The effect of periodontal treatment on serum leptin, interleukin-6, and C-reactive protein.

BACKGROUND: Previous studies suggest that periodontitis is closely related to obesity and metabolic syndrome. Leptin, a pleiotrophic hormone produced by adipose tissue, has been reported to be related to periodontitis. This study investigates the effects of periodontal treatment on the serum levels of leptin and other cytokines in patients with chronic periodontitis (CP). METHODS: Serum samples were taken from 33 CP patients (22 non-smokers, 11 smokers) and 18 healthy subjects. The serum leptin, adiponectin, tumor necrosis factor-alpha, interleukin (IL)-6, and C-reactive protein (CRP) levels were measured before and after non-surgical periodontal treatment. RESULTS: Significant differences between healthy and CP patients were found in serum leptin, IL-6, and CRP levels (P = 0.0018, P = 0.0064, and P = 0.0095, respectively). The serum leptin level was associated with mean probing depth, mean clinical attachment level, mean alveolar bone loss, and body mass index. There were significant associations between serum leptin levels and IL-6 and CRP levels. After non-surgical periodontal treatment, serum leptin, IL-6, and CRP levels were significantly decreased (mean +/- SD before and after, P value, respectively: leptin, 8.02 +/- 5.5, 7.10 +/- 4.4, P = 0.015; IL-6, 1.73 +/- 1.02, 1.36 +/- 0.73, P = 0.048; and CRP, 802.0 +/- 1065, 491.2 +/- 479.3, P = 0.047). CONCLUSIONS: Periodontal treatment is effective in reducing serum leptin, IL-6, and CRP levels. The results suggest that leptin, IL-6, and CRP could be mediating factors that connect metabolic syndrome and periodontitis.

Assessment of chromosome 19 for genetic association in severe chronic periodontitis.

BACKGROUND: A genome-association study is a powerful tool for analyzing small gene effects in complex diseases such as chronic periodontitis (CP), although the cost of analysis is prohibitive. We designed a study using the DNA pooling method, which could be a breakthrough in lowering such costs. This study was conducted to assess the genetic association in severe CP in a Japanese population. METHODS: We adopted a DNA pooling method by genotyping 454 densely spaced microsatellite (MS) markers in chromosome 19 as a pilot study, with the possibility of future use in a whole-genome study. This can reduce the high cost and technical burden, which is generally unavoidable in a genomic association study. Pooled DNA samples from 300 case subjects, 300 control subjects, and 200 systemically healthy subjects were screened by genotyping MS markers. The case-control association in the candidate region was analyzed by individual typing of MS and single nucleotide polymorphisms (SNPs). RESULTS: The single MS marker allele 17 of 1902G31 was isolated in association with severe CP (P = 0.0012 for 2 x 2; P <0.046 for 2 x m, where m refers to the number of polymorphic alleles observed in a population). No other SNP or MS polymorphism hypothesized to affect biologic functions in the critical region was found in the linkage disequilibrium block analysis. CONCLUSIONS: We efficiently isolated the susceptible locus for severe CP in chromosome 19 and identified a useful marker to evaluate the risk for disease. This approach can be applied to a whole-genome study in severe CP.

Effects of ascorbic acid on gingival melanin pigmentation in vitro and in vivo.

BACKGROUND: Gingival melanin pigmentation may cause esthetic concerns, even if no serious medical problem is present. As an inhibitor of melanin formation, ascorbic acid is often used to treat skin melanin pigmentation. Thus, the present study investigated the effects of ascorbic acid on gingival melanin pigmentation in vitro and in vivo. METHODS: The effects of ascorbic acid on melanin formation were evaluated in vitro in B16 mouse melanoma cells and three-dimensional human skin models. In addition, a clinical trial was performed to investigate the inhibitory effects of a gel containing ascorbic acid 2-glucoside (AS-G gel) on gingival melanin pigmentation. This study used a double-masked, split-mouth design on 73 subjects with symmetric gingival melanin pigmentation. AS-G gel was applied to one side of the gingiva for 12 weeks, whereas placebo gel was applied to the other side as a control. Luminance (L*)-value, which describes the lightness of gingiva, was determined by spectrophotometry to obtain an objective measure of melanin pigmentation every 4 weeks. RESULTS: Ascorbic acid significantly inhibited tyrosinase activity and melanin formation in B16 mouse melanoma cells (P <0.01 and P <0.05, respectively). The inhibitory effects of ascorbic acid on melanin formation were also significant in three-dimensional human skin models (P <0.01). Moreover, in the clinical trial, a significant relative change in pigmentation was seen after 4 weeks with the application of AS-G gel compared to placebo (L*-value ratio). CONCLUSION: Ascorbic acid (AS-G) has potential for the treatment of gingival melanin pigmentation.

Effects of treatment on soluble tumour necrosis factor receptor type 1 and 2 in chronic periodontitis.

