MicroRNA-663 antagonizes apoptosis antagonizing transcription factor to induce apoptosis in epithelial cells.

MicroRNAs are small functional RNAs that modulate various biological processes in cells by interfering with gene translation. We have previously demonstrated that certain miRNAs play a crucial role in the innate immune responses of human oral epithelial cells to Porphyromonas gingivalis. While addressing the mechanisms of P. gingivalis induced apoptosis in these cells, we discovered that certain miRNAs are upregulated upon stimulation with live bacteria. These upregulated miRNAs include hsa-miR-584, hsa-miR-572, hsa-miR-210, hsa-miR-492, hsa-miR-623 and hsa-miR-663. Further analysis revealed an unexpected role for hsa-miR-663 (miR-663). To further evaluate miR-663 function, we overexpressed miR-663 in epithelial cells which resulted in cellular apoptosis. The bioinformatics analysis of the miR-663 target prediction, revealed a strong binding affinity to a 3' UTR region of Apoptosis Antagonizing Transcription Factor (AATF) mRNA. To demonstrate the binding of miR-663 to AATF mRNA, the putative miR-663 target site within the 3'-UTR region of AATF was cloned in luciferase vector and transfected to HEK293T cells. Luminescence data showed the downregulation of luciferase activity in cells that had the full length target region of the putative binding site, confirming that AATF is one of the targets for miR-663. This prompted us to further evaluate its role in a cancer cell line (MCF-7) to determine miR-663s' apoptotic function. The overexpression of miR-663 led to a significant increase in apoptosis of MCF-7 cells. Taken together, miR-663 may function as an 'apoptomiR' by inhibiting the anti-apoptotic gene AATF to induce apoptosis. These findings could have therapeutic implications for epithelial cell targeting in cancer therapy.

Salivary and serum adiponectin and C-reactive protein levels in acute myocardial infarction related to body mass index and oral health.

BACKGROUND AND OBJECTIVE: Adiponectin is produced by adipose cells and is considered an anti-inflammatory molecule. In contrast, C-reactive protein (CRP) has been identified as a hallmark of systemic inflammation and used as a risk marker of cardiovascular disease (CVD). Of interest was the relationship of these two biomarkers to oral health and CVD risk. MATERIAL AND METHODS: This investigation examined these two molecules in serum and unstimulated whole saliva of patients within 48 h of an acute myocardial infarction (AMI) compared to control subjects. We hypothesized a differential response in these biomolecules resulting from the heart attack that would be affected by both the body mass index and oral health characteristics of the individuals. RESULTS: Significantly lower adiponectin levels were observed in the serum of patients with AMI. Serum adiponectin in both groups and salivary adiponectin in patients with AMI decreased with increasing body mass index. Oral health was significantly worse in patients with AMI, and both serum and salivary adiponectin were elevated with better oral health in control subjects. Serum CRP levels were increased in patients with AMI regardless of their oral health, and both serum and salivary CRP were significantly elevated in S-T wave elevated patients with MI. CONCLUSIONS: These initial data provide evidence relating obesity and oral health to salivary and serum analyte levels that occur in association with cardiac events. Relationships have been described between CVD risk and periodontal disease. Additionally, various systemic inflammatory biomarkers appear to reflect both the CVD risk and the extent/severity of periodontitis. Our findings indicated that oral health and obesity contribute to altering levels of these salivary and serum analytes in association with cardiac events. The potential that serum and/or salivary biomarkers could aid in evaluating CVD risk requires knowledge regarding how the oral health of the individual would impact the effectiveness of these biological measures.

The effect of periodontal scaling and root polishing on serum IL-17E concentrations and the IL-17A:IL-17E ratio.

