva applied to teeth with alveolar bone loss and calibrated PPAs, and (2) a pig jaw model with alveolar bone loss and surgically-induced PPAs The PPAs were filled with controlled amounts of radiopaque micro-particle filler using volumetric pipetting Inter-method and intra-method agreement tests were then used to compare the PPA depths and volumes obtained from CBCT images with values obtained by masked examiners using calibrated manual methods. RESULTS: Significant inter-method agreement (0.938-0.991) and intra-method agreement (0.94-0.99) were obtained when comparing analog manual data to digital CBCT measurements enabled by the radiopaque filler. SIGNIFICANCE: CBCT imaging with radiopaque micro-particle fillers is a plausible means of visualizing and digitally assessing the depths, volumes, and 3-D shapes of PPs This approach could transform the diagnosis and treatment planning of periodontal disease, with particular initial utility in complex cases Efforts to confirm the clinical practicality of these fillers are currently in progress.

Development of radiopaque, biocompatible, antimicrobial, micro-particle fillers for micro-CT imaging of simulated periodontal pockets.

OBJECTIVES: Approximately 109 bacteria can be harbored within periodontal pockets (PP) along with inflammatory byproducts implicated in the pathophysiology of systemic diseases linked to periodontitis (PD). Calculation of this inflammatory burden has involved estimation of total pocket surface area using analog data from conventional periodontal probing which is unable to determine the three-dimensional (3-D) nature of PP. The goals of this study are to determine the radiopacity, biocompatibility, and antimicrobial activity of transient micro-particle fillers in vitro and demonstrate their capability for 3-D imaging of artificial PP (U.S. Patent publication number: 9814791 B2). METHODS: Relative radiopacity values of various metal oxide fillers were obtained from conventional radiography and micro-computed tomography (µCT) using in vitro models. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were used to measure the biocompatibility of calcium tungstate (CaWO4) particles by determination of viable keratinocytes percentage (%) after exposure. After introducing an antibacterial compound (K21) to the radiopaque agent, antimicrobial tests were conducted using Porphyromonas gingivalis (P. gingivalis) and Streptococcus gordonii (S. gordonii) strains and blood agar plates. RESULTS: CaWO4 micro-particle-bearing fillers exhibited an X-ray radiopacity distinct from tooth structures that enabled 3-D visualization of an artificial periodontal pocket created around a human tooth. MTT assays indicated that CaWO4 micro-particles are highly biocompatible (increasing the viability of exposed keratinocytes). Radiopaque micro-particle fillers combined with K21 showed significant antimicrobial activity for P. gingivalis and S. gordonii. SIGNIFICANCE: The plausibility of visualizing PP with 3-D radiographic imaging using new radiopaque, biocompatible, transient fillers was demonstrated in vitro. Antibacterial (or other) agents added to this formula could provide beneficial therapeutic features along with the diagnostic utility.

Periodontal and other oral manifestations of immunodeficiency diseases.

The list of immunodeficiency diseases grows each year as novel disorders are discovered, classified, and sometimes reclassified due to our ever-increasing knowledge of immune system function. Although the number of patients with secondary immunodeficiencies (SIDs) greatly exceeds those with primary immunodeficiencies (PIDs), the prevalence of both appears to be on the rise probably because of scientific breakthroughs that facilitate earlier and more accurate diagnosis. Primary immunodeficiencies in adults are not as rare as once thought. Globally, the main causes of secondary immunodeficiency are HIV infection and nutritional insufficiencies. Persons with acquired immune disorders such as AIDS caused by the human immunodeficiency virus (HIV) are now living long and fulfilling lives as a result of highly active antiretroviral therapy (HAART). Irrespective of whether the patient's immune-deficient state is a consequence of a genetic defect or is secondary in nature, dental and medical practitioners must be aware of the constant potential for infections and/or expressions of autoimmunity in these individuals. The purpose of this review was to study the most common conditions resulting from primary and secondary immunodeficiency states, how they are classified, and the detrimental manifestations of these disorders on the periodontal and oral tissues.

An essential role of Ffar2 (Gpr43) in dietary fibre-mediated promotion of healthy composition of gut microbiota and suppression of intestinal carcinogenesis.

