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It has been shown that inadequate sleep has deleterious effects on health by suppressing immunity and promoting inflammation. The aim of this study was to investigate the effect of sleep and salivary glucose levels on the development of gingivitis in a prospective longitudinal study of Kuwaiti children. Data were collected from 10-y-old children ( N = 6,316) in 2012 and again in 2014. Children were approximately equally distributed from 138 elementary schools representing the 6 governorates of Kuwait. Calibrated examiners conducted oral examination, self-reported sleep evaluation interviews, anthropomorphic measurements, and unstimulated whole saliva sample collection. Salivary glucose levels were measured by a florescent glucose oxidase method; values of salivary glucose ≥1.13 mg/dL were defined as high glucose levels. A multilevel random intercept and slope analysis was conducted to determine the relationship between sleep duration and gingivitis on 3 levels: within schools, among children, and over time. The outcome was the progression of the extent of gingival inflammation in children over time. The main independent variables were the number of daily sleep hours and salivary glucose levels. Other explanatory variables and confounders assessed were governorate, dental caries and restorations, and obesity by waist circumference (adjusted for snacking and sex). Gingivitis increased over time in children who had shorter sleep duration ( P < 0.05). Salivary glucose levels >1.13 mg/dL predicted gingivitis ( P < 0.05). Children who had more decayed or filled teeth had more gingivitis ( P < 0.05). No significant association was found between gingivitis and obesity. The level of gingivitis was different among the 6 governorates of Kuwait. Additionally, there was a strong clustering effect of the observations within schools and among children across time. Longitudinal analysis of 6,316 Kuwaiti children revealed that shorter sleep duration and higher salivary glucose levels were both associated with increased gingival inflammation.

Bacterial infections are known to alter glucose metabolism within tissues via mechanisms of inflammation. We conducted this study to examine whether insulin response genes are differentially expressed in gingival tissues, comparing samples from experimental gingivitis and periodontitis subjects to those from healthy individuals. Total RNA was extracted from gingival biopsies from 26 participants: 8 periodontally healthy, 9 experimental gingivitis, and 9 periodontitis subjects. Gene expression patterns were evaluated with a polymerase chain reaction array panel to examine 84 candidate genes involved with glucose metabolism, insulin resistance, and obesity. Array data were evaluated with a t test adjusted by the false discover rate (P < 0.05), and ingenuity pathway analysis was performed for statistical testing of pathways. Although tissue samples were not sufficient to enable protein quantification, we confirmed the upregulation of the key gene using lipopolysaccharide-stimulated primary gingival epithelial cells by Western blot. The mRNA expression patterns of genes that are associated with insulin response and glucose metabolism are markedly different in experimental gingivitis subjects compared with healthy controls. Thirty-two genes are upregulated significantly by at least 2-fold, adjusted for false discover rate (P < 0.05). Periodontitis subjects show similar but attenuated changes in gene expression patterns, and no genes meet the significance criteria. Ingenuity pathway analysis demonstrates significant activation of the carbohydrate metabolism network in experimental gingivitis but not in periodontitis. G6PD protein increases in response to lipopolysaccharide stimulation in primary gingival epithelial cells, which is in the same direction as upregulated mRNA in tissues. Acute gingival inflammation may be associated with tissue metabolism changes, but these changes are not evident in chronic periodontitis. This study suggests that acute gingival inflammation may induce localized changes that modify tissue insulin/glucose metabolism., (© International & American Associations for Dental Research.)

