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.

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.

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.

Loss-of-function mutations in the cathepsin C gene result in periodontal disease and palmoplantar keratosis.

Papillon-Lefèvre syndrome, or keratosis palmoplantaris with periodontopathia (PLS, MIM 245000), is an autosomal recessive disorder that is mainly ascertained by dentists because of the severe periodontitis that afflicts patients. Both the deciduous and permanent dentitions are affected, resulting in premature tooth loss. Palmoplantar keratosis, varying from mild psoriasiform scaly skin to overt hyperkeratosis, typically develops within the first three years of life. Keratosis also affects other sites such as elbows and knees. Most PLS patients display both periodontitis and hyperkeratosis. Some patients have only palmoplantar keratosis or periodontitis, and in rare individuals the periodontitis is mild and of late onset. The PLS locus has been mapped to chromosome 11q14-q21 (refs 7, 8, 9). Using homozygosity mapping in eight small consanguineous families, we have narrowed the candidate region to a 1.2-cM interval between D11S4082 and D11S931. The gene (CTSC) encoding the lysosomal protease cathepsin C (or dipeptidyl aminopeptidase I) lies within this interval. We defined the genomic structure of CTSC and found mutations in all eight families. In two of these families we used a functional assay to demonstrate an almost total loss of cathepsin C activity in PLS patients and reduced activity in obligate carriers.

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.

[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.

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.

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.

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.

Functional Cathepsin C mutations cause different Papillon-Lefèvre syndrome phenotypes.

AIM: The autosomal-recessive Papillon-Lefèvre syndrome (PLS) is characterized by severe aggressive periodontitis, combined with palmoplantar hyperkeratosis, and is caused by mutations in the Cathepsin C (CTSC) gene. This study aimed to identify CTSC mutations in different PLS phenotypes, including atypical forms and isolated pre-pubertal aggressive periodontitis (PAP). MATERIAL AND METHODS: Thirteen families with different phenotypes were analysed by direct sequencing of the entire coding region and the regulatory regions of CTSC. The function of novel mutations was tested with enzyme activity measurements. RESULTS: In 11 of 13 families, 12 different pathogenic CTSC mutations were found in 10 typical PLS patients, three atypical cases and one PAP patient. Out of four novel mutations, three result in protein truncation and are thus considered to be pathogenic. The homozygous c.854C>T nucleotide exchange (p.P285L) was associated with an almost complete loss of enzyme activity. The observed phenotypic heterogeneity could not be associated with specific genotypes. CONCLUSIONS: The phenotypic variability of the PLS associated with an identical genetic background may reflect the influence of additional genetic or environmental factors on disease characteristics. CTSC mutation analyses should be considered for differential diagnosis in all children suffering from severe aggressive periodontitis.

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.

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.

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.

Impaired polymorphonuclear leukocyte 15-lipoxygenase activity in juvenile and rapidly progressive periodontitis.

15-lipoxygenase activity was investigated in sonicated polymorphonuclear leukocytes (PMNLs) from patients with juvenile and rapidly progressive periodontitis and adult periodontitis. The group with juvenile and rapidly progressive periodontitis had 17 patients (6 male, 11 female, mean age 27.4 years), and the age matched control group had 18 normal individuals (11 male, 7 female, mean age 26.3 years). The group with adult periodontitis had 14 patients with 9 male, 5 female, mean age 45.2 years and the age-matched control group had 6 normal subjects with 5 male, 1 female, mean age 43.7 years. 15-hydroxyeicosa-tetraenoic acid (15-HETE) synthesized in the group with juvenile and rapidly progressive periodontitis was 0.219 +/- 0.102 ng/mg protein (mean +/- S.D.), while it was 0.410 +/- 0.138 ng/mg protein in the age-matched control group. There was a significant difference between the two groups. The group with adult periodontitis produced 0.358 +/- 0.124 ng/mg protein and the age matched control group produced 0.448 +/- 0.176 ng/mg protein (no significant difference). These results are relevant to reports that PMNLs of patients with juvenile and rapidly progressive periodontitis have abnormal functions, while those of patients with adult periodontitis are normal.

