Effects of paraoxonase 1 on the cytodifferentiation and mineralization of periodontal ligament cells.

BACKGROUND AND OBJECTIVE: Single nucleotide polymorphisms (SNPs) of paraoxonase 1 (PON1) are known to be associated with the pathogenesis of osteoporosis and periodontitis. However, the effects of PON1 on the osteoblastic differentiation of periodontal ligament (PDL) cells are unclear. In this study, we examined the effects of PON1 on the osteoblastic differentiation of PDL cells, and analysed the role of PON1 SNPs on the pathogenesis of aggressive periodontitis (AgP) in the Japanese population. MATERIAL AND METHODS: Human PDL (HPDL) cells were exposed to the PON1 plasmid and PON1 inhibitor, 2-hydroxyquinoline, and cultured in mineralization medium. Expression of calcification-related genes and calcified nodule formation were assessed by real-time PCR, an alkaline phosphatase (ALPase) activity assay and Alizarin red staining. Sanger sequencing was performed to evaluate whether PON1 SNPs are associated with the pathogenesis of AgP in Japanese people. RESULTS: During osteoblastic differentiation of HPDL cells, expression of PON1 mRNA increased in a time-dependent manner. PON1 stimulated an increase in expression of mRNA for calcification-related genes, as well as ALPase activity. In contrast, 2-hydroxyquinoline clearly inhibited the expression of calcification-related genes, ALPase activity and calcified nodule formation in HPDL cells. Moreover, there was a statistically significant difference in the minor allele frequency of PON1 SNP rs854560 between the Japanese control database and patients with AgP in the Japanese population (P = .0190). CONCLUSION: PON1 induced cytodifferentiation and mineralization of HPDL cells, and PON1 SNP rs854560 may be associated with the pathogenesis of AgP in the Japanese population.

Sphingomyelin Phosphodiesterase 3 Enhances Cytodifferentiation of Periodontal Ligament Cells.

Sphingomyelin phosphodiesterase 3 ( Smpd3), which encodes neutral sphingomyelinase 2 (nSMase2), is a key molecule for skeletal development as well as for the cytodifferentiation of odontoblasts and alveolar bone. However, the effects of nSMase2 on the cytodifferentiation of periodontal ligament (PDL) cells are still unclear. In this study, the authors analyzed the effects of Smpd3 on the cytodifferentiation of human PDL (HPDL) cells. The authors found that Smpd3 increases the mRNA expression of calcification-related genes, such as alkaline phosphatase (ALPase), type I collagen, osteopontin, Osterix (Osx), and runt-related transcription factor (Runx)-2 in HPDL cells. In contrast, GW4869, an inhibitor of nSMase2, clearly decreased the mRNA expression of ALPase, type I collagen, and osteocalcin in HPDL cells, suggesting that Smpd3 enhances HPDL cytodifferentiation. Next, the authors used exome sequencing to evaluate the genetic variants of Smpd3 in a Japanese population with aggressive periodontitis (AgP). Among 44 unrelated subjects, the authors identified a single nucleotide polymorphism (SNP), rs145616324, in Smpd3 as a putative genetic variant for AgP among Japanese people. Moreover, Smpd3 harboring this SNP did not increase the sphingomyelinase activity or mRNA expression of ALPase, type I collagen, osteopontin, Osx, or Runx2, suggesting that this SNP inhibits Smpd3 such that it has no effect on the cytodifferentiation of HPDL cells. These data suggest that Smpd3 plays a crucial role in maintaining the homeostasis of PDL tissue.

Effect of nonsurgical periodontal treatment in conjunction with either systemic administration of amoxicillin and metronidazole or additional photodynamic therapy on the concentration of matrix metalloproteinases 8 and 9 in gingival crevicular fluid in patients with aggressive periodontitis.