Obesity is a risk factor for periodontitis, but the pathogenic mechanism involved is unclear. We studied the effects of insulin in periodontal tissues during the state of obesity-induced insulin resistance. Gingival samples were collected from fatty (ZF) and lean (ZL, control) Zucker rats. Endothelial nitric oxide synthase (eNOS) expression was decreased, and activities of protein kinase C (PKC) α, ß2, δ, and ϵ isoforms were significantly increased in the gingiva from ZF rats compared with those from ZL rats. Expression of oxidative stress markers (mRNA) and the p65 subunit of NF-κB was significantly increased in ZF rats. Immunohistochemistry revealed that NF-κB activation was also increased in the gingival endothelial cells from transgenic mice overexpressing NF-κB-dependent enhanced green fluorescent protein (GFP) and on a high-fat vs. normal chow diet. Analysis of the gingiva showed that insulin-induced phosphorylation of IRS-1, Akt, and eNOS was significantly decreased in ZF rats, but Erk1/2 activation was not affected. General PKC inhibitor and an anti-oxidant normalized the action of insulin on Akt and eNOS activation in the gingiva from ZF rats. This provided the first documentation of obesity-induced insulin resistance in the gingiva. Analysis of our data suggested that PKC activation and oxidative stress may selectively inhibit insulin-induced Akt and eNOS activation, causing endothelial dysfunction and inflammation., (© International & American Associations for Dental Research.)

Periodontal diseases, such as gingivitis and periodontitis, are characterized by bacterial plaque accumulation around the gingival crevice and the subsequent inflammation and destruction of host tissues. To test the hypothesis that cellular metabolism is altered as a result of host-bacteria interaction, we performed an unbiased metabolomic profiling of gingival crevicular fluid (GCF) collected from healthy, gingivitis, and periodontitis sites in humans, by liquid and gas chromatography mass spectrometry. The purine degradation pathway, a major biochemical source for reactive oxygen species (ROS) production, was significantly accelerated at the disease sites. This suggests that periodontal-disease-induced oxidative stress and inflammation are mediated through this pathway. The complex host-bacterial interaction was further highlighted by depletion of anti-oxidants, degradation of host cellular components, and accumulation of bacterial products in GCF. These findings provide new mechanistic insights and a panel of comprehensive biomarkers for periodontal disease progression.

We used an experimental gingivitis study design to compare crevicular fluid concentrations of Migration Inhibitory Factor (MIF) and Prostaglandin E(2) (PGE(2)) in younger (18 to 30 yrs) and older (46 to 77 yrs) healthy adults. PGE(2) increased after 1 wk in younger participants, whereas it decreased in older individuals after 1 wk of plaque accumulation. A significant interaction between age and time was observed for PGE(2) (p = 0.04). High concentrations of MIF were identified in both age groups at baseline. MIF increased in the younger participants, whereas in the older individuals a decrease over time was observed. MIF concentration was positively correlated with plaque index and gingival index in the older age group. Total counts of bacteria, Parvimonas micra and Prevotella intermedia, were significantly correlated with MIF concentration in older participants. In conclusion, MIF and PGE(2) production in response to bacterial accumulation seems to be modified by age.

Tumor necrosis factor-alpha-converting enzyme (TACE) is a metalloprotease which can shed several cytokines from the cell membrane, including receptor activator of NF-kappaB ligand (RANKL). This study aimed to investigate the hypothesis that TACE would be elevated in the gingival crevicular fluid (GCF) of persons with periodontitis. Total TACE amounts in GCF were higher in persons with chronic and aggressive periodontitis than in those with gingivitis or in healthy persons. TACE concentrations in GCF were higher in persons with chronic and aggressive periodontitis than in those with gingivitis, although not significantly higher than in healthy persons. Persons with chronic periodontitis receiving immunosuppressive treatment exhibited over 10-fold lower TACE levels than the other periodontitis groups. TACE was positively correlated with probing pocket depth, clinical attachment levels, and RANKL concentrations in GCF. In conclusion, the increased GCF TACE levels in persons with periodontitis and their positive correlation with RANKL may indicate an association of this enzyme with alveolar bone loss, and may warrant special attention in future therapeutic approaches.

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.

Cell-surface proteoglycans are involved in lymphocyte migration and activation. This study investigated the expression of syndecan-1, syndecan-4, and glypican in peripheral blood lymphocytes and by lymphocytes in variously inflamed periodontal tissues. Gingival specimens from healthy, gingivitis, or chronic periodontitis sites were stained by means of antibodies against B- and T-lymphocytes and also syndecan-1, syndecan-4, and glypican. Syndecan-1 expression by peripheral blood mononuclear cells (PBMC) from healthy, gingivitis, and chronic periodontitis subjects was assessed by flow cytometry. Syndecan-1 was expressed by B-cells/plasma cells but not T-cells in both gingivitis and chronic periodontitis lesions. Both B-cells/plasma cells and T-cells in gingivitis and chronic periodontitis expressed syndecan-4. Glypican was expressed only by macrophages. Stimulation of PBMC with mitogens and growth factors modulated syndecan-1 expression in both the T- and B-cells. Thus, cell-surface proteoglycan expression by lymphocytes in periodontal inflammation is cell-type-specific and may be modulated by inflammation.