Composition of the gut microbiota has profound effects on intestinal carcinogenesis. Diet and host genetics play critical roles in shaping the composition of gut microbiota. Whether diet and host genes interact with each other to bring specific changes in gut microbiota that affect intestinal carcinogenesis is unknown. Ability of dietary fibre to specifically increase beneficial gut microbiota at the expense of pathogenic bacteria in vivo via unknown mechanism is an important process that suppresses intestinal inflammation and carcinogenesis. Free fatty acid receptor 2 (FFAR2 or GPR43) is a receptor for short-chain fatty acids (acetate, propionate and butyrate), metabolites of dietary fibre fermentation by gut microbiota. Here, we show FFAR2 is down modulated in human colon cancers than matched adjacent healthy tissue. Consistent with this, Ffar2(-/-) mice are hypersusceptible to development of intestinal carcinogenesis. Dietary fibre suppressed colon carcinogenesis in an Ffar2-dependent manner. Ffar2 played an essential role in dietary fibre-mediated promotion of beneficial gut microbiota, Bifidobacterium species (spp) and suppression of Helicobacter hepaticus and Prevotellaceae. Moreover, numbers of Bifidobacterium is reduced, whereas those of Prevotellaceae are increased in human colon cancers than matched adjacent normal tissue. Administration of Bifidobacterium mitigated intestinal inflammation and carcinogenesis in Ffar2(-/-) mice. Taken together, these findings suggest that interplay between dietary fibre and Ffar2 play a key role in promoting healthy composition of gut microbiota that stimulates intestinal health.

High-throughput sequencing reveals key genes and immune homeostatic pathways activated in myeloid dendritic cells by Porphyromonas gingivalis 381 and its fimbrial mutants.

The human microbiome consists of highly diverse microbial communities that colonize our skin and mucosal surfaces, aiding in maintenance of immune homeostasis. The keystone pathogen Porphyromonas gingivalis induces a dysbiosis and disrupts immune homeostasis through as yet unclear mechanisms. The fimbrial adhesins of P. gingivalis facilitate biofilm formation, invasion of and dissemination by blood dendritic cells; hence, fimbriae may be key factors in disruption of immune homeostasis. In this study we employed RNA-sequencing transcriptome profiling to identify differentially expressed genes (DEGs) in human monocyte-derived dendritic cells (MoDCs) in response to in vitro infection/exposure by Pg381 or its isogenic mutant strains that solely express minor-Mfa1 fimbriae (DPG3), major-FimA fimbriae (MFI) or are deficient in both fimbriae (MFB) relative to uninfected control. Our results yielded a total of 479 DEGs that were at least two-fold upregulated and downregulated in MoDCs significantly (P ≤ 0.05) by all four strains and certain DEGs that were strain-specific. Interestingly, the gene ontology biological and functional analysis shows that the upregulated genes in DPG3-induced MoDCs were more significant than other strains and associated with inflammation, immune response, anti-apoptosis, cell proliferation, and other homeostatic functions. Both transcriptome and quantitative polymerase chain reaction results show that DPG3, which solely expresses Mfa1, increased ZNF366, CD209, LOX1, IDO1, IL-10, CCL2, SOCS3, STAT3 and FOXO1 gene expression. In conclusion, we have identified key DC-mediated immune homeostatic pathways that could contribute to dysbiosis in periodontal infection with P. gingivalis.

Dendritic cells at the oral mucosal interface.

The mucosal lining of the respiratory and digestive systems contains the largest and most complex immune system in the body, but surprisingly little is known of the immune system that serves the oral mucosa. This review focuses on dendritic cells, particularly powerful arbiters of immunity, in response to antigens of microbial or tumor origin, but also of tolerance to self-antigens and commensal microbes. Although first discovered in 1868, the epidermal dendritic Langerhans cells remained enigmatic for over a century, until they were identified as the most peripheral outpost of the immune system. Investigators' ability to isolate, enrich, and culture dendritic cells has led to an explosion in the field. Presented herein is a review of dendritic cell history, ontogeny, function, and phenotype, and the role of different dendritic cell subsets in the oral mucosa and its diseases. Particular emphasis is placed on the mechanisms of recognition and capture of microbes by dendritic cells. Also emphasized is how dendritic cells may regulate immunity/tolerance in response to oral microbes.