OBJECTIVES: The serum IL-17A:IL-17E ratio has previously been demonstrated to be a clinical marker of periodontitis. The aim of this study was to determine the effects of non-surgical periodontal treatment on the serum IL-17A:IL-17E ratio. MATERIALS AND METHODS: Forty chronic periodontitis patients completed this study and received periodontal treatment comprising scaling and root planing plus ultrasonic debridement. Clinical data were recorded at baseline, 6 weeks (R1) after treatment completion (full-mouth or quadrant-scaling and root planing) and 25 weeks after baseline (R2). Serum samples were taken at each time point and cytokines concentrations determined by ELISA. RESULTS: Following treatment, statistically significant reductions were noted in clinical parameters. However, IL-17A and IL-17E concentrations were significantly greater than baseline values before- and after-adjusting for smoking. The IL-17A:IL-17E ratio was lower at R1 and R2. Serum IL-6 and TNF levels were significantly lower at R1 only. Also exclusively at R1, serum IL-17A and IL-17E correlated positively with clinical parameters, while the IL-17A:IL-17E ratio correlated negatively with probing pocket depth and clinical attachment. CONCLUSION: Increased serum IL-17E and a reduced IL-17A:IL-17E ratio may be indicative and/or a consequence of periodontal therapy. Therefore, the role of IL-17E in periodontal disease progression and the healing process is worthy of further investigation. CLINICAL RELEVANCE: IL-17E may be a valuable biomarker to monitor the healing process following periodontal treatment as increased IL-17E levels and a reduced IL-17A:IL-17E ratio could reflect clinical improvements post-therapy. Therefore, monitoring serum IL-17E might be useful to identify individuals who require additional periodontal treatment.

Experimental gingivitis, bacteremia and systemic biomarkers: a randomized clinical trial.

BACKGROUND AND OBJECTIVE: Bacteremia and systemic inflammatory markers are associated with periodontal and systemic diseases and may be linking mechanisms between these conditions. We hypothesized that in the development of gingival inflammation, systemic markers of inflammation and bacteremia would increase. MATERIAL AND METHODS: To study the effect of bacteremia on systemic inflammatory markers, we recruited 80 subjects to participate in an experimental gingivitis study. Subjects were stratified based on gender, smoking and the number of bleeding sites and then randomized to one of two groups: control group (n = 40) or experimental gingivitis group (n = 40). Subjects in the control group conducted an oral hygiene regimen: brushing twice daily with a regular sodium fluoride cavity protection dentifrice and a standard manual toothbrush, flossing twice daily, and mouth rinsing with an anti-cavity fluoride rinse once daily. The experimental group stopped brushing and flossing, and used only the fluoride anti-cavity mouth rinse for 21 d. RESULTS: Seventy-nine of 80 subjects were evaluable. One subject in the control group was excluded from the results due to antibiotic use during the study. Our data showed the experimental gingivitis group exhibited a significant (p < 0.05) increase in dental plaque level and gingival inflammatory indices relative to baseline and the control group but a decrease in bacteremia and soluble intercellular adhesion molecule-1 levels vs. baseline. Bacteremia was negatively correlated with gingival inflammatory indices and soluble intercellular adhesion molecule-1 levels in the experimental gingivitis group, thus negating our hypothesis. CONCLUSION: We conclude that there are marked differences in systemic cytokine levels over the course of short-term experimentally induced gingivitis and further conclude that a long-term periodontitis study must be considered to address mechanisms whereby oral diseases may affect systemic diseases.

Is obesity a possible modifier of periodontal disease as a chronic inflammatory process? A case-control study.

BACKGROUND AND OBJECTIVE: This cross-sectional case-control study was conducted to provide a comparative evaluation of clinical periodontal measurements, together with serum levels of certain bioactive peptides and inflammatory cytokines, in relation to obesity. For this purpose, clinical periodontal measurements and the levels of serum leptin, adiponectin, interleukin-6 (IL-6), C-reactive protein and soluble intercellular adhesion molecule-1 of obese female individuals and their nonobese counterparts were compared. MATERIAL AND METHODS: Sixty obese (body mass index (BMI) > 30) and 31 nonobese (BMI < 30) female subjects were recruited for the present study. Before any periodontal intervention, serum samples were obtained and full-mouth clinical periodontal measurements were recorded at six sites per tooth. ELISA was used for the biochemical analysis. Data were tested statistically. RESULTS: Clinical attachment level was significantly higher in the obese group compared with the nonobese control group (p < 0.05). Serum levels of leptin and IL-6 were significantly higher in the obese group (p < 0.05). BMI correlated with the serum levels of inflammatory molecules (p < 0.05), but not with clinical periodontal parameters, in the obese group. CONCLUSION: In conclusion, obesity does not seem to have a prominent effect on clinical periodontal parameters but it does have many correlations with circulating inflammatory molecules. As suggested in the literature, increased levels of leptin and IL-6 in the obese group might be one explanation for a possible relationship between obesity and periodontal disease. A prospective study is warranted to clarify, in greater detail, the effects of obesity on periodontal health.