BACKGROUND: To evaluate in patients with aggressive periodontitis (AgP) the effect of nonsurgical periodontal treatment in conjunction with either additional administration of systemic antibiotics (AB) or application of photodynamic therapy (PDT) on the gingival crevicular fluid (GCF) concentration of matrix metalloproteinases 8 and 9 (MMP-8 and -9). METHODS: Thirty-six patients with AgP were included in the study. Patients were randomly assigned to treatment with either scaling and root planing (SRP) followed by systemic administration of AB (e.g. Amoxicillin + Metronidazole) or SRP + PDT. The analysis of MMP-8 and -9 GCF concentrations was performed at baseline and at 3 and 6 months after treatment. Nonparametric U-Mann-Whitney test was used for comparison between groups. Changes from baseline to 3 and 6 months were analyzed with the Friedman's ANOVA test with Kendall's index of consistency. RESULTS: In the AB group, patients showed a statistically significant (p = 0.01) decrease of MMP-8 GCF level at both 3 and 6 months post treatment. In the PDT group, the change of MMP-8 GCF level was not statistically significant. Both groups showed at 3 and 6 months a decrease in MMP-9 levels. However, this change did not reach statistical significance. CONCLUSIONS: Within the limits of the present study, it may be suggested that in patients with AgP, nonsurgical periodontal therapy in conjunction with adjunctive systemic administration of amoxicilin and metronidazole is more effective in reducing GCF MMP-8 levels compared to the adjunctive use of PDT.

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.

Telomerase expression in individuals with chronic and aggressive periodontitis.

BACKGROUND: The aim of this study is to evaluate the expression of human telomerase reverse transcription (hTERT) enzyme in chronic periodontitis (CP) and aggressive periodontitis (AgP) compared with healthy individuals. METHODS: A total of 79 individuals consented to participate in the study. The study sample comprised healthy individuals (n = 30), patients with CP (n = 30), and patients with AgP (n = 19). Gingival tissue was collected and evaluated for hTERT expression by Western blot and immunohistochemical methods. Reverse transcription polymerase chain reaction was performed using the gingival crevicular fluid (GCF) samples. RESULTS: The hTERT messenger RNA (mRNA) and protein expression was significantly higher in AgP compared with CP (P <0.001). In GCF, 53.33% of patients with CP and 68.42% of patients with AgP were showing hTERT mRNA expression, but it was not detected in the control group. The AgP tissue showed higher hTERT expression compared with CP (P <0.001). The hTERT mRNA expression did not show a correlation with gingival index (GI), plaque index (PI), probing depth (PD), and clinical attachment loss (AL) in patients with AgP, whereas hTERT protein expression was strongly correlated with GI, PI, PD, and AL in patients with AgP. The protein expression of hTERT shows significant but moderate correlation with GI and AL in patients with CP. CONCLUSION: High expression of hTERT might be associated with periodontal disease progression, suggesting that hTERT could be a potential prognostic marker.

Matrix metalloproteinase (MMP)-8 and tissue inhibitor of MMP-1 (TIMP-1) gene polymorphisms in generalized aggressive periodontitis: gingival crevicular fluid MMP-8 and TIMP-1 levels and outcome of periodontal therapy.

BACKGROUND: The aim of the present study is to investigate matrix metalloproteinase (MMP)-8 and tissue inhibitor of MMP-1 (TIMP-1) gene polymorphisms in generalized aggressive periodontitis (GAgP) and to assess the effects of MMP-8 and TIMP-1 genotypes on the outcomes of non-surgical periodontal therapy. METHODS: Genomic DNA was obtained from peripheral blood of 100 patients with GAgP and 167 periodontally healthy controls. MMP-8 +17 C/G, -799 C/T, -381 A/G and TIMP-1 372 T/C, *429 T/G polymorphisms were determined by polymerase chain reaction-restriction fragment length polymorphism. Patients with GAgP received non-surgical periodontal therapy and were followed for 6 months. Clinical periodontal parameters and gingival crevicular fluid (GCF) samples were collected at baseline and at follow-up visits. GCF biomarkers were analyzed by immunofluorescence assay and enzyme-linked immunosorbent assay. RESULTS: Distribution of the MMP-8 -799 C/T genotypes was significantly different between the GAgP and control groups (P <0.005). TIMP-1 372 T/C and *429 T/G genotypes in males were also significantly different between study groups (P <0.004). GCF MMP-8 levels decreased until 3 months after non-surgical therapy compared with baseline in T and G alleles, as well as G and C allele carriers (P <0.0125), whereas no significant decreased was observed in non-carriers (P >0.0125). CONCLUSION: On the basis of the present findings, it can be suggested that MMP-8 -799 C/T and TIMP-1 372 T/C, *429 T/G gene polymorphisms in males may be associated with the susceptibility to GAgP in the Turkish population.

mRNA expression of MMP-28 (Epilysin) in gingival tissues of chronic and aggressive periodontitis patients: a reverse transcriptase PCR study.

