Phytocannabinoids regulate inflammation in IL-1ß-stimulated human gingival fibroblasts.

OBJECTIVES: Billions of individuals worldwide suffer from periodontal disease, an inflammatory disease that results in hard-tissue and soft-tissue destruction. A viable therapeutic option to treat periodontal disease may be via cannabinoids that exert immunomodulatory effects, and the endocannabinoid system (ECS) is readily present in periodontal tissues that exhibit cannabinoid type 1 and 2 receptors (CB1R and CB2R). Phytocannabinoids (pCBs), which are a part of a heterogeneous group of molecules acting on cannabinoid receptors (CBR) derived from the cannabis plants, have been attributed to a wide variety of effects including anti-inflammatory activity and some pro-inflammatory effects depending on the cell type. Thus, this study aims to examine the effects of pCBs on primary human gingival fibroblasts (HGFs) in IL-1ß stimulated (simulated periodontal disease) HGFs. MATERIALS AND METHODS: Human gingival fibroblasts (HGFs) obtained from ATCC were cultured per the manufacturer's recommendation. The functional activity of cannabinoid receptors was measured using ACTOne (cAMP)-based CB1R and CB2R assay. The effects of three pCBs (0.1-10 µg/ml or 10-4.5 -10-6.5  M) on cell viability were assessed using the CCK-8 cellular dehydrogenase assay. IL-1ß (1 ng/ml) was added an hour before the treatment to stimulate inflammation in the HGFs before the addition of cannabinoid ligands. After 24-h incubation, the production of INF-γ, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, and TNF-α was measured using Mesoscale Discovery (MSD) Human Pro-Inflammatory kit. To measure prostaglandin E 2 levels (PGE2), Cisbio HTRF PGE2 assay kit was used per the manufacturer's recommendation to measure after 24-h incubation. The data were analyzed using GraphPad Prism 6.0. The analytes for each group were compared using a one-way ANOVA test with Bonferroni's correction. RESULTS: Cannabidivarin (CBVN or CBDV) (EC50  = 12 nM) and cannabigerol (CBG) (EC50  = 30 nM) exhibited agonist activity on CB2R with intermediate efficacy. Cannabidiol (CBD) did not exhibit activation of the CB2R, and the CB1R activation was not observed with any of the pCBs. Cytotoxicity results showed that concentrations of 2.50 µg/ml or greater for the pCBs were toxic except for CBVN. Lower concentrations of CBD and CBG (0.1-0.75 µg/ml), and CBVN at 2.50 µg/ml exhibited significant effects on HGF proliferation. In IL-1ß-stimulated HGFs, prostaglandin E2 (PGE2) production was significantly suppressed only by CBG and CBVN. CBD and CBG treatment alone did, however, elevate PGE2 production significantly compared to control. IL-1ß stimulation resulted in a robust increase in the production of all cytokines tested. Treatment of IL-ß-stimulated HGF with the three pCBs (1 µg/ml) significantly reduced INF-É£, TNF-α, and IL-2. The significant suppression of IL-4 was seen with CBD and CBVN, while only CBVN exerted suppression of IL-13. The three pCBs significantly increased IL-6, IL-10, and IL-12 levels, while none of the pCBs reduced the expression of IL-8 in IL-1ß-stimulated HGF. CONCLUSION: The effective inhibition of IL-1ß-stimulated production of PGE2 and cytokines by the pCB in HGFs suggests that targeting the endocannabinoid system may lead to the development of therapeutic strategies for periodontal therapy. However, each pCB has its unique anti-inflammatory profile, in which certain pro-inflammatory activities are also exhibited. The pCBs alone or in combination may benefit and aid in improving public oral health.

Cannabinoid type-2 receptor agonist, inverse agonist, and anandamide regulation of inflammatory responses in IL-1ß stimulated primary human periodontal ligament fibroblasts.