Chlorhexidine decreases the risk of ventilator-associated pneumonia in intensive care unit patients: a randomized clinical trial.

BACKGROUND AND OBJECTIVE: The aim was to evaluate whether oral swabbing with 0.2% chlorhexidine gluconate (CHX) decreases the risk of ventilator-associated pneumonia (VAP) in intensive care unit (ICU) patients. MATERIAL AND METHODS: Sixty-one dentate patients scheduled for invasive mechanical ventilation for at least 48 h were included in this randomized, double-blind, controlled study. As these patients were variably incapacitated, oral care was provided by swabbing the oral mucosa four times/d with CHX in the CHX group (29 patients) and with saline in the control group (32 patients). Clinical periodontal measurements were recorded, and lower-respiratory-tract specimens were obtained for microbiological analysis on admission and when VAP was suspected. Pathogens were identified by quantifying colonies using standard culture techniques. RESULTS: Ventilator-associated pneumonia developed in 34/61 patients (55.7%) within 6.8 d. The VAP development rate was significantly higher in the control group than in the CHX group (68.8% vs. 41.4%, respectively; p = 0.03) with an odds ratio of 3.12 (95% confidence interval = 1.09-8.91). Acinetobacter baumannii was the most common pathogen (64.7%) of all species identified. There were no significant differences between the two groups in clinical periodontal measurements, VAP development time, pathogens detected or mortality rate. CONCLUSION: The finding of the present study, that oral care with CHX swabbing reduces the risk of VAP development in mechanically ventilated patients, strongly supports its use in ICUs and indeed the importance of adequate oral hygiene in preventing medical complications.

The host cytokine response to Porphyromonas gingivalis is modified by gingipains.

BACKGROUND/AIMS: Clinical studies indicate that primary proinflammatory cytokines, such as interleukin-1beta (IL-1beta) are elevated in the gingival crevice around teeth with periodontitis but the secondary cytokines and chemokines, IL-6 and IL-8, are not. The human gingival epithelial cells (HGECs) lining the gingival sulcus respond to perturbation by microbes of dental plaque by releasing a wide range of cytokines. Porphyromonas gingivalis, a putative periodontal pathogen, possesses numerous virulence factors some of which directly impact on the host response. In the present study, we sought to determine how P. gingivalis influences the inflammatory cytokine responses. METHODS: HGECs were challenged with P. gingivalis and other putative periodontal pathogens, and the resultant production of IL-1beta, IL-6, and IL-8 was assayed by enzyme-linked immunosorbent assay (ELISA). Culture supernatants and recombinant human cytokines were challenged with live P. gingivalis wild-type and gingipain-deficient strains and the resultant cytokine profile was assessed by ELISA and Western blot. RESULTS: We show here that primary HGECs challenged with live P. gingivalis result in high levels of IL-1beta but not the related secondary cytokines IL-6 and IL-8. We further demonstrate that cytokine response differences are the result of the action of P. gingivalis proteases, with lysine gingipain being the most effective. CONCLUSION: We conclude that P. gingivalis, through lysine gingipain, can subvert the protective host proinflammatory response by direct cytokine degradation. Changes in the crevicular cytokine profile have consequences in periodontal disease pathogenesis that should be considered in the development of diagnostic and therapeutic modalities.

TLR2 promoter hypermethylation creates innate immune dysbiosis.

