Caseinolytic activity in gingival crevicular fluid from periodontitis patients

Introduction: Protease activity is essential for the progression of periodontal disease and several studies have shown that gingival crevicular fluid (GCF) proteases are associated with the attachment loss and bone destruction associated with periodontial disease. In addition to measuring protease levels using ELISA, it is also important to consider enzyme activity which can be measured using appropriate substrates. Aim: The aim of this work was to measure the proteolyitc activity in gingival crevicular fluid (GCF) from periodontitis patients using zymography and a fluorogenic protease substrate. Materials and Methods: Twenty four GCF samples were collected from patients with established periodontitis who had not received any periodontal treatment in the previous six months. A strip of perio-paper was inserted into the gingival crevice until light resistance was felt. After 30 seconds the perio-paper was removed and placed into 500 ul ice cold 0.01M sodium phosphate buffer, pH 7.2, containing 0.15M sodium chloride, vortex mixed for 30 seconds and stored at -80°C until required. GCF samples (10 ul) were electrophoresed on 4-16% Blue casein zymogram gels at 125V constant voltage for 90 min. Following electrophoresis the gel was washed in renaturation buffer for 30 min and then placed in developing buffer overnight. Areas of protease activity appeared as clear bands against a blue background. The total caseinolytic activity of each GCF sample was measured using a fluorescent assay with resorufin-labelled casein as the substrate. Results: The results showed that both casein zymography and fluorogenic assay methods were suitable for analysing caseinolytic activity in GCF samples from periodontitis patients. Caseinolytic activity was variable in the periodontitis samples studied and may reflect the episodic nature of the disease. Conclusion: Casein zymography and fluorogenic assay methods may be useful in future attempts to measure active episodes of periodontal disease.

Serum Cytokines and Adipokines in Periodontitis

Objective: To investigate whether there was an association between serum levels of pro-inflammatory cytokines, adipokines and periodontitis in 60-70 year-old men in Northern Ireland. Methods: Test subjects (n=61) with established periodontitis, categorised by loss of periodontal attachment (PAL) and pocketing of at least 5mm, were matched for age, smoking and BMI with controls (n=60) who were periodontitis resistant (no PAL or pocketing over 3mm). Serum levels of IL 1 alpha, TNF alpha, IL 6, adiponectin, resistin, leptin and C-reactive protein (CRP) were measured from serum using a multiplex array (Randox, UK). Mann Whitney analysis was used with the level of significance set at p<0.05. Results: There was considerable inter-individual variability in all the analytes measured. There was a significantly higher level of IL 6 in men with periodontitis (3.25 pg/ml, SD 2.29) than in those with no periodontitis (2.38 pg/ml, SD1.62), p=0.0041 (after Bonferroni correction for multiple testing p=0.029). Serum IL 6 was strongly correlated with CRP: r=0.52 (95% CI 0.38-0.64), p<0.0001. There was a lower serum level of adiponectin in men with periodontitis (3662.1 ng/ml, SD 2383.4) compared with those who were periodontitis resistant (4265.9 ng/ml, SD 2266.0), which just escaped statistical significance at p=0.068. There were no significant differences in the serum levels of any of the other analytes measured in relation to periodontal status. Conclusions: There was evidence in the 60-70 year-old male population investigated that periodontitis was associated with an increased serum level of IL 6.

ANRIL Variants and Aggressive Periodontitis in European and African Populations

Introduction: The chromosome 9p21 locus has been identified as a marker of coronary artery disease. In this locus studies have focused on variations in the ANRIL gene that has also been identified as a strong candidate for association with aggressive periodontitis (AgP).
Objective: To investigate possible associations between gene variants of ANRIL and AgP in European and African populations.
Methods: European AgP cases (n= 213) and age-matched periodontally healthy controls (n= 81) were recruited from centres in the United Kingdom (Belfast, Glasgow, Newcastle and London). African AgP cases (n= 95) and controls (n= 105) were recruited in Khartoum, Sudan. Five single nucleotide polymorphisms (SNPs) in ANRIL were genotyped using Sequenom and analysed using Haploview with permutation testing to correct for multiple candidates. Odds ratios (OR) and 95% confidence intervals (95%CI) were calculated.
Results: In the European subjects there was a significant association between rs518394 (p=0.0013; OR = 1.81, 95%CI 1.26-2.61) and rs1333049 (p=0.0028; OR = 1.75, 95%CI 1.21-2.52) and AgP. These associations remained significant after permutation testing. In addition there was an association between rs 1360590 (p=0.035) and AgP in females. In the African subjects there was a significant association between only one SNP rs1537415 and AgP (p=0.036; OR = 1.59, 95%CI 1.04-2.43), however, this was not significant following permutation testing. There were no significant associations with rs3217992 in either population.
Conclusions: SNP variants in the ANRIL locus were shown to be significantly associated with AgP in a European population and for the first time in an African population confirming this as the best replicated locus for aggressive periodontitis.

