Genome-wide association study of periodontal pocketing in Finnish adults.

BACKGROUND: A genome-wide association study is an analytical approach that investigates whether genetic variants across the whole genome contribute to disease progression. The aim of this study was to investigate genome-wide associations of periodontal condition measured as deepened periodontal pockets (≥ 4 mm) in Finnish adults. METHODS: This study was based on the data of the national Health 2000 Survey (BRIF8901) in Finland and the Northern Finland Birth Cohort 1966 Study totalling 3,245 individuals. The genotype data were analyzed using the SNPTEST v.2.4.1. The number of teeth with deepened periodontal pockets (≥ 4 mm deep) was employed as a continuous response variable in additive regression analyses performed separately for the two studies and the results were combined in a meta-analysis applying a fixed effects model. RESULTS: Genome-wide significant associations with the number of teeth with ≥ 4 mm deep pockets were not found at the p-level of < 5 × 10-8, while in total 17 loci reached the p-level of 5 × 10-6. Of the top hits, SNP rs4444613 in chromosome 20 showed the strongest association (p = 1.35 × 10-7). CONCLUSION: No statistically significant genome-wide associations with deepened periodontal pockets were found in this study.

Analysis of matrix metalloproteinases, especially MMP-8, in gingival creviclular fluid, mouthrinse and saliva for monitoring periodontal diseases.

Matrix metalloproteinase-8 is a promising candidate biomarker for oral fluid (gingival crevicular fluid, peri-implant sulcular fluid and saliva) and mouthrinse chair-side/point-of-care diagnostics to predict, diagnose and determine the progressive phases of episodic periodontitis and peri-implantitis, as well as to monitor the treatments and medications. Matrix metalloproteinase-8 can be used alone or together with interleukin-1beta and Porphyromonas gingivalis to calculate cumulative risk score at the subject level as a successful diagnostic tool, especially in large-scale public health surveys, in which a thorough periodontal examination is not feasible.

Matrix metalloproteinases and myeloperoxidase in gingival crevicular fluid provide site-specific diagnostic value for chronic periodontitis.

AIM: To identify the diagnostic accuracy of gingival crevicular fluid (GCF) candidate biomarkers to discriminate periodontitis from the inflamed and healthy sites, and to compare the performance of two independent matrix metalloproteinase (MMP)-8 immunoassays. MATERIALS AND METHODS: Cross sectional study. GCF (N = 58 sites) was collected from healthy, gingivitis and chronic periodontitis volunteers and analysed for levels of azurocidin, chemokine ligand 5, MPO, TIMP-1 MMP-13 and MMP-14 by ELISA or activity assays. MMP-8 was assayed by immunofluorometric assay (IFMA) and ELISA. Statistical analysis was performed using linear mixed-effects models and Bayesian statistics in R and Stata V11. RESULTS: MMP-8, MPO, azurocidin and total MMP-13 and MMP-14 were higher in periodontitis compared to gingivitis and healthy sites (p < 0.05). A very high correlation between MPO and MMP-8 was evident in the periodontitis group (r = 0.95, p < 0.0001). MPO, azurocidin and total levels of MMP-8, MMP-13 and MMP-14 showed high diagnostic accuracy (≥0.90), but only MMP-8 and MPO were significantly higher in the periodontitis versus gingivitis sites. MMP-8 determined by IFMA correlated more strongly with periodontal status and showed higher diagnostic accuracy than ELISA. CONCLUSIONS: MPO and collagenolytic MMPs are highly discriminatory biomarkers for site-specific diagnosis of periodontitis. The comparison of two quantitative MMP-8 methods demonstrated IFMA to be more accurate than ELISA.

Correlations between different protein species of oral rinse MMP-8 and patient-related factors.