Periodontitis is a common chronic inflammatory disease that is initiated by a complex microbial biofilm that poses significant health and financial burdens globally. Porphyromonas gingivalis is a predominant pathogen that maintains chronic inflammatory periodontitis. Toll-like receptors (TLRs) play an important role in periodontitis by recognizing pathogens and maintaining tissue homeostasis. Deficiencies in TLR expression and downstream signaling may reduce the host's innate defenses against pathogens, leading to bacterial persistence and exacerbated inflammation, which are now being better appreciated in disease pathologies. In the case of periodontitis, gingival epithelial cells form the first line of defense against pathogens. Innate immune dysregulation in these cells relates to severe disease pathology. We recently identified a blunted TLR2 expression in certain gingival epithelial cells expressing diminished cytokine signaling upon P. gingivalis stimulation. Upon detailed analysis of the TLR2 promoter CpG Island, we noted higher CpG methylation in this dysregulated cell type. When these cells were treated with DNA methyltransferase inhibitor, TLR2 mRNA and cytokine expression were significantly increased. If TLR2 expression plasmid was ectopically expressed in dysfunctional cells prior to P. gingivalis stimulation, the cytokine expression was increased, confirming the requirement of TLR2 in the P. gingivalis-mediated inflammatory response. We designed a chronic in vitro infection model to test if P. gingivalis can induce DNA methylation in normal gingival epithelial cells that express higher TLR2 upon agonist stimulation. Chronic treatment of normal epithelial cells with P. gingivalis introduced de novo DNA methylation within the cells. In addition, increased DNA methylation was observed in the gingiva of mice infected with P. gingivalis in a periodontitis oral gavage model. Moreover, tissues obtained from periodontitis patients also exhibited differential TLR2 promoter methylation, as revealed by bisulfite DNA sequencing. Taken together, DNA methylation of TLR2 can modulate host innate defense mechanisms that may confer increased disease susceptibility.

Scientific basis of a matrix metalloproteinase-8 specific chair-side test for monitoring periodontal and peri-implant health and disease.

Matrix metalloproteinases (MMPs), especially collagenase-2 (MMP-8), are key mediators of irreversible tissue destruction associated with periodontitis and peri-implantitis. MMP-8 is known to exist in elevated amounts and in active form in the gingival crevicular fluid (GCF) and peri-implant sulcular fluid (PISF) from progressing periodontitis and peri-implantitis lesions and sites, respectively. (Sorsa et al. Ann. N.Y. Acad. Sci. 737: 112-131 [1994]; Teronen et al. J. Dent. Res. 76: 1529-1537 [1997]). We have developed monoclonal antibodies to MMP-8 (Hanemaaijer et al. J. Biol. Chem. 272: 31504-31509 [1997]) that can be used in a chair-side dipstick test to monitor the course and treatment of periodontitis and peri-implantitis. Monoclonal and polyclonal antibody tests for MMP-8 coincided with the classical functional collagenase activity test from GCF and PISF (Sorsa et al. J. Periodont. Res. 22: 386-393 [1988]) in periodontal and peri-implant health and disease. In future a chair-side functional and/or immunological MMP-test can be useful to diagnose and monitor periodontal and peri-implant disease and health.

Clinical and genetic analysis of a large North European Caucasian family affected by early-onset periodontitis.

Genetic studies of early-onset periodontitis (EOP) are hampered by several factors. These include delayed onset of the trait, an upper age limit of expression of the disease, and lack of phenotypic information for edentulous family members. Segregation analyses of families with EOP support a major locus hypothesis but fail to define clearly the criteria used for diagnosis of the relatives. Confirmation of a proposed mode of inheritance and the identification of risk genes is awaited by means of family linkage studies. It is suggested that a system can be developed for the current and retrospective diagnosis of relatives of EOP probands. In addition, it is hypothesized that the large family presented here is suitable for a linkage study. Relatives of the proband who were unavailable for a full periodontal examination, were edentulous, or were deceased, were diagnosed by means of documented clinical evidence of periodontal disease or from reported case histories. Segregation analysis was performed. Analysis of the power of the pedigree to detect linkage was carried out by means of the SIMLINK program. Three different categories were defined according to the reliability of diagnosis of EOP. Segregation analysis indicated either autosomal-dominant or X-linked-dominant inheritance in this family. The simulations showed lod scores above 3.0 for all locations of the disease gene, and for each category of diagnosis. In conclusion, a method has been developed which can be used for the diagnosis of relatives of EOP probands when ideal clinical data are unavailable. The simulations suggest that this family is suitable for a genetic linkage study with the aim of identifying the location of one or more susceptibility genes.

Cytokine responses of oral epithelial cells to Porphyromonas gingivalis infection.