BACKGROUND AND OBJECTIVES: Matrix metalloproteinases degrade extracellular membrane and also release bioactive fragments and growth factors, thus influencing fundamental biological and pathological processes. Epilysin (MMP-28) differs from most other MMPs as it is expressed in a number of normal tissues, suggestive of functions in tissue homeostasis. The aim of the present study was to quantitatively evaluate and compare the mRNA expression of epilysin (MMP-28) in gingival tissues of healthy patients and of patients affected by chronic or aggressive periodontitis. METHODS: A total of 60 subjects, 20 periodontally healthy subjects, 20 with chronic periodontitis, and 20 with aggressive periodontitis, were included in this study. Periodontal status was evaluated by measuring gingival index, probing depth and clinical attachment level. mRNA expression of MMP-28 was determined by quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) in gingival tissue samples collected. RESULTS: Relative quantification of mRNA expression of MMP-28 was highest in healthy tissues (RQ = 0.97) when compared to subjects with chronic periodontitis (RQ = 0.37) and aggressive periodontitis (RQ = 0.23), but the difference was not statistically significant. CONCLUSION: mRNA expression of MMP-28 was highest in healthy tissues when compared to diseased periodontal tissues suggesting that MMP-28 could act as a biomarker for periodontal health.

Periodontal treatment reduces matrix metalloproteinase levels in localized aggressive periodontitis.

BACKGROUND: Matrix metalloproteinases (MMPs) are a family of host-derived proteinases reported to mediate multiple functions associated with periodontal breakdown and inflammation. High MMP levels in African-American children with localized aggressive periodontitis (LAgP) have been reported previously by the present authors. However, little is known about MMP reductions in gingival crevicular fluid (GCF) after therapy. This study aims to evaluate MMP levels in the GCF after treatment of LAgP and to correlate these levels with clinical response. METHODS: GCF samples were collected from 29 African-American individuals diagnosed with LAgP. GCF was collected from one diseased site (probing depth [PD] >4 mm, bleeding on probing [BOP], and clinical attachment level ≥ 2 mm) and one healthy site (PD ≤ 3 mm, no BOP) from each individual at baseline and 3 and 6 months after periodontal treatment, which consisted of full-mouth scaling and root planing (SRP) and systemic antibiotics. The volume of GCF was controlled using a calibrated gingival fluid meter, and levels of MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-12, and MMP-13 were assessed using fluorometric kits. RESULTS: MMP-1, MMP-8, MMP-9, MMP-12, and MMP-13 levels were reduced significantly up to 6 months, comparable to healthy sites at the same point. Significant correlations were noted between MMP-2, MMP-3, MMP-8, MMP-9, MMP-12, and MMP-13 levels and percentage of sites with PD >4 mm. MMP-3, MMP-12, and MMP-13 levels also correlated with mean PD of affected sites. CONCLUSION: Treatment of LAgP with SRP and systemic antibiotics was effective in reducing local levels of specific MMPs in African-American individuals, which correlated positively with some clinical parameters.

Non-surgical periodontal therapy decreases serum elastase levels in aggressive but not in chronic periodontitis.

AIM: Assessment of the effect of non-surgical periodontal therapy (SRP) on serum inflammatory parameters in patients with untreated aggressive (AgP) and chronic (ChP) periodontitis. METHODS: Overall, 31 ChP and 29 AgP were examined clinically prior to and 12 weeks after SRP (subgingival scaling of all pockets within 2 days) with systemic antibiotics for patients positive for Aggregatibacter actinomycetemcomitans (14 AgP, 9 ChP). Blood was sampled prior to, one day, 6, and 12 weeks after the first SRP visit. Serum elastase, C-reactive protein (CRP), lipopolysaccharide-binding protein (LBP), interleukin (IL) 6, 8, and leukocyte counts were assessed. RESULTS: At baseline, serum elastase, CRP, and LBP were significantly (p < 0.01) higher in AgP than ChP. Serum elastase, CRP, LBP, and IL-6 were significantly (p < 0.001) elevated one day after scaling in both groups. Both groups showed significant clinical improvement (p < 0.001). A significant difference was observed regarding change of serum elastase 12 weeks after SRP between AgP and ChP (p = 0.015). Multiple regression analysis revealed AgP, African origin, and bleeding on probing to be associated with more pronounced elastase reduction. CRP reduction was associated with African origin, systemic antibiotics, and baseline probing pocket depth. CONCLUSION: SRP results in serum elastase reduction in AgP but not in ChP.