OBJECTIVE: The aim of this study is to understand the role of cannabinoid type 2 receptor (CB2R) during periodontal inflammation and to identify anti-inflammatory agents for the development of drugs to treat periodontitis (PD). BACKGROUND: Cannabinoid type 2 receptor is found in periodontal tissue at sites of inflammation/infection. Our previous study demonstrated anti-inflammatory responses in human periodontal ligament fibroblasts (hPDLFs) via CB2R ligands. METHODS: Anandamide (AEA), HU-308 (agonist), and SMM-189 (inverse agonist) were tested for effects on IL-1ß-stimulated cytokines, chemokines, and angiogenic and vascular markers expressed by hPDLFs using Mesoscale Discovery V-Plex Kits. Signal transduction pathways (p-c-Jun, p-ERK, p-p-38, p-JNK, p-CREB, and p-NF-kB) were investigated using Cisbio HTRF kits. ACTOne and Tango™ -BLA functional assays were used to measure cyclic AMP (cAMP) and ß-arrestin activity. RESULTS: IL-1ß stimulated hPDLF production of 18/39 analytes, which were downregulated by the CB2R agonist and the inverse agonist. AEA exhibited pro-inflammatory and anti-inflammatory effects. IL-1ß increased phosphoproteins within the first hour except p-JNK. CB2R ligands attenuated p-p38 and p-NFĸB, but a late rise in p-38 was seen with HU-308. As p-ERK levels declined, a significant increase in p-ERK was observed later in the time course by synthetic CB2R ligands. P-JNK was significantly affected by SMM-189 only, while p-CREB was elevated significantly by CB2R ligands at 180 minutes. HU-308 affected both cAMP and ß-arrestin pathway. SMM-189 only stimulated cAMP. CONCLUSION: The findings that CB2R agonist and inverse agonist may potentially regulate inflammation suggest that development of CB2R therapeutics could improve on current treatments for PD and other oral inflammatory pathologies.

Vitamin D attenuates human gingival fibroblast inflammatory cytokine production following advanced glycation end product interaction with receptors for AGE.

BACKGROUND AND OBJECTIVES: Vitamin D [1,25(OH)2 D3 or 1,25D3] is critical in musculoskeletal health, inflammation, immune response, and glucose metabolism. Patients with vitamin D deficiency may be at higher risk of diabetes and periodontitis. Diabetic patients exhibit exacerbated inflammation and more periodontal destruction. Advanced glycation end products (AGEs), formed during diabetic hyperglycemia, activate inflammatory pathways in periodontitis. Human gingival fibroblasts (HGFs) express receptors for AGEs (RAGEs) and can contribute to inflammation. OBJECTIVES: Determine whether glycated human serum albumin (G-HSA) augments HGF IL-6 and IL-8 production, and whether treatment with 1,25D3 attenuates cytokine production following stimulation with G-HSA + IL-1ß and/or IL-17. MATERIAL AND METHODS: HGFs were incubated ±G-HSA or normal human serum albumin (HSA), ±IL-1ß and/or IL-17, ±1,25D3. Cytokines were measured by ELISA. Neutralizing anti-RAGE was used to assess AGE-RAGE interaction. Endotoxin was measured using the ToxinSensor™ System. Data were expressed as mean ± standard deviation and analyzed using a one-way analysis of variance (ANOVA) and Scheffe's F procedure for post hoc comparisons. RESULTS: G-HSA or IL-1ß, but not HSA, significantly stimulated IL-6 and IL-8 production. G-HSA or HSA when combined with IL-1ß or IL-1ß + IL-17 synergistically stimulated IL-6 and IL-8. Neutralizing anti-RAGE inhibited IL-6 and IL-8 produced by cells stimulated with IL-1ß + G-HSA but not (+HSA). Synergism caused by HSA did not appear to be mediated by endotoxin since its levels in G-HSA and HSA were not sufficient to stimulate fibroblasts. Vitamin D inhibited IL-6 and IL-8 production stimulated by G-HSA or HSA + IL-1ß or IL-1ß + IL-17. CONCLUSIONS: Results suggest that the "perioprotective" effects of vitamin D are related to its ability to regulate inflammatory cytokine production by HGFs following AGE-RAGE interaction.

Microleakage of Cements in Prefabricated Zirconia Crowns.