LL-37 quantification in gingival crevicular fluid (GCF) from periodontitis patients

Background: LL-37, an anti-microbial peptide belonging to the cathelicidin family, derives its name from two N-terminal leucine residues and the 37 amino acids comprising the peptide. LL-37 is the only known cathelicidin to exist in humans. It exhibits both anti-bacterial and immunomodulatory properties. Objectives: In the current study, LL-37 was quantified in GCF from periodontitis patients. Previous studies have relied on qualitative results from Western blotting to detect LL-37 in GCF. This study aims to quantitatively determine LL-37 levels in GCF. Methods: GCF and bacterial plaque samples, pre- and post non-surgical periodontal treatment, were collected from 4 sites in 12 patients presenting with advanced periodontitis. Plaque samples were analysed by QPCR for the presence or absence of the periodontopathic bacterium Porphyromonas gingivalis (P. gingivalis). The concentrations of LL-37 in patient samples pre- and post-treatment were deduced by indirect enzyme linked immunosorbent assay (ELISA). Results: Concentrations of LL-37 in samples varied between a minimum and maximum of 1 and 40 ng/ml. LL-37 levels were shown, pre-treatment, to be higher in deep pockets (6-9 mm) compared with shallower pockets (3-5 mm) and highest in those sites which were positive for P. gingivalis. Non-surgical therapy resulted in a significant improvement in clinical indices while expression levels of P. gingivalis were reduced. Following treatment, LL-37 levels in GCF decreased from an average of 6.5 ± 1 - 5.8 ± 1.2 ng/ml. The most interesting observation however was the reduction in LL-37 levels, from an average of 7 ± 1.3 – 2.5 ± 1.1 ng/ml in those sites where P. gingivalis infection was eradicated post-treatment. Conclusions: LL-37 levels are increased at sites showing advanced periodontal disease, reduce following treatment and appear to be linked to the presence of P. gingivalis. This study will further our knowledge of host defence in chronic diseases such as periodontitis.

Microbiological Profile of Aggressive Periodontitis in a Sudanese Population

Background: Several bacterial species have been identified as being associated with aggressive periodontitis (AgP) notably Aggregatibacter actinomycetemcomitans (Aa) and Porphyromonas gingivalis (Pg). There are limited data on bacterial associations with AgP in African populations. Objective: To investigate possible associations between specific bacteria and AgP in a Sudanese population. Methods: Subgingival plaque samples were collected from 93 (20 male, 73 female) Sudanese patients diagnosed with AgP and from 72 (23 male, 48 female) periodontally healthy Sudanese controls. Quantitative PCR was used to identify Aa, Pg, Treponema denticola (Td) and Fusobacterium nucleatum (Fn). The prevalence of these bacterial species was compared using Chi-square analysis. Odds ratios (OR) were calculated using standard methods. Results: The cases with AgP were well matched in age with the controls: 24.8 (SD 5.1) compared with 23.5 (SD 3.7) years, p=0.07. There was a significantly higher prevalence of Pg in AgP (73%) than in the controls (33%), p<0.0001. The OR for Pg to be associated with AgP was 5.44 (95% confidence intervals 2.78-10.64). In 26 (38%) of the AgP cases positive for Pg there were low levels of this bacterium (<100 copies). Both Td and Fn were identified in virtually all (>95%) the plaque samples studied from both AgP and controls. Aa was the least frequently identified species and was present in only 28% of AgP and 18% of controls, p=0.14. The OR for Aa to be associated with AgP was slightly increased at 1.76 (95% CI 0.83-3.74), however, this was not significant (p=0.14). Conclusion: In the Sudanese subjects studied Pg but not Aa was associated with AgP. There were very low levels of Pg in many of the plaque samples from AgP.

Changes in substance P and neurokinin A in gingival crevicular fluid in response to periodontal treatment

Background: The study of periodontitis provides a unique model for assessing the involvement of neuropeptides in inflammatory disease.

Carboxypeptidase-mediated metabolism of calcitonin gene-related peptide in human gingival crevicular fluid - a role in periodontal inflammation?