Interleukin-6 (IL-6) binds to human gingival fibroblasts (HGF) in the presence of a soluble form of IL-6 receptor (sIL-6R). We investigated the effects of IL-6 on the functions of HGF in the presence of sIL-6R. HGF changed their morphology from spindle-shaped to round, and detached from the culture dish by stimulation with IL-6/sIL-6R. In this condition, a signal transducer gp130 and a transcription factor Stat3 were phosphorylated, resulting in activation of transcription factors Stat3 and C/EBPbeta. Cytoskeletal beta-actin and adhesion molecule integrin-alpha5, a subunit of alpha5beta1 integrin (VLA-5), were found to possess potential binding domains for these transcription factors in their promoters. Accumulation of beta-actin and integrin-alpha5 mRNA decreased, contrary to the expectation of the induction of gene transcription. Furthermore, the decrease in their mRNAs was associated with reduced expression of both actin and VLA-5 proteins. These results suggest that the expression of VLA-5 and actin was down-regulated in HGF through an IL-6 signaling pathway, resulting in impairment of HGF adherence.

The effect of inflammation on the distribution of chondroitin sulfate and dermatan sulfate proteoglycans was assessed after normal and inflamed human gingivae were stained with monoclonal antibodies against these extracellular matrix macromolecules. The tissues were obtained following periodontal surgery and reacted with specific antibodies after pre-treatment with chondroitinase ACII or chondroitinase ABC, and staining was visualized by the immunoperoxidase technique. The results indicated that these two proteoglycans were present in both the 4-sulfated and 6-sulfated isomeric forms. While chondroitin sulfate appeared to be uniformly distributed throughout the connective tissue, dermatan sulfate showed greater intensity of staining in the areas immediately subjacent to the epithelium. Positive staining for chondroitin sulfate was noted in the intercellular spaces of the epithelium. In inflamed tissues, there was significant staining associated with 4-sulfated dermatan sulfate and chondroitin sulfate, but this had lost the structured pattern of staining noted in normal sections. The 6-sulfated isomeric forms were greatly reduced in inflamed tissues and tended to show a predilection to be localized within the perivascular tissues. In the inflamed tissues, there was intense staining for chondroitin sulfate associated with the infiltrating inflammatory cells. These findings corroborate earlier biochemical studies on normal and inflamed gingival tissues. The specific tissue localization of dermatan sulfate and chondroitin sulfate in tissues damaged by inflammation indicates that, as opposed to the large loss of collagenous material noted during inflammation, there is not a corresponding large loss of proteoglycan. Indeed, at specific inflammatory foci, the intensity of staining for these macromolecules may intensify.

Crevicular fluid from gingivitis patients contains significant levels of a cysteine protease which was characterized as the lysosomal protease cathepsin B, as judged by substrate specificity, thiol dependence, pH optimum, kinetic parameters, pH stability, and inhibitor sensitivities. A highly-sensitive fluorometric assay procedure was used to establish the mean level of cathepsin B activity for 25 gingivitis patients.

The levels of cAMP were measured by means of radioimmunoassay in 20 gingival samples free from inflammatory cells and 43 samples with moderately dense and dense aggregations of inflammatory cells. The average values were found to be 340 +/- 44 and 552 +/- 59 pmol/gm wet tissue, respectively. The increase in cAMP levels in inflammation is suggested to be modulated by an increase in endogenous prostaglandin synthesis and could be one of the natural mechanisms by which the host protects itself from dangerous consequences of unregulated immune response.