Oral mucosal expression of HIV-1 receptors, co-receptors, and alpha-defensins: tableau of resistance or susceptibility to HIV infection?

The basic premise of whether transmission of HIV-1 through the oral mucosa actually occurs, and through what route, is a topic of intense interest. Our work has focused on HIV-1 receptors/co-receptors and alpha-defensin-1 in situ in human gingiva. Regardless of HIV-1 infection, the role that C-type lectin receptors might play in periodontal pathogenesis is of great interest. We have shown that the gingival lamina propria, when inflamed, becomes increasingly infiltrated with DC-SIGN+MR+ dermal dendritic cells (DDCs), while the inflamed epithelium shows a decrease in Langerin+ Langerhans cells (LCs). Moreover, DDCs and LCs contribute to the mature CD83+ DC pool in situ, and form immune conjugates with CD4+ T-cells in the lamina propria (Jotwani and Cutler, 2003). This raises the intriguing possibility that oral mucosal DCs may be involved in HIV-1 transfer to T-cells in situ. However, this possibility is tendered by the challenges faced by the virus in gaining access to oral mucosal immune cells, including their ability to survive the salivary defenses, cross the mucosal barrier, resist inactivation by alpha-defensins, and overcome the paucity of co-receptor CCR5 in (healthy) oral mucosa (i.e., required for productive infection [Jotwani et al., 2004]). To date, there is little evidence of direct infection by HIV-1 of oral mucosal DCs/T cells and other cells in situ. Abbreviations used in this paper: CP, chronic periodontitis; CCR5, chemokine receptor 5; CXCR4, C-X-C receptor 4; DCs, dendritic cells; DC-SIGN, DC-specific ICAM-3 grabbing non-integrin; DDC, dermal dendritic cells; LCs, Langerhans cells; LP, lamina propria; MR, mannose receptor.

Increase in HIV receptors/co-receptors/alpha-defensins in inflamed human gingiva.

Transmission of HIV-1 through the oral cavity is considered to be a rare event. To identify factors in resistance/susceptibility to oral HIV-1 infection, we analyzed expression in human gingiva of HIV-1 receptors Langerin, DC-SIGN, MR, and GalCer, HIV-1 co-receptors CCCR5, CXCR4, and anti-microbial protein alpha-defensin-1. Our results show that healthy gingiva is infiltrated with cells expressing all HIV-1 receptors tested; however, there are very few CCR5(+) cells and a complete absence of CXCR4(+) cells in the lamina propria. In chronic periodontitis (CP), DC-SIGN, MR, CXCR4, and CCR5 increase, but this was accompanied by a ten-fold increase in alpha-defensin-1 mRNA. The CCR5(+) cells were revealed to be T-cells, macrophages, and dermal dendritic cells. Moreover, epithelial expression of GalCer and CXCR4 together was not apical and showed no trend with underlying inflammation. Thus, low expression of HIV-1 co-receptors in health and high expression of alpha-defensin during CP may comprise endogenous factors that provide protection from oral HIV-1 infection.

Multiple dendritic cell (DC) subpopulations in human gingiva and association of mature DCs with CD4+ T-cells in situ.

Gingival epithelium is a site of active trafficking of Langerhans cells (LCs), while the lamina propria in chronic periodontitis (CP) contains CD83+ mature dendritic cells (mDCs) and CD4+ T-cells. The immune cells that contribute to the mDCs, and whether mDCs engage with T-cells in situ, are unclear. Using several immunohistochemical approaches, combined with fluorescence-, light-, and scanning laser confocal-microscopy, we show that, in addition to LCs, the gingiva contains dermal DCs (DDCs) in the lamina propria; moreover, DDCs increase in number during CP. Furthermore, DDCs, LCs, and B-cells co-express CD83 in CP and contribute to the mDC pool. Double-staining for CD83 and CD4 revealed that mDCs associate with clusters of CD4+ T-cells in the lamina propria. Analysis of these data suggests that multiple DC subsets mature in the gingiva and that mature DCs engage in antigen presentation with T-cells in chronic periodontitis.

Prior exposure of mice to Fusobacterium nucleatum modulates host response to Porphyromonas gingivalis.