PURPOSE: The purpose of this study was to investigate and compare microleakage of prefabricated posterior NuSmile® ZR and EZCrowns when cemented with BioCem and Ketac Cem dental cements. METHODS: Forty extracted permanent teeth (n equals 10 per group) were fitted for either NuSmile or EZCrowns prefabricated posterior zirconia crowns and cemented with BioCem or Ketac Cem, for a total of four groups. Crowned teeth were placed in Dulbecco's Phosphate Buffered Saline solution at 37 degrees Celsius for 24 hours. Teeth were thermocycled between five degrees Celsius and 55 degrees Celsius for 6,000 cycles, stained with two percent basic fuchsin, sectioned, and visually inspected for microleakage utilizing stereomicroscopy on a four-point scale. Data were statistically analyzed using one-way analysis of variance, SNK (P<.05). RESULTS: All samples in this study had microleakage. NuSmile® ZR crowns demonstrated significantly less microleakage when cemented with BioCem and compared to Ketac Cem (P=.002). NuSmile® ZR cemented with BioCem had significantly less microleakage compared to EZ-Crowns with Ketac Cem (P=.002). CONCLUSIONS: In this in vitro investigation, BioCem cement had significantly less microleakage in zirconia pre-fabricated crowns compared to Ketac Cem, and NuSmile® ZR crowns cemented with BioCem resulted in significantly less microleakage than EZ-Crowns cemented with Ketac Cem.

Anti-inflammatory activity of cannabinoid receptor 2 ligands in primary hPDL fibroblasts.

OBJECTIVES: Approximately 65 million adults in the US have periodontitis, causing tooth loss and decreased quality of life. Cannabinoids modulate immune responses, and endocannabinoids are prevalent during oral cavity inflammation. Targets for intervention in periodontal inflammation are cannabinoid type 1 and 2 receptors (CB1R, CB2R), particularly CB2R because its levels increase during inflammation. We previously demonstrated that SMM-189 (CB2R inverse agonist) decreased pro-inflammatory cytokine production in primary microglial cells. The hypothesis of this study was that cannabinoids anandamide (AEA), HU-308 (CB2R selective agonist), and SMM-189 decrease pro-inflammatory IL-6 and MCP-1 production by primary human periodontal ligament fibroblasts (hPDLFs) stimulated with P. gingivalis LPS, TNF-α, or IL-1ß. DESIGN: Cytotoxic effects of cannabinoid compounds (10-4-10-6.5 M), LPS (1-1000 ng/ml), TNFα (10 ng/ml) and IL-1ß (1 ng/ml) were assessed by measuring effects on cellular dehydrogenase activity. IL-6 and MCP-1 production were measured using Mesoscale Discovery (MSD) Human Pro-Inflammatory IL-6 and MSD Human Chemokine MCP-1 kits and analyzed using MSD Sector 2400 machine. RESULTS: EC50 values for AEA, SMM-189, and HU-308 were 16 µM, 13 µM, and 7.3 µM respectively. LPS (1 µg/ml), TNF-α (10 ng/ml), and IL-1ß (1 ng/ml) increased IL-6 and MCP-1 production, which were inhibited by AEA, SMM-189, and HU-308. AEA alone significantly increased IL-6, but not MCP-1 levels, but the other cannabinoids alone had no effect. CONCLUSION: The effective inhibition of LPS, TNF-α, IL-1ß stimulated IL-6 and MCP-1 production by CB2R ligands in hPDLFs suggests that targeting the endocannabinoid system may lead to development of novel drugs for periodontal therapy, aiding strategies to improve oral health.

The Potential Role of Curcumin in Periodontal Therapy: A Review of the Literature.