Variables of host response in saliva of patients with periodontitis: a pilot study.

OBJECTIVE: Periodontitis is an infection that results from an imbalance between periodontopathic microorganisms and the local and systemic host defense. This study analyzed saliva samples of patients with periodontitis for several biomarkers of host response. METHOD AND MATERIALS: Saliva was collected from 13 patients with chronic periodontitis, seven patients with aggressive periodontitis, and 13 periodontally healthy control subjects. Diverse markers of host response representing innate and adaptive immune response as well as antioxidative variables were determined. RESULTS: Patients with aggressive periodontitis had significantly higher values of lipid peroxidation and cathepsin C activity in saliva. The highest activities of neutrophil elastase, proteinase 3, and superoxide dismutase were measured in chronic periodontitis patients. Levels of antimicrobial peptides HNPs 1-3 were significantly highest in chronic periodontitis patients than in aggressive periodontitis or control subjects. Immunoglobulin G levels directed against Aggregatibacter actinomycetemcomitans were highest in aggressive periodontitis patients, while those directed against Porphyromonas gingivalis were highest in chronic periodontitis patients. Immunoglobulin A levels directed against these periodontopathogens did not differ among the groups. CONCLUSION: Chronic periodontitis patients showed higher levels of markers primarily associated with combating infection. The levels of markers known mainly for tissue damage were higher in aggressive periodontitis patients. Neutrophil-related markers may be able to identify and differentiate patients with periodontitis.

[Granulocyte elastase levels in saliva and gingival crevicular fluid of subjects with various periodontal conditions].

OBJECTIVE: To compare the granulocyte elastase (EA) levels in saliva and/or gingival crevicular fluid (GCF) of subjects with various periodontal conditions and analyze the relation between EA levels in GCF and in saliva. METHODS: GCF and salivary samples were collected from 17 subjects with healthy periodontium, 14 with gingivitis, 24 with chronic periodontitis (CP) and 24 with aggressive periodontitis (AgP). The EA levels in GCF and saliva were analyzed. RESULTS: The GCF-EA level in AgP were significantly higher than that in CP (0.485 3 ± 0.225 0 vs. 0.288 4 ± 0.193 1, P<0.01); the levels of EA in saliva of periodontitis patients (AgP and CP) were higher than those of healthy and gingivitis subjects (0.844 5 ± 0.660 6, 0.637 3 ± 0.648 9 vs. 0.031 6 ± 0.020 6, 0.012 2 ± 0.005 8, P<0.001). A positive correlation was found between EA levels in saliva and those in GCF (r=0.660). CONCLUSION: GCF-EA level may serve as a marker for clinical assessment of periodontal conditions. The measurement of EA levels in saliva may facilitate to overall screen periodontitis patients in epidemiological study or to monitor periodontal conditions in clinical practice.

Periodontal pathogens affect the level of protease inhibitors in gingival crevicular fluid.

In periodontitis, an effective host-response is primarily related to neutrophils loaded with serine proteases, including elastase (NE) and protease 3 (PR3), the extracellular activity of which is tightly controlled by endogenous inhibitors. In vitro these inhibitors are degraded by gingipains, cysteine proteases produced by Porphyromonas gingivalis. The purpose of this study was to determine the level of selected protease inhibitors in gingival crevicular fluid (GCF) in relation to periodontal infection. The GCF collected from 31 subjects (nine healthy controls, seven with gingivitis, five with aggressive periodontitis and 10 with chronic periodontitis) was analyzed for the levels of elafin and secretory leukocyte protease inhibitor (SLPI), two main tissue-derived inhibitors of neutrophil serine proteases. In parallel, activity of NE, PR3 and arginine-specific gingipains (Rgps) in GCF was measured. Finally loads of P. gingivalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythia and Treponema denticola were determined. The highest values of elafin were found in aggressive periodontitis and the lowest in controls. The quantity of elafin correlated positively with the load of P. gingivalis, Ta. forsythia and Tr. denticola, as well as with Rgps activity. In addition, NE activity was positively associated with the counts of those bacterial species, but not with the amount of elafin. In contrast, the highest concentrations of SLPI were found in periodontally healthy subjects whereas amounts of this inhibitor were significantly decreased in patients infected with P. gingivalis. Periodontopathogenic bacteria stimulate the release of NE and PR3, which activities escape the control through degradation of locally produced inhibitors (SLPI and elafin) by host-derived and bacteria-derived proteases.