OBJECTIVES: The aim of this study is to examine correlations between different oral rinse matrix metalloproteinase (MMP)-8 protein species in western blot (WB) analysis, quantitative MMP-8 measurements, and patient-related factors. Elevated activated MMP-8 (aMMP-8) associate with periodontitis and a diagnostic point-of-care technology has been developed based on aMMP-8. In WB, different MMP-8 protein species can be analyzed. Relative abundancy of fragmented 20-25 kDa forms in WB has been associated with and reflects MMP-8 activation and related fragmentation and elevated quantitative aMMP-8 measurements. MATERIAL AND METHODS: A random sample of 192 participants from a periodontal disease screening study was used for this study. Oral rinse samples for biomarker analyses were collected before clinical periodontal examinations. aMMP-8 immunofluorometric (IFMA) and WB analysis (utilizing the same monoclonal antibody, 8708), polymorphonuclear leukocyte (PMN) elastase activity test and tissue inhibitor of metalloproteinases (TIMP)-1 ELISA levels were performed from the oral rinse samples. Distinct MMP-8 protein species were differentiated in the WB analysis. Principal component (PC) analysis was conducted to explore correlation patterns between the different species. Adjusted correlation analysis between the extracted PCs of WB and aMMP-8 IFMA levels and multilevel regression analysis were conducted to explore if the other periodontal disease-related biomarkers and clinical surrogate measures and patient-related factors are co-variating with the extracted components. RESULTS: Distinct correlation patterns between the MMP-8 protein species were observed. The first four PCs explained 89% of the whole variance in PC analysis. Statistically significant correlation (p < 0.05) were observed as follows: PC1 positively with 21 kDa (r = .69) and 25 kDa fragments (r = .55) and negatively with 150 kDa complexes (r = -.46). PC2 correlated with 45 (r = .70) and 55 kDa (r = .65) activated forms, PC3 with 70-80 kDa latent proforms (r = .63) and 90-100 kDa complexes (r = .67), and PC4 with 35 kDa fragments (r = .81). There were significant correlations between quantitative (IFMA) aMMP-8 measurements and PC1 (p < 0.001), PC2 (<0.05) and PC3 (<0.05) but not with PC4. In multilevel regression models age, PMN elastase activity, TIMP-1 levels, and a number of 4-5 mm periodontal pockets were associated with PC1, nonsmoking with PC2, age and PMN elastase activity with PC3, and age and smoking with PC4. CONCLUSIONS: Relative abundancy of fragmented 21-25 kDa protein species was correlated with the quantitative aMMP-8 (IFMA) measurements, which is in line with previous results. Different patient-related factors (smoking, age, proteolytic activity) may modify the formation of different MMP-8 protein species in oral rinse samples and may cause variability in quantitative aMMP-8 measurement.

Collagenase-2 (MMP-8) as a point-of-care biomarker in periodontitis and cardiovascular diseases. Therapeutic response to non-antimicrobial properties of tetracyclines.

Neutrophil collagenase or collagenase-2 (matrix metalloproteinase [MMP]-8) belongs to the collagenase subgroup of the MMP superfamily of calcium- and zinc-dependent neutral proteinases. MMP-8 is catalytically the most competent proteinase to initiate type I collagen and extracellular matrix degradation associated with periodontal and peri-implant tissue destruction leading to tooth and dental implant loss. Regarding cardiovascular diseases, pathologically excessive MMP-8 has been implicated in atherosclerotic plaque destabilization and rupture probably through its proteolytic ability to thin the protecting collagenous fibrous cap lining coronary and other arteries. During the initiation and course of inflammatory responses in periodontitis, peri-implantitis and cardiovascular diseases, proinflammatory mediators including especially MMP-8 are up-regulated not only in affected tissues but also in the secreted, disease-affected, oral fluids (gingival crevicular fluid [GCF], peri-implant sulcular fluid [PISF], mouthrinse and saliva) as well as in serum and plasma. Regarding periodontitis, peri-implantitis and cardiovascular diseases, the oral fluid and serum MMP-8 analysis has proven to be a sensitive and an objective biomarker as an indicator of health, pathologic processes and pharmacologic response to therapeutic intervention including doxycycline medication as an MMP inhibitor. Oral fluids, i.e., GCF, PISF, mouthrinse and saliva are easily and non-invasively collected for the site- and patient-specific diagnostic analysis in periodontitis and peri-implantitis, whereas serum and/or plasma sample collection is required for diagnosis and monitoring of cardiovascular diseases. Research in periodontology and cardiology has identified a need for the development of innovative point-of-care diagnostic tests for MMP-8. We summarize and review the recent studies on these topics.