Periodontitis is a chronic inflammatory disease in the oral cavity caused by bacterial biofilm attached to tooth surfaces. The periodontal pathogenic microorganisms trigger the disease process; however, the destruction of the periodontium is mostly caused by the host's immune response to the bacterial insults. The main thrust of periodontal therapy has been centered traditionally on reducing the microbial load by mechanical and antimicrobial means. This approach has been reported to be effective for the majority of patients and sites. However, modulating the host response by anti-inflammatory agents could provide another viable pathway to managing poorly responding periodontal patients. The overall objective of this paper is to review current data pertinent to curcumin and its dual anti-inflammatory and antimicrobial properties and to explore its potential in managing patients with periodontal diseases. Curcumin has a wide biological spectrum that could provide clinicians with an alternative anti-inflammatory and antimicrobial agent for managing a variety of maladies including periodontal diseases. However, large-scale longitudinal randomized clinical trials are needed to prove efficacy and effectiveness of curcumin in managing periodontitis. Furthermore, its structure requires modification in order to improve its bioavailability and its clinical effectiveness. Further research aiming at improving its delivery and formulation will enhance its dual potential as an important anti-inflammatory and anti-microbial agent in periodontology.

Effects of cranberry components on IL-1ß-stimulated production of IL-6, IL-8 and VEGF by human TMJ synovial fibroblasts.

OBJECTIVE: Osteoarthritis (OA) in the TMJ is characterized by deterioration of articular cartilage and secondary inflammatory changes. Interleukin-1ß (IL-1ß) stimulates IL-6, IL-8, and vascular endothelial growth factor (VEGF) in synovial fluid of TMJ with internal derangement and bony changes. The cranberry (Vaccinium macrocarpon) contains polyphenolic compounds that inhibit production of pro-inflammatory molecules by gingival cells in response to several stimulators. This study examined effects of cranberry components on IL-1ß-stimulated IL-6, IL-8, and VEGF production by human TMJ synovial fibroblast-like cells. DESIGN: Cranberry high molecular weight non-dialyzable material (NDM) was derived from cranberry juice. Human TMJ synovial fibroblast-like cells from joints with degenerative OA and an ankylosed TMJ without degeneration were incubated with IL-1ß (0.001-1nM)±NDM (25-250µg/ml) (2h preincubation). Viability was assessed via activity of a mitochondrial enzyme. IL-6, IL-8, and VEGF in culture supernatants were measured by ELISA; NF-κB and AP-1 transcription factors were measured in nuclear extracts via binding to specific oligonucleotides. DATA ANALYSIS: ANOVA and Scheffe's F procedure for post hoc comparisons. RESULTS: NDM did not affect cell viability but inhibited IL-1ß stimulated IL-6, IL-8, and VEGF production in all cell lines (p<0.05). NDM partially reduced nuclear levels of NF-κB and AP-1 (p<0.04), depending upon cell line and time of exposure to IL-1ß+NDM. CONCLUSION: Cranberry NDM inhibition of IL-1ß-stimulated IL- 6, IL-8, and VEGF production by TMJ synovial fibroblast-like cells suggests that cranberry components may be useful as a host modulatory therapeutic agent to prevent or treat inflammatory arthropathies of the TMJ.

The effect of long-term aspirin intake on the outcome of non-surgical periodontal therapy in smokers: a double-blind, randomized pilot study.

OBJECTIVE: The objective of this parallel, double-blind, randomized pilot study was to determine the effect of a daily dose of 325 mg of aspirin (ASA) on the clinical outcomes of scaling and root planing in a selected group of adult smokers. BACKGROUND: The response to periodontal therapy is inferior among smokers compared to non-smokers. Long-term intake of ASA has been shown to exert a positive impact on reducing both the prevalence and severity of periodontitis, among high-risk groups of subjects such as heavy smokers and diabetics. It is reasonable to assume that systemic administration of ASA in conjunction with reduction of the bacterial load by scaling and root planing may improve and prolong the benefits of periodontal therapy. To date, only few prospective interventional clinical studies have specifically addressed the periodontal needs of smokers. METHODS: The study includes 24 smokers. The following clinical parameters were measured preoperatively and at 3, 6, 9 and 12 mo postoperatively: (i) gingival index; (ii) plaque index; (iii) probing depth; (iii) probing attachment level; (iv) gingival recession; and (v) bleeding scores. Study subjects received scaling and root planing over several visits and were randomly assigned into two equal groups; a control group (C), which received a placebo and a test group (T), which took a daily dose of 325 mg ASA. No additional therapy was provided over the 1 year observation period. RESULTS: There were more statistically significant differences (p < 0.05; one- tailed) between pretest and posttest scores in the T group than in the C group. Mean percent increase in sites with probing depth 1-3 mm (T: 8.78; C: 7.21); mean percent reduction in sites with probing depth 4-6 mm (T: -7.25; C: -5.09 not statistically significant, NS); mean percent reduction in sites with probing depth ≥ 7 mm (T: -1.42; C: -02.09); mean percent reduction in sites with probing attachment level 3-4 mm (T: -3.63; C: 0.48 NS); mean percent reduction in sites with bleeding on probing (T: -12.37; C: -2.59 NS) (p < 0.05, NS). CONCLUSIONS: Daily intake of 325 mg of ASA following scaling and root planing improved treatment outcomes in smokers, without an increase in gingival bleeding tendency. ASA promoted a higher incidence of shallow pockets and more gain in attachment level.