Accumulating evidence indicates that epithelia are not merely mechanical barriers but also important elements of the innate immune system. The present study was performed to examine cytokine responses of oral epithelial cells after infection with the periodontal pathogen Porphyromonas gingivalis. The KB-cell line and primary cultures of periodontal pocket epithelium were infected with P. gingivalis for assessment of bacterial invasion by an antibiotic protection assay, and examination of expression of interleukin-1 beta, interleukin-6, interleukin-8, and tumor necrosis factor-alpha by in situ hybridization and immunohistochemistry. We observed that P. gingivalis induces a strong cytokine response, positively correlated with the adhesive/invasive potential of the infecting strain, in both KB cells and primary cultures. These findings indicate that the epithelial cells of the periodontal pocket are an integral part of the immune system, eliciting cytokine responses to a bacterial challenge. In this context, the adhesive/invasive phenotype of P. gingivalis appears to contribute to pathogenicity.

The effect of smoking on mechanical and antimicrobial periodontal therapy.

The aim of this investigation was to evaluate the effect of smoking on the outcome of periodontal therapy. The study consisted of 54 patients who participated in a 4-group parallel-arm clinical trial on the efficacy of three locally delivered antimicrobial systems as adjuncts to scaling and root planing in the treatment of sites with persistent pocketing after a course of scaling and root planing. These groups included scaling and root planing either alone (S) (n = 3), or in conjunction with the application of 25% tetracycline fibers (S&T) (n = 13), 2% minocycline gel (S&Mi) (n = 14), or 25% metronidazole gel (S&Me) (n = 14). In each patient four pockets > 5 mm with bleeding on probing (BOP) and/or suppuration were studied. The number of subjects who smoked was: 8 (61.5%) in the S&T group, 8 (57.1%) in the S&Mi group, 6 (42.9%) in the S&Me group, and 6 (46.2%) in the S group. The probing depth, attachment level and other clinical parameters were assessed at baseline and 6 weeks after treatments. The clinical results of this comparative study have been previously reported. Regardless of the type of treatment, the change in the probing depth (delta PD) and attachment gain (delta AL) were greater in non-smoker subjects than smoker subjects. delta PD was 1.14 mm versus 0.76 mm (P = 0.019), and delta AL was 0.52 mm versus 0.50 mm at (P = 0.845) for non-smokers and smokers respectively. The analysis of variance using the general linear model (GLM) was used for delta PD and delta AL and took into account the variations in the treatments, number of smoker subjects per group, and baseline probing depth. There was a significant interaction between the "smoking" and the "baseline PD." Further analysis using linear regression indicated that, while there was a significant relationship between the baseline PD and the delta PD or delta AL among the non-smokers, weak and insignificant relationship existed among the smoker subjects. Thus, smoking may have an important role in determining the prognosis of periodontal treatment, particularly in persistent and deep pockets.

A six-month comparison of three periodontal local antimicrobial therapies in persistent periodontal pockets.

BACKGROUND: Currently, several local antimicrobial delivery systems are available to periodontists. The aim of this 6-month follow-up parallel study was to evaluate the efficacy of three commercially available local delivery systems as adjuncts to scaling and root planing in the treatment of sites with persistent periodontal lesions. METHODS: Seventy-nine patients with 4 pockets > or = 5 mm and bleeding on probing and/or suppuration were randomized into 4 treatment groups which included: scaling and root planing alone (S) (20 patients), or in conjunction with the application of 25% tetracycline fibers (S+Tet) (19 patients), or 2% minocycline gel (S+Min) (21 patients), or 25% metronidazole gel (S+Met) (19 patients). Clinical measurements were taken at baseline, 6 weeks, 3 months, and 6 months after antimicrobial application. Treatments were applied using the distributors' recommended protocols. RESULTS: All 4 therapies resulted in significant improvements from baseline in probing depth, attachment level, bleeding on probing, and the Modified Gingival Index (MGI) scores. The improvements in clinical parameters were greater in all 3 adjunctive treatment groups than scaling and root planing alone. The mean probing depth reductions at 6 months were: scaling + tetracycline = 1.38 mm; scaling + metronidazole = 0.93 mm; scaling + minocycline = 1.10 mm; and scaling alone = 0.71 mm. The probing depth reduction at all time points was significantly greater in the scaling plus tetracycline fiber group than the scaling and root planing alone group (P<0.01). There was also a significant improvement for scaling plus tetracycline fiber application over scaling and metronidazole at both 6 weeks and 3 months, although this did not remain significant at the 6-month visit. While the frequency of sites with suppuration was markedly reduced following all antimicrobial treatments, the most effective reductions were seen in the scaling plus tetracycline fiber group, followed by the minocycline group. CONCLUSIONS: Although all 3 locally applied antimicrobial systems seem to offer some benefit over scaling and root planing alone, a treatment regimen of scaling and root planing plus tetracycline fiber placement gave the greatest reduction in probing depth over the 6 months after treatment.