Intracytoplasmic enzymes in gingival crevicular fluid of patients with aggressive periodontitis.

BACKGROUND AND OBJECTIVE: Biochemical parameters of crevicular fluid could provide evidence of periodontal tissue disease. The aim of this study was to analyze enzymes in crevicular fluid in aggressive localized and generalized periodontitis. MATERIAL AND METHODS: One hundred and twenty-four subjects were classified as having localized (n = 36) or generalized aggressive periodontitis (n = 38) and subclassified into moderate and severe groups. Controls were 50 periodontitis-free subjects. Activities of the enzymes lactate dehydrogenase, neutrophil elastase, alkaline phosphatase and aspartate aminotransferase were determined. Data were analyzed using one-way ANOVA and Tukey's test. RESULTS: Among the subjects with localized aggressive periodontitis, values of lactate dehydrogenase and alkaline phosphatase increased notably in moderate and severe periodontitis compared with control subjects. Values for aspartate aminotransferase increased with the severity of the disease, and neutrophil elastase was increased in the moderate and severe states. In generalized aggressive periodontitis, lactate dehydrogenase showed higher values than in control subjects in both periodontal subgroups. Alkaline phosphatase and neutrophil elastase showed higher significant differences between moderate and severe periodontitis compared with the control group. Aspartate aminotransferase showed differences between the severe and moderate periodontitis groups compared with the control group. Of all the enzymes analyzed, only lactate dehydrogenase showed higher values in localized than in generalized aggressive periodontitis. CONCLUSION: Lactate dehydrogenase may distinguish localized and generalized aggressive periodontitis. Alkaline phosphatase increases from moderate to severe states in both types of periodontitis. Aspartate aminotransferase and neutrophil elastase only increase with strong evidence of periodontal destruction.

Evaluation of the influence of ozonotherapy on the clinical parameters and MMP levels in patients with chronic and aggressive periodontitis.

PURPOSE: A comparison of the clinical status and salivary MMP levels after SRP alone or with ozonotherapy in patients with aggressive and chronic periodontitis. MATERIAL/METHODS: The study was performed in 52 generally healthy subjects with chronic or aggressive periodontitis. Group CP-S consisted of 12 patients with chronic periodontitis, who underwent scaling and root planing (SRP). In group CP-O there were 25 patients with chronic periodontitis who additionaly to SRP underwent ozonotherapy. The same therapy was performed in group AP, containing 15 patients with aggressive periodontitis. Plaque index, approximal plaque index, bleeding on probing, sulcus bleeding index, probing pocket depth and clinical attachment loss were measured at baseline, at two weeks and two months post-therapy. The levels of MMP-1, MMP-8 and MMP-9 were estimated in non-stimulated saliva with an ELISA method. RESULTS: All the clinical parameters assessed in the study groups were reduced after treatment. SRP with additional ozonotherapy provided an increase in MMP levels in patients with chronic periodontitis and a reduction in MMP levels in patients with aggressive periodontitis. CONCLUSIONS: SRP followed by ozonotherapy does not lead to further improvement in clinical periodontal parameters in patients with AP and CP.

Purine nucleoside phosphorylase activity and expression are upregulated in sites affected by periodontal disease.