We investigated the effect of non-steroidal anti-inflammatory drugs on prostaglandins (PGs) and 12-hydroxyeicosatetraenoic acid (12-HETE) formation by inflamed human gingival tissues. Gingival tissue homogenates were incubated with 14C-arachidonic acid in the presence of indomethacin, piroxicam, or ibuprofen, and the organic solvent extracts were chromatographed on silica gel plates with standards for radiometric assay. There was a significant negative trend between the doses (10(-7)-10(-3) M) of each of indomethacin, piroxicam, and ibuprofen, and the amounts of PGF2 alpha, PGE2, PGD2, and 15-keto-PGE2 produced. All three drugs have a significant inhibitory effect on PGs and 12-HETE production at 10(-3) M when compared with the control. The rank order effectiveness of the drugs, at 10(-3) M, on PG inhibition was indomethacin greater than piroxicam greater than ibuprofen, and on 12-HETE inhibition was indomethacin greater than ibuprofen greater than piroxicam.

Collagen degradation in different stages of gingival inflammation was examined. Higher collagenolytic activity and nonspecific protease activity were found in the severely inflamed gingiva, as compared to mildly inflamed or healthy gingiva. This was shown by electrophoretic studies and measurement of peptide-bound hydroxyproline in the culture.

Incubation of [14C]-arachidonic acid with slices of inflamed human gingivae in the absence of glutathione resulted in formation of 6-keto-PGF1 alpha, the stable hydrolytic product of prostacyclin. Significantly smaller amounts of this product were obtained when normal gingivae were treated in a similar manner. The formation of prostacyclin in inflamed gingivae may play an important role in alveolar bone resorption and as a mediator of inflammation in periodontal disease.

Collagens, solubilized by pepsin-digestion of diphenylhydantoin-induced overgrown gingiva, appeared similar to collagens solubilized from inflamed gingiva with regard to: ratio of type I to type III collagen, ratio of alpha1 to alpha2 of type I collagen, and degree of hydroxylation of type I collagen.

The fibrinolytic activity originated by activation of plasminogen was found to be greater in the inflamed gingiva than in the normal gingiva. The activator of plasminogen present in the normal and inflamed gingiva of the beagle dog was demonstrated histochemically. Plasminogen activator was found adjacent to the crevicular epithelium and blood vessels.

A water-soluble lipopolysaccharide from Salmonella enteritidis and a phenol-soluble lipopolysaccharide from Leptotrichia buccalis were applied topically to the healthy marginal gingivae of beagle dogs. Saline was applied to contralateral areas as an internal control. Increases in vascular permeability were monitored by measurement of gingival fluid, and the collected gingival fluid samples were assayed for kininogenase and kinin activities. Both lipopolysaccharides induced an inflammatory response, as indicated by increased gingival fluid flow. Kininogenase-kinin activities paralleled the increases in gingival fluid flow, with the highest values being associated with peak increases in gingival fluid. The results indicate that both lipopolysaccharides, although different in lipid solubility, penetrate healthy sulcular epithelium and initiate an inflammatory response which is mediated in part by the kallikrein-kinin system. Interrelationships between this system and other inflammatory mediators suggest that kinin generation not only plays a role in the early phases of acute gingival inflammation, but may also contribute to the activation of other mediators appearing later in the response and in chronic inflammatory lesions.

Metronidazole concentrations were estimated in four human volunteers after a single dose of 750 mg taken orally. Samples of blood, saliva, and gingival crevice fluid were collected before intake and during the following 24 hours. The concentrations of metronidazole in plasma and saliva were measured by high-performance liquid chromatography (HPLC). The concentrations in gingival fluid were estimated by a capillary agar-diffusion assay. The results of the metronidazole measurements as obtained by both methods were significantly correlated. The peak concentrations of metronidazole in plasma and saliva were in the same range, 8.7-13.8 micrograms/mL, and similar concentrations were found in the gingival fluid samples. It is concluded that metronidazole taken orally has similar pharmacokinetics in both saliva and plasma, and that a single oral dose of 750 mg metronidazole leads to a concentration of the drug in the gingival crevice fluid that exceeds the minimal inhibitory concentration for most anaerobic oral micro-organisms.