Multiple periodontal pathogens sequentially colonize the subgingival niche during the conversion from gingivitis to destructive periodontal disease. An animal model of sequential immunization with key periodontal pathogens has been developed to determine whether T- and B-lymphocyte effector functions are skewed and fail to protect the host from pathogenic challenge. The present study was performed to evaluate the immunomodulatory effect of exposure to Fusobacterium nucleatum prior to Porphyromonas gingivalis. Group 1 (control) mice were immunized with phosphate-buffered saline, group 2 were immunized with F. nucleatum prior to P. gingivalis and group 3 were immunized with P. gingivalis alone. All the T-cell clones derived from group 2 demonstrated type 2 helper T-cell clone (Th2 subsets), whereas those from group 3 mice demonstrated Th1 subsets. Exposure of mice to F. nucleatum prior to P. gingivalis interfered with the opsonophagocytosis function of sera against P. gingivalis. In adoptive T-cell transfer experiments, in vivo protective capacity of type 2 helper T-cell clones (Th2) from group 2 was significantly lower than type 1 helper T-cell clones (Th1) from group 3 against the lethal dose infection of P. gingivalis. Western blot analysis indicated a different pattern of recognition of P. gingivalis fimbrial proteins between sera from group 2 and group 3. In conclusion, these studies suggest that exposure of a host to F. nucleatum prior to the periodontal pathogen P. gingivalis modulates the host immune responses to P. gingivalis at the humoral, cellular and molecular levels.

Lipopolysaccharides from distinct pathogens induce different classes of immune responses in vivo.

The adaptive immune system has evolved distinct responses against different pathogens, but the mechanism(s) by which a particular response is initiated is poorly understood. In this study, we investigated the type of Ag-specific CD4(+) Th and CD8(+) T cell responses elicited in vivo, in response to soluble OVA, coinjected with LPS from two different pathogens. We used Escherichia coli LPS, which signals through Toll-like receptor 4 (TLR4) and LPS from the oral pathogen Porphyromonas gingivalis, which does not appear to require TLR4 for signaling. Coinjections of E. coli LPS + OVA or P. gingivalis LPS + OVA induced similar clonal expansions of OVA-specific CD4(+) and CD8(+) T cells, but strikingly different cytokine profiles. E. coli LPS induced a Th1-like response with abundant IFN-gamma, but little or no IL-4, IL-13, and IL-5. In contrast, P. gingivalis LPS induced Th and T cell responses characterized by significant levels of IL-13, IL-5, and IL-10, but lower levels of IFN-gamma. Consistent with these results, E. coli LPS induced IL-12(p70) in the CD8alpha(+) dendritic cell (DC) subset, while P. gingivalis LPS did not. Both LPS, however, activated the two DC subsets to up-regulate costimulatory molecules and produce IL-6 and TNF-alpha. Interestingly, these LPS appeared to have differences in their ability to signal through TLR4; proliferation of splenocytes and cytokine secretion by splenocytes or DCs from TLR4-deficient C3H/HeJ mice were greatly impaired in response to E. coli LPS, but not P. gingivalis LPS. Therefore, LPS from different bacteria activate DC subsets to produce different cytokines, and induce distinct types of adaptive immunity in vivo.

Mature dendritic cells infiltrate the T cell-rich region of oral mucosa in chronic periodontitis: in situ, in vivo, and in vitro studies.

Previous studies have analyzed the lymphoid and myeloid foci within the gingival mucosa in health and chronic periodontitis (CP); however, the principal APCs responsible for the formation and organizational structure of these foci in CP have not been defined. We show that in human CP tissues, CD1a(+) immature Langerhans cells predominantly infiltrate the gingival epithelium, whereas CD83(+) mature dendritic cells (DCs) specifically infiltrate the CD4(+) lymphoid-rich lamina propria. In vivo evidence shows that exacerbation of CP results in increased levels of proinflammatory cytokines that mediate DC activation/maturation, but also of counterregulatory cytokines that may prevent a Th-polarized response. Consistently, in vitro-generated monocyte-derived DCs pulsed with Porphyromonas gingivalis strain 381 or its LPS undergo maturation, up-regulate accessory molecules, and release proinflammatory (IL-1beta, PGE(2)) and Th (IL-10, IL-12) cytokines. Interestingly, the IL-10:IL-12 ratio elicited from P. gingivalis-pulsed DCs was 3-fold higher than that from Escherichia coli-pulsed DCs. This may account for the significantly (p < 0.05) lower proliferation of autologous CD4(+) T cells and reduced release of IFN-gamma elicited by P. gingivalis-pulsed DCs. Taken together, these findings suggest a previously unreported mechanism for the pathophysiology of CP, involving the activation and in situ maturation of DCs by the oral pathogen P. gingivalis, leading to release of counterregulatory cytokines and the formation of T cell-DC foci.