BACKGROUND AND OBJECTIVE: Purine nucleoside phosphorylase (PNP) is an enzyme that catalyzes the reversible phosphorolysis of purine nucleosides, playing a key role in the purine salvage pathway. Activated T cells seem to rely heavily on PNP to remain functionally active and are particularly sensitive to PNP deficiency. The role of PNP in periodontal tissues has not been characterized thus far. The aim of this study therefore was to assess the activity and expression of PNP in the gingival tissues of periodontitis patients. MATERIAL AND METHODS: Ten patients consecutively admitted for treatment had their periodontal clinical variables recorded and their gingival crevicular fluid collected. After periodontal treatment the patients were seen once a month for plaque and bleeding control, and had their periodontal variables recorded and gingival crevicular fluid collected at 90 and 180 d. Purine nucleoside phosphorylase-specific activity was assessed using a spectrophotometer through the addition of the PNP substrate analog 2-amino-6mercapto-7-methyl purine riboside to the gingival crevicular fluid. In parallel, PNP expression was assessed by immunohistochemistry and real-time PCR in gingival biopsies and cell culture. RESULTS: Purine nucleoside phosphorylase activity was higher in the gingival crevicular fluid of periodontally diseased sites, which was positively correlated with improvements of the clinical variables. Treatment of periodontal disease induced a striking decrease of PNP activity in periodontally diseased sites. Expression of PNP was more pronounced in mononuclear cells and endothelial cells of the gingiva, and the mRNA levels were 5.7-fold higher in inflamed tissues compared with control samples. CONCLUSION: Purine nucleoside phosphorylase activity and expression are upregulated in periodontally diseased sites and can be detected in the gingival crevicular fluid.

Genetic variation of myeloperoxidase gene contributes to aggressive periodontitis: a preliminary association study in Turkish population.

Myeloperoxidase (MPO) is a lysosomal enzyme found in the azurophilic granules of polymorphonuclear leukocytes. It is involved in the defense against periodontal bacteria, and is also able to mediate inflammatory tissue destruction in aggressive and chronic periodontitis. The aim of this study was to explore the association between MPO-463G/A gene polymorphism and aggressive periodontitis (AgP) and chronic periodontitis (CP). The study included 147 subjects. Probing depth (PD), clinical attachment loss (CAL), plaque index (PI), and gingival index (GI) were recorded as the clinical parameters. Genomic DNA was obtained from the peripheral blood of 32 subjects with AgP, 25 with CP, and 90 reference controls. We genotyped the MPO-463G/A polymorphism using the PCR-RFLP method. All data were analyzed using SPSS version 13.0 for windows. There were no significant differences between the CP patients and controls regarding MPO-463A/G gene polymorphism either in terms of allele frequency or genotype frequency of MPO-463A/G. However, either in terms of allele frequency or genotype frequency of MPO-463A/G, there were significant differences between the AgP patients and the controls. In conclusion, our data suggest that MPO-463G/A may be associated with increased risk of aggressive periodontitis in Turkish patients.

COX-2 is associated with periodontitis in Europeans.

COX-2 plays an important role in periodontitis by mediating inflammatory reactions in periodontal tissues, and the COX-2 polymorphisms rs20417 and rs689466 have been reported to be associated with periodontitis in populations of Taiwanese and Chinese ethnicity. To test whether these variants were associated with periodontitis in populations of European ethnicity, we genotyped the single-nucleotide polymorphisms (SNPs) rs689466 and rs6681231, the latter a haplotype tagging SNP (htSNP) for rs20417 (r2>0.95), in our large-analysis population of individuals with aggressive (n = 532) and chronic periodontitis (n = 1052), and 2873 healthy control individuals. The rare G allele of htSNP rs6681231 was associated with aggressive periodontitis prior to and after adjustment for the covariates smoking, diabetes, and gender, with an odds ratio of 1.57 (95% confidence interval 1.18-2.08; p = 0.002). The validation of the association of rs20417 by the htSNP rs6681231 provides evidence for a general genetic risk of COX-2 variants in the pathogenesis of periodontitis.

A genome-wide association study identifies GLT6D1 as a susceptibility locus for periodontitis.