Cytotoxicity and alkaline phosphatase activity evaluation of endosequence root repair material.

INTRODUCTION: The purpose of this in vitro study was to evaluate the cytotoxicity and alkaline phosphatase (ALP) activity of a new bioceramic root repair material, EndoSequence Root Repair Material (ESRRM; Brasseler USA, Savannah, GA), and to compare these characteristics with those of ProRoot MTA (Dentsply Tulsa Dental, Tulsa, OK) and Geristore (GR; Den-Mat LLC, Santa Maria, CA). METHODS: Human Saos-2 osteoblast-like cells were exposed to 1-, 3-, and 7-day elutes of the materials (100% and 50% strength) for 24 hours after which the bioactivity and ALP activity of the cells were evaluated using a methylthiazol sulfophenyl (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay and para-Nitrophenylphosphate colorimetric assay, respectively. In the positive control group, Triton X-100 (Boehringer Mannheim Corp, Indianapolis, IN) was used to lyse the cells, representing 100% cytotoxicity, and in the negative control group cells received fresh culture medium only. Data were statistically analyzed using the unpaired t test and 1-way analysis of variance. RESULTS: The results revealed that the bioactivity of the cells as well as ALP activity were significantly decreased after exposure to ESRRM elutes in almost all time periods, both in 100% and 50% concentrations, with the exception of ALP activity of day 1 elutes of ESRRM at 50% concentration. MTA did not change the bioactivity or ALP activity of the cells. GR elutes of 100% concentration reduced the bioactivity on days 1 and 3, whereas GR elutes of 50% concentration affected the cells only on day 1. None of the GR elutes had any effect on ALP activity of the cells. CONCLUSIONS: It was concluded that ESRRM elutes of all time periods in general reduced the bioactivity and ALP activity of osteoblast-like cells. GR reduced bioactivity only, whereas MTA had no effect on the cells.

Periodontal and oral manifestations of marijuana use.

UNLABELLED: Marijuana, prepared from the plant Cannabis sativa, is the most widely used illicit drug in the United States. Marijuana use has been associated with adverse psychosocial and health effects, including effects on oral tissues. Periodontal literature has limited references to the periodontal effects of cannabis use. In this report, we present two cases of marijuana-associated gingival enlargement and review the literature on oral complications of marijuana use. METHODS: Two asymptomatic males, aged 23 and 42 years, presented independently for oral prophylaxis. Both had an unremarkable medical history and related a history of significant marijuana use of 2-16 years duration. Common findings following oral and periodontal examination were nicotinic stomatitis-like lesions, uvulitis and gingival enlargement. Marginal and papillary gingiva of the anterior dentition were the areas primarily affected by gingival enlargement, while some of these areas exhibited a nodular or "pebbly" appearance. RESULTS: Marijuana-associated gingival enlargement was diagnosed in the reported cases. A review of the literature revealed two other reports of marijuana-associated gingival enlargement, all in young adult males with chronic (2 or more years) cannabis use. These authors reported a resemblance to phenytoin-induced enlargement. Biochemical similarities between phenytoin and cannabis active compounds suggest possible common pathogenetic mechanisms. Uvulitis and nicotinic stomatitis appear to be the two most common of the several oral manifestations of marijuana use. CONCLUSIONS: Chronic marijuana use may result in gingival enlargement with clinical characteristics similar to phenytoin-induced enlargement.