Associations Between Glutathione-S-Transferase Genotypes and Bronchial Hyperreactivity Patients With Di-isocyanate Induced Asthma. A Follow-Up Study.

Introduction: Di-isocyanates TDI (toluene di-isocyanate), MDI (diphenylmethane di-isocyanate), and HDI (hexamethylene di-isocyanate) are the most common chemicals causing occupational asthma. Di-isocyanate inhalation has been reported to induce oxidative stress via reactive oxygen and nitrogen species leading to tissue injury. Glutathione transferases (GSTs) and N-acetyltransferases (NATs) are detoxifying enzymes whose general function is to inactivate electrophilic substances. The most important genes regulating these enzymes, i.e., GSTM1, GSTP1, GSTT1, NAT1, and NAT2 have polymorphic variants resulting in enhanced or lowered enzyme activities. Since inability to detoxify harmful oxidants can lead to inflammatory processes involving activation of bronchoconstrictive mechanisms, we studied whether the altered GST and NAT genotypes were associated with bronchial hyperreactivity (BHR) in patients with di-isocyanate exposure related occupational asthma, irrespective of cessation of di-isocyanate exposure, and adequacy of asthma treatment. Methods: Polymerase chain reaction (PCR) based methods were used to analyze nine common polymorphisms in GSTM1, GSTM3, GSTP1, GSTT1, NAT1, and NAT2 genes in 108 patients with diagnosed occupational di-isocyanate-induced asthma. The genotype data were compared with spirometric lung function and BHR status at diagnosis and in the follow-up examination on average 11 years (range 1-22 years) after the asthma diagnosis. Serum IgE and IL13 levels were also assessed in the follow-up phase. Results: An association between BHR and GSTP1 slow activity (Val105/Val105) genotype was demonstrated in the subjects at the follow-up phase but not at the diagnosis phase. Moreover, the patients with the GSTP1 slow activity genotype exhibited characteristics of Th-2 type immune response more often compared to those with the unaltered GSTP1 gene. Interestingly, all 10 patients with the GSTP1 slow activity genotype had both the GSTM3 slow activity genotype and the unaltered GSTT1 gene. Discussion: The results suggest associations of the low activity variants of the GSTP1 gene with BHR. The fact that these associations came up only at the follow-up phase when the subjects were not any more exposed to di-isocyanates, and used asthma medication, suggest that medication and environmental factors influence the presentation of these associations. However, due to the exploratory character of the study and relatively small study size, the findings remain to be confirmed in future studies with larger sample sizes.

Diagnosis of Newly Delivered Mothers for Periodontitis with a Novel Oral-Rinse aMMP-8 Point-of-Care Test in a Rural Malawian Population.