Determination of pseudocholinesterase activity in the gingival crevicular fluid, saliva, and serum from patients with juvenile periodontitis and rapidly progressive periodontitis.

By use of a spectrophotometric method, pseudocholinesterase (PCE) activities were determined in gingival crevicular fluid (GCF), saliva, and serum from patients with juvenile periodontitis (JP) and rapidly progressive periodontitis (RPP) and from controls. The PCE activity in the GCF samples was 181 +/- 48 U/L in the JP group, 588 +/- 135 U/L in the RPP group, and 88.5 +/- 29.1 U/L in the control group. Saliva PCE activity levels were 9.1 +/- 1.7 U/L in the JP group, 21.8 +/- 4.5 U/L in the RPP group, and 12.7 +/- 0.8 U/L in the control group. GCF contained a higher PCE activity than saliva but a lower one than that of serum. The RPP group had a significantly higher PCE activity in both the GCF and saliva samples. No significant differences could be found regarding serum enzyme levels. Also, no significant correlations were present between biochemical values and the severity of periodontal disease. GCF may be an important source for the PCE content of saliva. It is suggested that the increased PCE activity seen in RPP patients might be caused by either the direct production of esterases by bacteria or the induction of esterases during periodontal destruction.

The in vivo expression of the collagenolytic matrix metalloproteinases (MMP-2, -8, -13, and -14) and matrilysin (MMP-7) in adult and localized juvenile periodontitis.

Periodontal inflammation is characterized by irreversible degradation of periodontal ligament collagen fibers leading to loss of tooth attachment. Cultured gingival keratinocytes and fibroblasts express, in vitro, various matrix metalloproteinases (MMPs) which can degrade fibrillar collagens. We hypothesized that several MMPs are also synthesized in vivo by sulcular epithelium, and analyzed the collagenolytic MMPs (MMP-2, -8, -13, and -14) and matrilysin (MMP-7) in gingival tissue specimens and gingival crevicular fluid from adult and localized juvenile periodontitis patients by in situ hybridization, immunohistochemistry, and Western immunoblotting. MMP-2, -7, -8, and -13 were expressed in gingival sulcular epithelium. MMP-7 and -13 were also located in fibroblasts and macrophages, and MMP-8 in neutrophils. MMP-8- and -13-positive cells/mm2 were higher in periodontitis gingiva when compared with healthy control tissue (p < 0.01). In periodontal diseases, gingival sulcular epithelium expresses several, rather than a single, collagenolytic MMPs, and this proteolytic cascade is evidently responsible for the tissue destruction characteristic of adult and juvenile periodontitis.

Novel mutations in the cathepsin C gene in patients with pre-pubertal aggressive periodontitis and Papillon-Lefèvre syndrome.

Aggressive periodontitis (AP) in pre-pubertal children is often associated with genetic disorders like Papillon-Lefèvre syndrome (PLS). PLS is caused by mutations in the cathepsin C (CTSC) gene. We report a novel CTSC mutation (c.566-572del) in an otherwise healthy AP child and two novel compound heterozygous mutations (c.947T>G, c.1268G>C) in a PLS patient. We conclude that at least a subset of pre-pubertal AP is due to CTSC mutations and therefore may be an allelic variant of PLS.

Human neutrophil gelatinase and associated lipocalin in adult and localized juvenile periodontitis.

In search of direct in vivo evidence of matrix metalloproteinases (MMPs) in periodontal tissue destruction, we studied the presence and localization of MMP-9 and neutrophil gelatinase-associated lipocalin (NGAL) in adult periodontitis (AP) and localized juvenile periodontitis (LJP) gingival tissue specimens by immunohistochemistry, and the activities of gelatinases by Western blot, enzymography, and activity measurements, using radioactive gelatin as substrate in gingival crevicular fluid (GCF) and saliva. In gingival tissue obtained from AP and LJP patients, polymorphonuclear leukocyte (PMN) 92-kDa MMP-9 and NGAL were seen in the connective tissue, but both the sulcular and the oral epithelia were consistently negative. Whereas PMNs located in the gingival blood vessels showed strictly cytoplasmic MMP-9 and NGAL immunoreactivities, in the case of PMN extravasation the staining reactions extended extracellularly. Gelatinase activities consisting mainly of 92-kDa gelatinase were increased in AP GCF relative to LJP GCF and periodontally healthy control GCF. Western blot with specific anti-NGAL antibodies revealed the presence of 25-kDa NGAL and its high-molecular-weight forms in AP and LJP GCF and saliva and in culture medium of oral keratinocytes, but not in gingival fibroblast culture medium. We conclude that extravasated degranulating PMNs are the major source of MMP-9 and NGAL in periodontitis gingiva, GCF, and saliva.