Pathophysiological relationships between periodontitis and systemic disease: recent concepts involving serum lipids.

Periodontitis has been traditionally regarded as a chronic inflammatory oral infection. However, recent studies indicate that this oral disease may have profound effects on systemic health. The search for cellular/molecular mechanisms linking periodontitis to changes in systemic health and systemic physiology has resulted in the evolution of a new area of lipid research establishing linkages between existing multidisciplinary biomedical literature, recent observations concerning the effects of serum lipids on immune cell phenotype/function, and a heightened interest in systemic responses to chronic localized infections. There appears to be more than a casual relationship between serum lipid levels and systemic health (particularly cardiovascular disease, diabetes, tissue repair capacity, and immune cell function), susceptibility to periodontitis, and serum levels of pro-inflammatory cytokines. In terms of the potential relationship between periodontitis and systemic disease, it is possible that periodontitis-induced changes in immune cell function cause metabolic dysregulation of lipid metabolism through mechanisms involving proinflammatory cytokines. Sustained elevations of serum lipids and/or pro-inflammatory cytokines may have a serious negative impact on systemic health. The purpose of this paper is to present the background, supporting data, and hypotheses related to this concept. As active participants in this emerging and exciting area of investigation, we hope to stimulate interest and awareness among biomedical scientists and practitioners.

Clinical benefits of oral irrigation for periodontitis are related to reduction of pro-inflammatory cytokine levels and plaque.

BACKGROUND: Although a growing body of evidence indicates that oral irrigation with water has therapeutic benefits in periodontitis, the mechanisms of action have not been elucidated. The aims of this study were: (1) to analyze the effects of oral irrigation (Water Pik Oral Irrigator) on the clinical signs of adult periodontitis (AP) and on the levels of interleukin-1 beta (IL-beta), prostaglandin-E2 (PGE2), interleukin-10 (IL-10) and interferon-gamma (IFN-gamma) in GCF, and (2) to analyze the influence of the periodontitis-related IL-1 genotype (IL-1GT) on these variables. METHOD: A single-center, blinded study in otherwise healthy humans (n= 52) with localized mild to moderate AP was carried out, using the following groups: group A (n= 12), no oral hygiene for 14 days; group B (n=20), routine oral hygiene (ROH) for 14 days; group C (n=20), supra-gingival oral irrigation plus ROH for 14 days. Group A patients were crossed-over to group C for 14 days (=day 28) after a professional prophylaxis. Group assignment was randomized by a coin toss, with the exception of group A subjects, who were self-selected as per recommendations of the internal review board for human subjects. GCF was sampled from 3 study teeth per patient and analyzed for IL-1 beta, PGE2, IL-10 and IFN gamma by ELISA on days 0, 7, 14 and 28. Probing pocket depths (PPD), clinical attachment levels (CAL), bleeding on probing (BOP), gingival index (GI) and plaque index (PI) were measured by a calibrated examiner (TWS) on days 0, 14 and 28. Analysis of covariance was performed using SAS 6.12 and Proc Mixed with group and IL-1GT as the factors and the baseline levels as the covariate, with output being least squares means and least significant difference (LSD). Significant differences were declared if the p-value for the F-statistic was < or =0.05. RESULTS: Oral irrigation plus ROH resulted in a significant reduction in PPD, BOP, GI and PI, as well as IL-beta levels by 7 days and PGE2 levels by 14 days, relative to ROH or no oral hygiene. Interestingly, decreased IL-1 beta levels in patients using oral irrigation plus ROH was accompanied by a trend for increased levels of the "anti-inflammatory" cytokine IL-10. ROH reduced GI, BOP and PI, and PGE2 levels by 14 days, but had no effect on IL-1 beta or IL-10 levels relative to no oral hygiene. The effects of no oral hygiene were reversed by a prophy followed by oral irrigation plus ROH for 14 days. No clinical differences were evident between IL-1 GT (+) patients (n= 1) and GT (-) patients (n=40), but the former had significantly elevated levels of GCF IL-10 and borderline increases in IL-1 beta (p=0.07). CONCLUSIONS: Oral irrigation with water for 14 days had an improved therapeutic benefit for AP over that of routine oral hygiene alone and this improvement was accompanied by a down-modulation of the pro-inflammatory cytokine profile in GCF.