A novel qualitative point-of-care test of activated matrix metalloproteinase-8 (aMMP-8) using noninvasive oral rinse sampling procedures has been developed for the early detection of collagen breakdown indicating periodontal tissue destruction. The main object of this study was to assess the reliability of the test in a low-income setting to identify participants with history of periodontal destruction detected as alveolar bone loss (ABL) in radiographs. This cross-sectional study included 486 women who had recently delivered in rural Malawi. The aMMP-8 test and dental panoramic radiographs were taken within 48 h of delivery. The performance of the test in comparison to radiological examinations was tested by following the standards for reporting of diagnostic accuracy studies protocol (STARD) with respective statistical measures and 95% confidence intervals. From the 486 eligible participants, 461 mothers with complete data, aged from 15 to 46 years (mean 24.8, SD 6.0) were included in the analysis. ABL was identified in 116 of 461 participants. There was 56% agreement between the aMMP-8 test results and detected ABL (yes or no) in radiographs. Calculated sensitivity of the test was 80% (72⁻87%), specificity 48% (43⁻54%), positive predictive value 34% (31⁻37%), negative predictive value 88% (83⁻91%), positive likelihood ratio 1.55 (1.35⁻1.77), and negative likelihood ratio 0.41(0.28⁻0.60). The aMMP-8 test sensitivity and negative predictive value to identify the ABL cases were relatively high, but there was additionally a high rate of test-positive results in participants without ABL, especially in young mothers, leading to low overall agreement between the test results and radiological bone loss. Further longitudinal studies are needed to examine if the test positive subjects are in risk of future bone loss before the detectable signs of periodontitis in radiographs.

The utility of gingival crevicular fluid matrix metalloproteinase-8 response patterns in prediction of site-level clinical treatment outcome.

BACKGROUND: Different gingival crevicular fluid (GCF) matrix metalloproteinase (MMP)-8 response patterns were studied among non-smoking and smoking patients with chronic periodontitis (CP) and generalized aggressive periodontitis (GAgP) to test the utility of GCF MMP-8 levels predicting the site-level treatment outcome. METHODS: Data from four independent longitudinal studies were combined. Altogether, the studies included 158 periodontal sites from 67 patients with CP and 32 patients with GAgP, and GCF samples were collected at baseline, after the treatment, and during the 6-month maintenance period. All GCF samples were analyzed by immunofluorometric assay for MMP-8. Different site-level MMP-8 response patterns were explored by the cluster analysis. Most optimal MMP-8 cutoff levels were searched with receiver operating characteristic analyses, and the predictive utility of defined levels was tested. RESULTS: Distinct types of MMP-8 response patterns were found in both smokers and non-smokers. MMP-8 levels exceeding the optimal cutoff levels separately defined for smokers and non-smokers indicated increased risk for compromised treatment outcome at baseline and during the maintenance period. Seventy-one percent of non-smokers (positive likelihood ratio of 4.22) and 88% of smokers (positive likelihood ratio of 5.00) with positive test results at both baseline and the maintenance period had compromised treatment outcome. The double-positive result indicated 46% and 39% point risk increase for the compromised outcome, respectively. CONCLUSION: GCF MMP-8 analysis with defined cutoff levels could be used to predict the site-level treatment outcome and for longitudinal monitoring of the disease status during the maintenance period.

Gingival crevicular fluid matrix metalloproteinase-8 levels predict treatment outcome among smokers with chronic periodontitis.

BACKGROUND: Molecular biomarkers are needed for diagnostic use in periodontal diseases. The aim of this study is to explore different gingival crevicular fluid (GCF) matrix metalloproteinase-8 (MMP-8) patterns in smokers and non-smokers with chronic periodontitis (CP) and test the utility of baseline GCF MMP-8 levels in predicting categorically assessed treatment outcomes. METHODS: The study population comprised 15 patients with CP (five non-smokers and 10 smokers). GCF sampling of five to seven periodontal sites per patient was done at baseline, post-treatment, and bimonthly during the maintenance period from 8 to 12 months. GCF MMP-8 levels were measured with an immunofluorometric assay. MMP-8 response patterns were explored by cluster analysis. The ability of baseline MMP-8 levels to predict categorical treatment outcomes was analyzed with receiver operating characteristic curves. RESULTS: GCF MMP-8 response patterns could be clustered into two different site profiles among both smokers and non-smokers. Smoker site profiles 1 and 2 had significantly different clinical attachment level and gingival recession changes by the end of the maintenance period. In smoker sites, baseline MMP-8 levels significantly predicted the categorical treatment outcome. CONCLUSIONS: Baseline GCF MMP-8 levels strongly predict how MMP-8 levels behave during the maintenance period. In smoker sites, high baseline MMP-8 levels indicate weak treatment response.