The importance of data presentation regarding gingival crevicular fluid myeloperoxidase and elastase-like activity in periodontal disease and health status.

BACKGROUND: The enzymatic profile of gingival crevicular fluid (GCF) is being analyzed with increasing interest, but related studies lack a general consensus on most methodological points, including the appropriate mode of data presentation. METHODS: GCF myeloperoxidase (MPO) and elastase-like activity (ELA) levels were spectrophotometrically determined on a total of 60 subjects who were divided into three equal subgroups as early-onset periodontitis (EOP), adult periodontitis (AP), and healthy. GCF enzyme levels were calculated and evaluated both as total enzyme activity and enzyme concentration. The correlations between these GCF enzymes and clinical periodontal status were also analyzed. RESULTS: With both modes of data presentation, the results regarding MPO activity were consistent. When presented either as total MPO activity or MPO concentration, the periodontally healthy group showed significantly lower MPO activity than the two patient groups (P<0.05). However, two modes of data presentation did not match when GCF ELA was concerned. When data were reported as total ELA, the healthy group exhibited lower enzyme activity (0.02 +/- 0.001 U) than EOP (0.04 +/- 0.01 U) and AP (0.06 +/- 0.02 U) groups; but when reported as concentration, the highest ELA levels were seen in the healthy group (221 +/- 31.53 nmol/min/ml), followed by AP (98.63 +/- 23.03 nmol/min/ml) and EOP (70.49 +/- 12.02 nmol/min/ml) (P<0.05). A strong-positive and significant correlation existed between GCF MPO and ELA. Correlations with clinical parameters were mostly observed with total activities. CONCLUSIONS: The findings of the present study confirm the relationship between GCF ELA and MPO activity and periodontal disease and also support the functional relativity between the two enzymes. Furthermore, based on these findings, it can be suggested that data presentation by use of total activity seems to be more sensitive in both the reflection of the actual enzymatic profile of GCF and also the existing clinical periodontal status. For each GCF component, the validity of different modes of data presentation should be considered.

Analysis of human gingival tissue and gingival crevicular fluid beta-glucuronidase activity in specific periodontal diseases.

BACKGROUND: Beta-glucuronidase (betaG) is one of the enzymes involved in the destruction of non-collagenous components of the extracellular matrix. It is also considered an indicator or predictor of periodontal disease activity. The present study was conducted to determine the presence and the levels of betaG activity in gingival tissue and gingival crevicular fluid (GCF) in periodontal disease and health status. The validity of 2 expressions of data, total betaG activity versus betaG concentration, and the correlations between clinical periodontal status and betaG profile was also evaluated. METHODS: betaG activities in gingival tissues and GCF samples from 57 individuals, divided into 3 equal groups of adult periodontitis (AP), early-onset periodontitis (EOP), and periodontally healthy subjects were spectrophotometrically examined. RESULTS: Both patient groups had higher betaG levels in both gingiva and GCF than controls. Significant differences were observed among all groups when total GCF betaG activities were examined (P <0.05). However, the difference between AP and controls was not significant when concentration values were compared (P >0.05). The highest GCF betaG activity, with both expressions, was detected in EOP group. No absolute correlations between clinical parameters and betaG activity were observed, except for random correlations in the patient groups with mean total betaG activities. Also GCF/gingiva betaG levels and the 2 expressions did not show absolute correlations. CONCLUSIONS: The findings of the present study confirm the relationship between betaG activity and periodontal diseases. The differences in data concerning GCF total betaG activity and betaG concentration may suggest that they are not matching measures. Data presentation seems to be an important factor in GCF/enzyme profile studies.