Comparison of 3 periodontal local antibiotic therapies in persistent periodontal pockets.

The aim of this study was to evaluate the efficacy of 3 commercially available periodontal systems for local delivery of antibiotics as adjuncts to scaling and root planing in treatment of sites with persistent periodontal lesions following a course of scaling and root planing. Fifty-four patients with 4 pockets > or = 5 mm and bleeding on probing and/or suppuration were randomized in 4 treatment groups including: scaling and root planing plus application of 25% tetracycline fiber (S + Tet) (13 patients), scaling and root planing plus application of 2% minocycline gel (S + Min) (14 patients), scaling and root planing plus application of 25% metronidazole gel (S + Met) (14 patients), and scaling and root planing alone (S) (13 patients). Clinical measurements were taken at baseline and 6 weeks after the end of treatment periods. All treatments were applied using the distributors' recommended protocols and resulted in significant improvement in probing depth, attachment level, bleeding on probing and the modified gingival index (MGI) scores. The improvements in clinical parameters were greater in all three adjunctive treatment groups than scaling and root planing alone. The mean probing depth reductions were: S + Tet = 1.35 mm, S + Met = 0.95 mm, S + Min = 0.87 mm and S = 0.60 mm. The probing depth reduction was significantly greater in the scaling plus tetracycline fiber group than the scaling and root planing alone group (P = 0.002). The difference between groups in improvement of attachment level or bleeding on probing was not significant Scaling plus tetracycline fiber treatment resulted in the greatest reduction in the MGI scores which was significantly greater than all other groups. While the frequency of sites with suppuration was markedly reduced following all treatments, it reached zero in the scaling plus tetracycline fiber group. No serious adverse effects were observed or reported for any treatment. While all three locally applied antimicrobial systems seem to offer some benefit over scaling and root planing alone, a treatment regimen of scaling and root planing plus tetracycline fiber replacement gave the greatest advantage in the treatment of persistent periodontal lesions at least during the 6-week period following treatment.

The effect of subgingival antimicrobial therapy on the levels of stromelysin and tissue inhibitor of metalloproteinases in gingival crevicular fluid.

Recent investigations imply that a key mechanism in the pathogenesis of periodontal disease may be the ability of oral microorganisms to induce production and/or activation of matrix metalloproteinases (MMPs) in the host tissues. It has been suggested that the pharmacologic inhibition of MMP activity could play an important role in achieving a desirable outcome in periodontal therapy. The efficacy of locally delivered antibiotics on the level of gingival crevicular fluid (GCF) stromelysin (SL) and tissue inhibitor of metalloproteinases (TIMP) on sites with a history of a poor response to mechanical treatment was studied. Fifty-two patients with 4 periodontal pockets > or = 5 mm and bleeding on probing were randomized into four groups of 13 patients. One group received scaling and root planing alone and the other three groups received scaling and root planing plus a locally delivered antimicrobial system. These included 25% tetracycline fiber, 2% minocycline gel, and 25% metronidazole gel. The GCF samples taken at baseline and 6 weeks after treatments were analyzed using an enzyme linked immunosorbent assay (ELISA). GCF SL levels significantly decreased after adjunctive tetracycline fiber (paired t-test, P = 0.020) and minocycline gel (paired t-test, P = 0.023) treatments whereas it remained almost unchanged in the other two groups. While the GCF TIMP level did not change significantly in the scaling and root planing alone group, it significantly increased for all three adjunctive antimicrobial treatments (for tetracycline fiber P < 0.001, minocycline gel P = 0.005, metronidazole gel P < 0.001). The use of adjunctive locally delivered antimicrobial systems, particularly the tetracycline family, may offer an advantage in changing the metalloproteinase profile of the GCF to one more compatible with periodontal health.