Comparative radiopacity of tetracalcium phosphate and other root-end filling materials.

AIM: This study compared the radiopacity of tetracalcium phosphate (TTCP) and 11 root-end filling materials relative to human dentine. METHODOLOGY: Specimens of 2 mm thickness and a graduated aluminium stepwedge were placed on dental X-ray films and exposed to an X-ray beam. The optical densities of the specimens and aluminium steps were measured. The optical densities of the specimens were correlated to the equivalent thickness of aluminium with a regression analysis equation. The equation was used to calculate the equivalent aluminium thickness of each of the specimens. RESULTS: Nine of the materials were found to be of acceptable radiopacity (at least 2 mm Al more radiopaque than dentine). TCCP and two of the glass-ionomer compounds were found to have insufficient radiopacity to be radiographically distinguishable from human dentine. CONCLUSIONS: All the materials were found to be distinguishable radiographically from dentine, except for Vitrebond, TTCP and Ketac-Fil. Amalgam was the most radiopaque material and Ketac-Fil was the least radiopaque material tested.

In vitro evidence that lipopolysaccharide of an oral pathogen leaks from root-end filled teeth.

AIM: The ability to achieve a complete apical seal of the root canal system is thought to be important in the success of non-surgical and surgical endodontics. The aim of this study was to establish whether or not root-end filled teeth allow leakage of lipopolysaccharide (LPS) from a known oral pathogen in vitro. METHODOLOGY: Lipopolysaccharide (LPS) from a virulent strain of Prophyromonas gingivalis (P. gingivalis) (A7436 from patient with refractory periodontitis), was isolated by the Westphall and Jann technique, dialysed extensively, lyophilyzed, resuspended in distilled water and analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Root canals from 10 teeth were instrumented endodontically and the apical 3 mm of resected roots were filled with gutta-percha. The teeth were mounted in 12 mL polypropylene vials by using sticky wax and root surfaces were covered with two layers of nail varnish. Teeth were filled with 3.3 mg mL-1 LPS and the vials filled with 11 mL of Tris Buffered Saline (TBS) containing 0.05% sodium azide. Both positive and negative controls were run in parallel with the experimental specimens. Aliquots were removed each day and subjected to slot blot analysis to quantitate the amount of LPS that had leaked into the bottom of the vials. The density of slots was analyzed using a laser densitometer and regression analysis was used to generate a standard curve, confidence intervals and experimental values. RESULTS: The data indicated that teeth obturated apically with gutta-percha leaked, whilst no LPS leakage was detected in teeth covered completely with nail varnish (P < 0.05). CONCLUSIONS: In vitro teeth with gutta-percha root-end fills can permit leakage of LPS from an identified oral pathogen.

Heightened gingival inflammation and attachment loss in type 2 diabetics with hyperlipidemia.