Background: Metabolism by peptidases plays an important role in modulating the levels of biologically-active neuropeptides. The metabolism of the anti-inflammatory neuropeptide calcitonin gene-related peptide (GCRP), but not the pro-inflammatory neuropeptides substance P (SP) and neurokinin A (NKA) by components of the gingival crevicular fluid (GCF), could potentiate the inflammatory process in periodontitis.

Quantitative analysis of MRP-8 in gingival crevicular fluid in periodontal health and disease using microbore HPLC.

BACKGROUND:

The protein components of GCF can be separated by reverse-phase microbore HPLC on a C18 column with detection on the basis of 214 nm absorbance. A single major symmetrical protein peak eluting with a retention time of 26 min (50% acetonitrile) was evident in gingival crevicular fluid (GCF) from periodontitis patients but not in healthy GCF. This protein was identified as human MRP-8 by N-terminal amino acid sequencing and liquid chromatography quadropole mass spectrometry.

AIMS:

To quantify the amount of MRP-8 detectable in GCF from individual healthy, gingivitis and periodontitis affected sites and to study the relationship, if any, between the levels of this responsive protein and periodontal health and disease.

METHODS:

GCF was sampled (30 s) from healthy, gingivitis, and periodontitis sites in peridontitis subjects (n=15) and from controls (n=5) with clinically healthy gingiva and no periodontitis. Purified MRP-8 was sequenced by Edmann degradation and the phenylthiohydantoin (PTH) amino acid yield determined (by comparison of peak area with external PTH amino acid standards). This value was subsequently used to calculate the relative amount of protein in the peak eluting with a retention time of 26.0 min (MRP-8) in individual GCF chromatograms.

RESULTS:

Higher levels of MRP-8 were detected in inflammatory sites: periodontitis 457.0 (281.0) ng; gingivitis 413.5 (394.5) ng compared with periodontally healthy sites in diseased subjects 14.6 (14.3) ng and in controls 18.6 (18.5) ng, p=0.003. There was at least 20-fold more MRP-8 in the inflammatory compared with the healthy sites studied.

CONCLUSIONS:

The preliminary data indicate that MRP-8 is present in GCF, with significantly greater amounts present at diseased than healthy sites. A systematic study of the relationship of this protein to periodontal disease could prove useful in further clarifying whether MRP-8 could be a reliable GCF biomarker of gingivitis and periodontitis.

Persistently raised C-reactive protein levels are associated with advanced periodontal disease

Objective: The aim was to investigate whether there was an association between periodontitis or tooth loss in a homogeneous group of 60-70-year-old Western European men and either a sustained high or low level of C-reactive protein (CRP).
Material and Methods: Men enrolled in a cohort study of cardiovascular disease in Northern Ireland were screened in 1990-1994 and rescreened in 2001-2004, when a periodontal examination was completed. High-sensitivity CRP was measured from fasting blood samples. There were 806 men with six or more teeth who had either a high level (>3 mg/l) or a lower level of CRP at both time points. Multivariate analysis was carried out using logistic regression with adjustment for possible confounders. Models were constructed with the CRP level as the outcome variable and various measures of periodontal status (low and high threshold periodontitis) or tooth loss as predictor variables. Confounders included in the analysis were known cardiovascular risk factors of age, smoking, diabetes, BMI and socioeconomic status.
Results: There were 67 men who had a high value of CRP (>3 mg/l) and 739 men who had a CRP value =3 mg/l at both time points. The unadjusted odds ratio (OR) for advanced periodontitis to be associated with high CRP was 3.62, p=0.0003. The association was somewhat attenuated but remained significant (OR=2.49, p=0.02) after adjustment for confounders. A high level of tooth loss was also associated with high CRP with an adjusted OR of 2.17, p=0.008. Low threshold periodontitis was not associated with the level of CRP.
Conclusion: There was an association between advanced periodontitis and elevated CRP levels as measured at two time points at a 10-year interval in the 60-70-year-old European males investigated. This association was adjusted for various cardiovascular risk factors. There was also an association between high levels of tooth loss and high CRP in the men studied.