Vitamin D and its impact on oral health--an update.

Vitamin D has been shown to regulate musculoskeletal health by mediating calcium absorption and mineral homeostasis. Evidence has demonstrated that vitamin D deficiency may place subjects at risk for not only low mineral bone density/osteoporosis and osteopenia but also infectious and chronic inflammatory diseases. Studies have shown an association between alveolar bone density, osteoporosis and tooth loss and suggest that low bone mass may be a risk factor for periodontal disease. Several recent reports demonstrate a significant association between periodontal health and the intake of vitamin D. An emerging hypothesis is that vitamin D may be beneficial for oral health, not only for its direct effect on bone metabolism but also due to its ability to function as an anti-inflammatory agent and stimulate the production of anti-microbial peptides.

Effects of a hindered amine light stabilizer and a UV light absorber used in maxillofacial elastomers on human gingival epithelial cells and fibroblasts.

STATEMENT OF PROBLEM: Ultraviolet light absorber (UVA) and hindered amine light stabilizer (HALS) retard sun-induced pigment degradation in silicone elastomeric maxillofacial prostheses. HALS inhibits polymer degradation and UVA dissipates UV radiation. Their effects on oral cells are unknown. PURPOSE: The purpose of this study was to evaluate the effects of UVA and HALS on membrane integrity, viability, and proliferation of human gingival fibroblasts and epithelial cells. MATERIAL AND METHODS: Tinuvin 123 (HALS) and Tinuvin 213 (UVA) were assessed for cytotoxicity, individually and in a 1:1 ratio (used in elastomers; HALS/UVA). The cells were exposed to HALS, UVA, or HALS/UVA (or control media containing only the diluent), and colorimetric assays measured membrane damage, viability, and proliferation. The data (% cytotoxicity or % control) were analyzed using 3-way cross-classified fixed effects ANOVA (alpha=.05). RESULTS: HALS did not negatively affect either cell type. UVA or HALS/UVA (>or= approximately 0.004%) decreased viability by >or=90% in both cell lines; lower concentrations decreased activity in epithelial cells while increasing it in fibroblasts. UVA or HALS/UVA damaged membranes of both cell lines, but epithelial cells were more resistant. UVA or HALS/UVA inhibited proliferation of both cell types similarly. There was a slight synergistic effect of HALS and UVA on membrane damage in both cell lines, but generally not on other parameters. CONCLUSIONS: Although it is unknown if HALS or UVA leaches from silicone elastomers in vivo, these data suggest that relative resistance of epithelial cells to UVA-induced membrane damage, and UVA's stimulation of fibroblast viability at some concentrations, might provide some protective effect.

Kennedy Space Center Cardiovascular Disease Risk Reduction Program evaluation.

This program evaluation examined the Kennedy Space Center (KSC) Cardiovascular Disease (CVD) Risk Reduction Program which aims to identify CVD risk factors and reduce these risk factors through health education phone counseling. High risk participants (those having two or more elevated lipid values) are identified from monthly voluntary CVD screenings and counseled. Phone counseling consists of reviewing lab values with the participant, discussing dietary fat intake frequency using an intake questionnaire, and promoting the increase in exercise frequency. The participants are followed-up at two-months and five-months for relevant metrics including blood pressure, weight, body mass index (BMI), total cholesterol, high density lipoprotein (HDL) and low density lipoprotein (LDL) cholesterol, triglycerides, dietary fat intake, and exercise frequency. Data for three years of the KSC CVD Program included 366 participants, average age of 49 years, 75% male, and 25% female. For those with complete two and five month follow-up data, significant baseline to two-month follow-up comparisons included decreases in systolic blood pressure (p = 0.03); diastolic blood pressure (p = 0.002); total cholesterol, LDL cholesterol and dietary fat intake (all three at p < 0.0001) as well as a significant increase in exercise frequency (p = 0.04). Significant baseline to five-month follow-up comparisons included decreases in triglycerides (p = 0.05); and total cholesterol, LDL cholesterol and dietary intake (all three at p < 0.0001). These program evaluation results indicate that providing brief phone health education counseling and information at the worksite to high risk CVD participants may impact CVD risk factors.