Comparative efficacy of the standard flossing procedure and a new floss applicator in reducing interproximal bleeding: a short-term study.

This was a parallel stratified study which examined the effect on gingival health of a new floss holder and applicator, designed to deliver a 25 microliters dose of 0.1% chlorhexidine solution to each interdental embrasure during the flossing procedure. Fifty-two patients with simple chronic gingivitis were stratified according to age, sex, and baseline interdental bleeding score and then assigned to one of three treatment groups. One of the following interdental cleaning agents was used once daily during a 2-week period: conventional floss; a flossing device with chlorhexidine; or a flossing device with placebo solution. Gingival health was assessed using the interdental bleeding index (IBI); i.e., the ratio of bleeding sites to the number of sites tested by stimulation with an interdental cleaner. The percentage reduction in bleeding amounted to 38.3% for conventional floss, 51.5% for the flossing device with chlorhexidine, and 51.4% for the flossing device with placebo. The reductions in both flossing device groups were significantly greater than that of the conventional floss group as determined by one-way ANOVA (F = 4.0; P = 0.024) and multiple range tests. There were no statistically significant differences between the two flossing device groups. There was no difference in patients' perception of ease of use of their respective materials; however, 72% of chlorhexidine users and 94% of placebo users, but only 24% of conventional floss users, felt that their interdental cleaning regimens left their mouths feeling fresher. It is therefore postulated that the pleasant tasting spray may have been an important stimulus to extended use of the new device and may explain its greater effectiveness.

Detection of IgA subclasses and J chain mRNA bearing plasma cells in human dental periapical lesions by in situ hybridization.

Humoral immune responses are implicated in the pathogenesis of human dental periapical lesions. To elucidate whether IgA-associated immune systems play a role in the lesions, the in situ hybridization technique was used to detect J chain mRNA expression, which is correlated with the secretion of dimeric IgA. In addition, IgA subclass mRNA-expressing cells were also investigated by double target in situ hybridization (ISH) methodology using digoxigenin- and biotin-labeled IgA subclass specific oligonucleotide probes. This double target ISH technique involved immunochemical detection with an alkaline phosphatase-conjugated antibody and a peroxidase conjugated avidin-biotin complex system to detect IgA subclass mRNA in the formalin-fixed, paraffin wax embedded periapical tissue sections. Twenty-four biopsy samples (16 periapical granulomas and 8 radicular cysts) were examined. IgA subclass mRNA positive plasma cells were detected in all samples. IgA1 mRNA-expressing cells were predominant both in granulomas and cysts (mean = 75.3 +/- 11.2%, 64.8 +/- 21.3%, respectively), and the IgA1 proportion was higher in granulomas than in cysts, although no significant difference was seen between the two lesions (p = 0.132). J chain mRNA positive cells were very sparsely detected in 21/24 cases. The median percentages of J chain mRNA positive cells/IgA mRNA positive plasma cells (4.7%, range 0.3-13.6%) in cysts were significantly higher than in granulomas (1.3%, range 0-7.7%; p = 0.03). This result supports the hypothesis that dimeric IgA may be more actively produced in radicular cysts than in granulomas. These features are thought to reflect the local activation of the periapical immune system in response to environmental factors and indicate that secretory IgA mediated immune defense systems appear to play little role in these lesions. Our results indicate that the IgA-associated immune response in periapical lesions is more similar to serum or systemic IgA responses than to mucosa-associated immune responses where dimeric IgA predominates.

Relative distribution of plasma cells expressing immunoglobulin G subclass mRNA in human dental periapical lesions using in situ hybridization.