Neuropeptide Y (NPY) and NPY Y1 receptor in periodontal health and disease

Abstract
OBJECTIVES:
Neuropeptide Y (NPY) coordinates inflammation and bone metabolism which are central to the pathogenesis of periodontitis. The present study was designed to determine whether NPY was quantifiable in human gingival crevicular fluid (GCF) and to test the null hypothesis that GCF levels of NPY were the same in periodontal health and disease. A subsidiary aim was to determine the potential functionality of released NPY by detecting the presence of NPY Y1 receptors in gingival tissue.
DESIGN:
The periodontitis group consisted of 20 subjects (10 females and 10 males) mean age 41.4 (S.D. 9.6 years). The control group comprised 20 subjects (10 females and 10 males) mean age 37.4 (S.D. 11.7 years). NPY levels in GCF were measured in periodontal health and disease by radioimmunoassay. NPY Y1 receptor expression in gingival tissue was determined by Western blotting of membrane protein extracts from healthy and inflamed gum.
RESULTS:
Healthy sites from control subjects had significantly higher levels of NPY than diseased sites from periodontitis subjects. NPY Y1 receptor protein was detected in both healthy and inflamed gingival tissue by Western blotting.
CONCLUSIONS:
The significantly elevated levels of NPY in GCF from healthy compared with periodontitis sites suggests a tonic role for NPY, the functionality of which is indicated by the presence of NPY Y1 receptors in local gingival tissue.

Tooth loss and implant outcomes in patients refractory to treatment in a periodontal practice

Aim: The aim of this study was to investigate the factors associated with continued significant tooth loss due to periodontal reasons during maintenance following periodontal therapy in a specialist periodontal practice in Norway.
Material and Methods: A case-control design was used. Refractory cases were patients who lost multiple teeth during a maintenance period of 13.4 (range 8-19) years following definitive periodontal treatment in a specialist practice. Controls were age- and gender-matched maintenance patients from the same practice. Characteristics and treatment outcomes were assessed, and all teeth classified as being lost due to periodontal disease during follow-up were identified. The use of implants in refractory cases and any complications relating to such a treatment were recorded.
Results: Only 27 (2.2%) patients who received periodontal treatment between 1986 and 1998 in a specialist practice met the criteria for inclusion in the refractory to treatment group. Each refractory subject lost 10.4 (range 4-16) teeth, which represented 50% of the teeth present at baseline. The rate of tooth loss in the refractory group was 0.78 teeth per year, which was 35 times greater than that in the control group. Multivariate analysis indicated that being in the refractory group was predicted by heavy smoking (p=0.026), being stressed (p=0.016) or having a family history of periodontitis (p=0.002). Implants were placed in 14 of the refractory patients and nine (64%) of these lost at least one implant. In total, 17 (25%) of the implants placed in the refractory group were lost during the study period.
Conclusions: A small number of periodontal maintenance patients are refractive to treatment and go on to experience significant tooth loss. These subjects also have a high level of implant complications and failure. Heavy smoking, stress and a family history of periodontal disease were identified as factors associated with a refractory outcome.

Impaired Immune Tolerance to Porphyromonas gingivalis Lipopolysaccharide Promotes Neutrophil Migration and Decreased Apoptosis.

Periodontitis, a chronic inflammatory disease of the tissues supporting the teeth, is characterized by an exaggerated host immune and inflammatory response to periopathogenic bacteria. Toll-like receptor activation, cytokine network induction, and accumulation of neutrophils at the site of inflammation are important in the host defense against infection. At the same time, induction of immune tolerance and the clearance of neutrophils from the site of infection are essential in the control of the immune response, resolution of inflammation, and prevention of tissue destruction. Using a human monocytic cell line, we demonstrate that Porphyromonas gingivalis lipopolysaccharide (LPS), which is a major etiological factor in periodontal disease, induces only partial immune tolerance, with continued high production of interleukin-8 (IL-8) but diminished secretion of tumor necrosis factor alpha (TNF-) after repeated challenge. This cytokine response has functional consequences for other immune cells involved in the response to infection. Primary human neutrophils incubated with P. gingivalis LPS-treated naïve monocyte supernatant displayed a high migration index and increased apoptosis. In contrast, neutrophils treated with P. gingivalis LPS-tolerized monocyte supernatant showed a high migration index but significantly decreased apoptosis. Overall, these findings suggest that induction of an imbalanced immune tolerance in monocytes by P. gingivalis LPS, which favors continued secretion of IL-8 but decreased TNF- production, may be associated with enhanced migration of neutrophils to the site of infection but also with decreased apoptosis and may play a role in the chronic inflammatory state seen in periodontal disease.

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

Papillon-Lefevre 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(1,2). 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(3-6). The PLS locus has been mapped to chromosome 11q14-q21 (refs 7-9). 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.