Effect of a cyclooxygenase-2 inhibitor on interleukin-1beta-stimulated activation of the transcription factor nuclear factor-kappa B in human gingival fibroblasts.

BACKGROUND: In previous work, the cyclooxygenase-2 inhibitor NS-398 inhibited interleukin (IL)-1beta-stimulated prostaglandin E(2) (PGE(2)) production almost completely while partially inhibiting IL-6 production in aggressive periodontitis (AgP) human gingival fibroblasts. PGE(2) and the transcription factor nuclear factor-kappa B (NF-kappaB) regulate IL-1beta-stimulated IL-6 production. Cytoplasmic NF-kappaB is bound to inhibitors (IkappaB proteins). IL-1beta initiates a cascade resulting in phosphorylation and degradation of IkappaB, allowing nuclear translocation of NF-kappaB and target gene activation. The purpose of this study was to determine whether NS-398 inhibited phosphorylation of IkappaB and NF-kappaB activation. METHODS: AgP fibroblasts (1 to 2 x 10(6)) were exposed to IL-1beta (1 x 10(11)M) with or without NS-398 (10 nM) in serum-free medium. The NF-kappaB subunit p65 and phospho-IkappaBalpha were measured in whole cell, cytoplasmic, or nuclear extracts, using colorimetric assays. Enzyme-linked immunosorbent assays were used to measure PGE(2) and IL-6 production by 2.5 x 10(4) cells after exposure to IL-1beta with or without NS-398 in serum-free medium. RESULTS: Consistent with previous results, NS-398 reduced IL-1beta-stimulated PGE(2) by approximately 98% (P <0.001) and IL-6 by approximately 65% (P <0.001). IL-1beta increased nuclear and cytoplasmic p65 ( approximately 8-fold [P <0.001] and approximately 2.5-fold [P <0.03], respectively) over control levels. NS-398 reduced IL-1beta-stimulated nuclear and cytoplasmic p65 to control levels. IL-1beta increased phospho-IkappaBalpha in whole cell extracts by a maximum of approximately 9.5 times (P = 0.0001), and this was inhibited significantly by NS-398 (P

Role of the c-myc proto-oncogene in the proliferation of hereditary gingival fibromatosis fibroblasts.

BACKGROUND: Hereditary gingival fibromatosis (HGF) is a fibrotic gingival enlargement. In previous work, HGF fibroblasts grew faster and produced more collagen and fibronectin (FN) than normal gingival (GN) fibroblasts. HGF FN and collagen production, but not proliferation, were under autocrine transforming growth factor (TGF)-beta control, suggesting other means of activation of HGF proliferation. Elevated/prolonged expression of the proto-oncogene c-myc is implicated in disregulation of cell growth. The objectives of this study were to: 1) determine if c-myc expression is abnormal in quiescent and serum-stimulated HGF and GN fibroblasts and 2) determine the relationship between c-myc expression and fibroblast proliferation using a c-myc antisense oligonucleotide (ODN). METHODS: Proliferation was determined by enzyme-linked immunosorbent assay (ELISA), measuring incorporation of bromodeoxyuridine into DNA. Expression of c-myc was determined by quantitative polymerase chain reaction (PCR), using incorporation of fluorescent dCTP and detection via electrophoresis. RESULTS: Proliferation was minimal until 24 hours or more after serum stimulation, when HGF proliferation was greater than GN (P < or = 0.02). All cells expressed c-myc mRNA at quiescence and > or = 1 hour after serum stimulation. Expression of c-myc in quiescent HGF fibroblasts was elevated, and it peaked and remained higher after serum stimulation than in GN cells. Proliferation of an HGF cell line was inhibited by 4 microM c-myc antisense ODN (14% decrease; P < or = 0.006) and 8 microM c-myc antisense ODN (approximately 80% decrease; P < or = 0.0001), but generally not by c-myc sense ODN. This effect was reversed by hybridizing the c-myc antisense and sense ODNs (P = 0.007). CONCLUSION: Data suggest that elevated proliferation of an HGF fibroblast cell line is related to elevated c-myc expression.