Immunoglobulin G (IgG)-producing plasma cells are the predominant immunoglobulin secreting plasma cells in human dental periapical lesions, compared with immunoglobulin A- and immunoglobulin M-producing plasma cells. In this study, the cells expressing mRNA, that encoded the distinct IgG subclasses, were detected using an in situ hybridization technique in 25 periapical lesions. These lesions consisted of 14 periapical granulomas and 11 radicular cysts. Four oligonucleotide probes were chemically synthesized from IgG subclass-specific hinge region genes to ensure specificity of the probes. Plasma cells expressing mRNA, which coded for the IgG subclasses, were detected in formalin-fixed/paraffin wax-embedded sections. Background staining was negligible in all of the sections tested. The in situ hybridization method used in this study was both specific and sensitive for the detection of mRNA encoding each of the four distinct IgG subclasses, whereas the cells retained good morphology. The relative proportions of plasma cells expressing each of the IgG subclass-specific mRNAs in both granulomas and cysts were as follows: IgG1 (57.4 and 55.5%); IgG2 (34.1 and 34.6%); IgG3 (4.0 and 4.3%); and IgG4 (4.0 and 5.5%). There were no significant differences between the percentages of plasma cells expressing each of the IgG subclass mRNAs between the two types of lesions. IgG1 producing plasma cells comprised the highest proportion of IgG-producing plasma cells in both types of periapical lesion. IgG2-producing plasma cells were next in abundance, followed by plasma cells for either IgG3 or IgG4, which were in roughly equivalent numbers.

Detection of interleukin-1 beta mRNA-expressing cells in human gingival crevicular fluid by in situ hybridization.

Interleukin-1 beta (IL-1 beta) mRNA-expressing cells in human gingival crevicular washings (GCW) obtained from patients with periodontitis and healthy controls were examined by in situ hybridization. GCW was done at 15 diseased sites [Gingival Index > 1; pocket depth > or = 5 mm] from five patients with adult periodontitis and eight clinically periodontal healthy sites from three volunteers GI < or = 1; PD < or = 3 mm), and then the samples were cytocentrifuged. In situ hybridization using digoxigenin-labelled oligonucleotide probe complementary to human IL-1 beta mRNA showed IL-1 transcripts in both polymorphonuclear leucocytes and mononuclear cells but not in epithelial cells in all GCW samples from diseased and healthy sites. Polymorphs were the predominant leucocytes in diseased and healthy sites, averaging 91.7 +/- 4.6 and 77.0 +/- 10.3%, respectively. The percentages of IL-1 beta mRNA-positive polymorphonuclear leucocytes in GCW samples from diseased and healthy sites were 92.3 +/- 4.7 and 80.9 +/- 10.3%, respectively. The IL-1 beta gene signals in individual cells were quantified in five samples (two healthy and three diseased sites). The mean amounts of IL-1 beta mRNA expression in polymorphonuclear leucocytes was higher than that of mononuclear cells in all samples and there was heterogeneity within the populations of polymorphonuclear and mononuclears cells in their ability to express the IL-1 beta gene. These findings indicate that IL-1 beta may be predominantly produced by polymorphonuclear leucocytes in the gingival crevice of patients with adult periodontitis and periodontally healthy controls.

Langerhans cells in human chronic gingivitis and phenytoin-induced gingival hyperplasia.

Langerhans cell numbers in oral epithelium increase as dental plaque accumulates. The anti-convulsant drug phenytoin predisposes to gingival hyperplasia in certain patients who take this medication for epilepsy and who also have poor oral hygiene. In this study 7 patients with phenytoin-induced gingival hyperplasia were compared with 5 subjects with chronic marginal gingivitis. On initial examination and on completion of the hygiene phase of periodontal therapy (a period ranging from 3.0 to 4.25 months), clinical indices of plaque and gingivitis were recorded and biopsies were taken from the lower anterior labial gingiva. Frozen sections were stained by an immunoperoxidase technique using the monoclonal antibody OKT6, and the number of Langerhans cells in a defined cross-sectional area was counted. In phenytoin-induced gingival hyperplasia there was a marked increase in Langerhans cells (13.8 +/- 0.45) when compared with chronic gingivitis (7.7 +/- 0.31; p less than 0.05). Both groups showed marked reductions in their plaque and gingival indices and numbers of Langerhans cells once treatment had been completed. However, levels of Langerhans cells in the drug-induced hyperplasia remained significantly higher (3.5 +/- 0.26) than in chronic gingivitis (1.5 +/- 0.22; p less than 0.05).