BACKGROUND: Our previous studies in diabetic (DB) rats suggest that hyperlipidemia may cause a dysregulation of the cellular and local cytokine response to periodontitis (AP). The objective of the present study was to determine if diabetes has a similar dysregulatory effect on the gingival response to AP in humans. METHODS: Peripheral blood, as well as gingival tissue (GT) and gingival crevicular fluid (GCF), was obtained from a total of 35 patients who were categorized into the following groups based on level of diabetic (type 2) control and presence or absence of adult periodontitis (AP): group 1, systemically and periodontally healthy (n = 6); group 2, systemically healthy with adult periodontitis (n = 7); group 3, well-controlled diabetes and periodontally healthy (n = 6); group 4, well-controlled diabetes with adult periodontitis (n = 5); group 5, poorly controlled diabetes and periodontally healthy (n = 5); group 6, poorly controlled diabetes and adult periodontitis (n = 6). All subjects were given a thorough periodontal examination, including probing depths (PD), clinical attachment levels (CAL), gingival index (GI), plaque index (PI), and vertical bitewing radiographs. Blood studies included levels of glycated hemoglobin (HbA1c), triglycerides (TG), cholesterol (CHL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL). The levels of interleukin-1 beta (IL-1beta) in GCF and GT, interleukin-6 (IL-6), and platelet-derived growth factor AB (PDGF-AB) in GT from patients in each experimental group were analyzed by enzyme-linked immunosorbent assay (ELISA). RESULTS: Our results indicate that all clinical indices except PI were significantly elevated in the poorly controlled and well-controlled diabetics, compared to systemically healthy patients, but only in the subjects without preexisiting AP (Tukey's multiple comparisons, P <0.05). Pairwise linear regression analysis revealed significant (P <0.01) positive associations between periodontal inflammation (PD, CAL, PI, GI) and levels of GCF IL-1beta, GT IL- 1beta GT IL-6, but not GT PDGF; moreover, GT IL-6 levels were significantly associated (P<0.05) with GT IL-1beta. As TG levels increased in the non-AP patients (group 1 < group 3 < group 5), there was a trend, not significant, for increased GCF IL-1beta levels and increased gingival inflammation. Interestingly, periodontitis resulted in increased PDGF-AB levels in the gingiva of systemically healthy and well-controlled diabetes patients, but this increase was obtunded in poorly controlled diabetes patients. CONCLUSIONS: This confirms our earlier work in the diabetic rat model. These studies indicate that decreased metabolic control in type 2 diabetics results in increased serum triglycerides and has a negative influence on all clinical measures of periodontal health, particularly in patients without preexisting periodontitis. Levels of the cytokine IL- 1beta showed a trend for increasing as diabetic control diminished. In contrast, levels of the growth factor PDGF, which normally increase in periodontitis, decreased in poorly controlled diabetics with periodontitis. These studies suggest a possible dysregulation of the normal cytokine/growth factor signaling axis in poorly controlled type 2 diabetics that may contribute to periodontal breakdown/diminished repair.

Association between periodontitis and hyperlipidemia: cause or effect?

BACKGROUND: Epidemiological studies suggest a relationship between periodontitis and coronary artery disease, but the mechanism has not been established. Recent studies in animals indicate that low dose endotoxin, as in a gram-negative infection, can induce hyperlipidemia and myeloid cell hyperactivity. The association between periodontitis, systemic exposure to Porphyromonas gingivalis, lipopolysaccharides (LPS), and hyperlipidemia has not been examined in humans. METHODS: Sera were obtained from 26 adult periodontitis patients and 25 healthy control (C) subjects selected from patients and staff. Serum antibodies against Porphyromonas gingivalis and its LPS were analyzed by enzyme-linked immunosorbent assay (ELISA) and Western blotting, respectively. Serum triglycerides (TG) and cholesterol (CHOL) were assayed by a commercial laboratory. The associations between AP and blood levels of TG, CHOL, and anti-P. gingivalis whole cells and LPS were examined by logistic regression analysis. Peripheral blood polymorphonuclear leukocytes (PMNs) from 6 healthy fasted donors were incubated with purified TG (0.1 mg/ml) for 2 hours at 37 degrees C, stimulated with 100 ng/ml P. gingivalis LPS, and the release of IL-1beta measured by ELISA. RESULTS: The presence of periodontitis was significantly associated with age (odds ratio = 3.5, P = 0.04), elevated TG levels (odds ratio = 8.6, P = 0.0009), elevated CHOL levels (odds ratio = 7, P = 0.004), elevated ELISA titer (odds ratio = 35, P = 0.003) and reactivity with P. gingivalis LPS (odds ratio = 41, P = 0.001). PMNs from all 6 healthy patients released modest levels of IL-1beta (10 to 60 pg/ml) when stimulated with 100 ng/ml P. gingivalis LPS. Addition of TG resulted in a significant increase (P <0.05) in IL- 1beta secreted that ranged from 7 to 150% over LPS alone. No IL-1beta was elicited by TG or vehicle alone. CONCLUSIONS: The results of this study indicate the presence of a significant relationship between periodontitis, hyperlipidemia, and serum antibodies against P. gingivalis LPS that warrants further examination in a larger patient population. Furthermore, these studies indicate that elevated triglycerides are able to modulate IL-1beta production by PMNs stimulated with P. gingivalis LPS.