Periodontitis is a widespread, complex inflammatory disease of the mouth, which results in a loss of gingival tissue and alveolar bone, with aggressive periodontitis (AgP) as its most severe form. To identify genetic risk factors for periodontitis, we conducted a genome-wide association study in German AgP patients. We found AgP to be strongly associated with the intronic SNP rs1537415, which is located in the glycosyltransferase gene GLT6D1. We replicated the association in a panel of Dutch generalized and localized AgP patients. In the combined analysis including 1758 subjects, rs1537415 reached a genome-wide significance level of P= 5.51 x 10(-9), OR = 1.59 (95% CI 1.36-1.86). The associated rare G allele of rs1537415 showed an enrichment of 10% in periodontitis cases (48.4% in comparison with 38.8% in controls). Fine-mapping and a haplotype analysis indicated that rs1537415 showed the strongest association signal. Sequencing identified no further associated variant. Tissue-specific expression analysis of GLT6D1 indicated high transcript levels in the leukocytes, the gingiva and testis. Analysis of potential transcription factor binding sites at this locus predicted a significant reduction of GATA-3 binding affinity, and an electrophoretic mobility assay indicated a T cell specific reduction of protein binding for the G allele. Overexpression of GATA-3 in HEK293 cells resulted in allele-specific binding of GATA-3, indicating the identity of GATA-3 as the binding protein. The identified association of GLT6D1 with AgP implicates this locus as an important susceptibility factor, and GATA-3 as a potential signaling component in the pathophysiology of periodontitis.

PDK1 regulates chemotaxis in human neutrophils.

Phosphoinositide-dependent kinase (PDK1) plays a central role in signal transduction mediated by phosphatidylinositol 3-kinases (PI3K) and regulates cellular functions in neutrophils. Neutrophils from individuals diagnosed with localized aggressive periodontitis (LAP) present an in vivo phenotype with depressed chemotaxis. The aim of this study was to test the hypothesis that PDK1 regulates chemotaxis in neutrophils and is responsible for the abnormal neutrophil chemotaxis LAP. Neutrophil chemotaxis was significantly suppressed by the PDK1 inhibitor staurosporine. When cells were transfected with PDK1 siRNA, there was a significant reduction in chemotaxis, while superoxide generation was not significantly affected. In primary neutrophils from persons with LAP, PDK1 expression and activation levels were significantly reduced, and this reduction was associated with the reduced phosphorylation of Akt (Thr308) and chemotaxis. Analysis of these data demonstrates that PDK1 is essential for the chemotactic migration of neutrophils, and in the absence of PDK1, neutrophil chemotaxis is impaired.

Increased expression of extracellular matrix metalloproteinase inducer is associated with matrix metalloproteinase-1 and -2 in gingival tissues from patients with periodontitis.

BACKGROUND AND OBJECTIVE: Extracellular matrix metalloproteinase inducer (EMMPRIN), an immunoglobulin-like cell surface glycoprotein, could promote collagenolytic balance in favor of the expression and activation of matrix metalloproteinases (MMPs). This study was to investigate the expression of EMMPRIN in gingival tissues from different periodontal conditions and to correlate it with the production of MMP-1 and MMP-2. MATERIAL AND METHODS: Gingival biopsies were collected from 15 patients with untreated advanced chronic periodontitis and 15 patients with aggressive periodontitis (AgP). The control group consisted of 12 subjects diagnosed either as periodontally healthy individuals or as individuals with a gingival index of one (H/G1). The peptides and mRNA of EMMPRIN, MMP-1 and MMP-2 were detected by immunohistochemistry and semi-quantitative reverse transcriptase-polymerase chain reaction, respectively. RESULTS: The expression of EMMPRIN, MMP-1 and MMP-2 peptides in periodontally healthy tissues was mainly confined to the gingival epithelium. The EMMPRIN was strongly expressed in the cell membrane of the basal layer. Immunoreactivity for EMMPRIN was more intensive and more widespread in periodontitis, extended from the epithelial layers to the underlying connective tissues, and was essential in both inflammatory and fibroblast-like cells. In addition, MMP-1 and MMP-2 showed the same localized expression. The chronic periodontitis group had a significantly higher mRNA expression of EMMPRIN and MMP-2 compared with the H/G1 subjects (p < 0.05). Production of MMP-1 and MMP-2 by gingival tissues was correlated with the mRNA level of EMMPRIN (r = 0.463, p = 0.013 for MMP-1 and r = 0.404, p = 0.033 for MMP-2). CONCLUSION: The expression of EMMPRIN in human normal and diseased gingiva might contribute to periodontal physiological and pathological processes; moreover, its increased production might be associated with MMP-1 and MMP-2 expression.