Cyclooxygenase-2 Inhibitors Decrease Interleukin-1ß-Stimulated Prostaglandin E2 and IL-6 Production by Human Gingival Fibroblasts.

BACKGROUND: Previous work showed that normal and aggressive periodontitis (AgP) gingival fibroblasts produce the boneresorbing cytokine IL-6. PGE2 is important in regulating IL-6 production. Non-steroidal anti-inflammatory drugs inhibit PG synthesis via COX-1 and/or COX-2 isoenzymes and may inhibit periodontal destruction. COX-2 is induced after cellular activation (i.e., by inflammatory cytokines such as IL-1ß). Little is known about IL-1ß-stimulated AgP fibroblast IL-6 and PGE2 production and their regulation by COX inhibitors. The objective of this study was to determine the effects of COX-2 inhibitors on amounts of PGE2 and IL-6 made by IL-1ß-stimulated gingival fibroblasts. METHODS: Gingival fibroblasts (2.5 × 104 ) from healthy or severe periodontitis patients were cultured in serum-free medium, with or without IL-1ß (10-11 M) for 24 hours, with or without the COX-1/2 inhibitor indomethacin or the selective COX-2 inhibitors NS-398, celecoxib, or rofecoxib. PGE2 and IL-6 in culture supernatants were determined by specific enzyme-linked immunosorbent assay (ELISA)s. RESULTS: All of the COX inhibitors caused dose-dependent decreases in IL-1ß-stimulated PGE2 , to a maximum of >90% in all cell lines (P ≤0.0001). The selective COX-2 inhibitors, but not indomethacin, caused partial (generally up to approximately 60%), dose-dependent decreases in IL-1ß-stimulated IL-6 in all cell lines (P ≤0.003). When exogenous PGE2 was added concurrently with COX-2 inhibitors before addition of IL-1ß, IL-6 production returned to levels at or approaching that produced by cells exposed only to IL-1ß (P ≤0.04). CONCLUSION: The results suggest that COX-2 inhibition may be useful in helping to control fibroblast production of IL-6 in patients with severe periodontitis. J Periodontol 2003;74:1754-1763.

Cyclooxygenase-2 inhibitors decrease interleukin-1beta-stimulated prostaglandin E2 and IL-6 production by human gingival fibroblasts.

BACKGROUND: Previous work showed that normal and aggressive periodontitis (AgP) gingival fibroblasts produce the bone-resorbing cytokine IL-6. PGE2 is important in regulating IL-6 production. Non-steroidal anti-inflammatory drugs inhibit PG synthesis via COX-1 and/or COX-2 isoenzymes and may inhibit periodontal destruction. COX-2 is induced after cellular activation (i.e., by inflammatory cytokines such as IL-1beta). Little is known about IL-1beta-stimulated AgP fibroblast IL-6 and PGE2 production and their regulation by COX inhibitors. The objective of this study was to determine the effects of COX-2 inhibitors on amounts of PGE2 and IL-6 made by IL-1beta-stimulated gingival fibroblasts. METHODS: Gingival fibroblasts (2.5 x 10(4)) from healthy or severe periodontitis patients were cultured in serum-free medium, with or without IL-1beta (10(-11)M) for 24 hours, with or without the COX-1/2 inhibitor indomethacin or the selective COX-2 inhibitors NS-398, celecoxib, or rofecoxib. PGE2 and IL-6 in culture supernatants were determined by specific enzyme-linked immunosorbent assay (ELISA)s. RESULTS: All of the COX inhibitors caused dose-dependent decreases in IL-1beta-stimulated PGE2, to a maximum of > 90% in all cell lines (P < or = 0.0001). The selective COX-2 inhibitors, but not indomethacin, caused partial (generally up to approximately 60%), dose-dependent decreases in IL-1beta-stimulated IL-6 in all cell lines (P < or = 0.003). When exogenous PGE2 was added concurrently with COX-2 inhibitors before addition of IL-1beta, IL-6 production returned to levels at or approaching that produced by cells exposed only to IL-1beta (P < or = 0.04). CONCLUSION: The results suggest that COX-2 inhibition may be useful in helping to control fibroblast production of IL-6 in patients with severe periodontitis.