AIM: We reported that soluble tumour necrosis factor receptor type 2 (sTNFR2)/type 1 (sTNFR1) ratios in gingival crevicular fluid (GCF) decreased as the severity of chronic periodontitis (CP) increased. This study investigated the effects of the periodontal treatment on TNF-alpha, sTNFR1 and R2 in GCF and serum of CP patients. MATERIAL AND METHODS: Thirty-five serum and 90 GCF samples were obtained from 35 CP patients (23 non-smokers and 12 smokers) at baseline and after treatment. The levels of TNF-alpha, sTNFR1 and R2 in serum and GCF were quantified by enzyme-linked immunosorbant assay. RESULTS: No significant differences were found in the serum levels of TNF-alpha, sTNFR1 and R2 and the ratio of sTNFR2/R1 between baseline and after treatment. After treatment, sTNFR1 and R2 levels in GCF of non-smokers and smokers were significantly decreased compared with baseline. However, the sTNFR2/R1 ratio was significantly increased (non-smoker: 0.56+/-0.03-0.84+/-0.03, p<0.0001; smoker: 0.59+/-0.06-0.85+/-0.04, p=0.0019). There were no significant differences between non-smoking and smoking CP groups in serum and GCF. CONCLUSION: The ratio of sTNFR2/R1 in GCF significantly increased after treatment, and could be related to the clinical state of CP.

Solubility screening on a series of structurally related compounds: cosolvent-induced changes on the activity coefficient of hydrophobic solutes.

A homologous series of solutes was chosen as a model for a group of structurally related compounds with different physicochemical properties, as is commonly the case during the screening of potential drug candidates. Thermal properties of the crystalline solutes and solubility determinations were used to quantify the two independent factors that determine the solubility of organic compounds: crystallinity and hydrophobicity. A solubility screening study was conducted on the series. By expressing the obtained solubility enhancement expressed as changes in the activity coefficient, it is possible to visually compare the effect of different cosolvents. The results show the importance of solute-solvent polarity match. Polarity match between water miscible cosolvents and hydrophobic compounds is not truly attainable, but comparison of the screening results points out the closest matches (optimal effect), facilitating the systematic evaluation of solubilization approaches.

The combined genotypes of stimulatory and inhibitory Fc gamma receptors associated with systemic lupus erythematosus and periodontitis in Japanese adults.

BACKGROUND: The pathobiology of systemic lupus erythematosus (SLE) is similar to that of periodontitis in that the immunoglobulin G Fc receptor (FcgammaR) and proinflammatory cytokines play an important role. Genetic variations of FcgammaR and interleukin (IL)-1 are associated with susceptibility to both diseases. Therefore, we evaluated whether the combination of FcgammaR or IL-1 polymorphic genes represents a common risk factor for SLE and periodontitis. METHODS: The study population consisted of Japanese adults with SLE and periodontitis (SLE+P group; n = 46), SLE only (SLE group; n = 25), periodontitis only (P group; n = 58), and healthy individuals with no systemic or oral disease (H group; n = 44). Clinical periodontal condition was evaluated by measurement of probing depth, clinical attachment level, and alveolar bone loss. Genomic DNA was isolated from peripheral blood and analyzed for determination of FcgammaR genotypes (FcgammaRIIA, FcgammaRIIB, FcgammaRIIIA, and FcgammaRIIIB) and IL-1 genotypes (IL-1A +4845 and IL-1B +3954) by allele-specific polymerase chain reactions or DNA sequencing. RESULTS: A significant overrepresentation of the R131 allele of stimulatory FcgammaRIIA and the 232T allele of inhibitory FcgammaRIIB was found in the SLE+P group compared to the H group (P = 0.01 and P = 0.0009, respectively). The combination of FcgammaRIIA-R131 and FcgammaRIIB-232T alleles yielded a strong association with SLE and periodontitis (SLE+P group versus P group: P = 0.01, odds ratio: 3.3; SLE+P group versus H group: P = 0.0009, odds ratio: 11.2). Furthermore, SLE patients with the combined FcgammaR risk alleles exhibited more severe periodontal tissue destruction compared to other SLE patients. The frequencies of IL-1 polymorphic alleles were too low to assess the association with SLE or periodontitis. CONCLUSION: The combination of stimulatory FcgammaRIIA and inhibitory FcgammaRIIB genotypes may increase susceptibility to SLE and periodontitis in the Japanese population.

Salivary enzyme levels after scaling and interleukin-1 genotypes in Japanese patients with chronic periodontitis.

BACKGROUND: Saliva has been used as a diagnostic fluid in medicine and dentistry. It is easy to collect using non-invasive methods. The intracellular enzymes present in saliva have been studied as markers of periodontal disease. The purpose of this study was to determine the salivary enzyme levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) after scaling and to clarify the influence of interleukin (IL)-1 genotypes on these enzyme levels. METHODS: Forty-nine Japanese patients with chronic periodontitis (24 men and 25 women; mean age: 55.1 years) were enrolled in this study. Measurements of clinical parameters including probing depth (PD), clinical attachment level (CAL), and bleeding on probing (BOP) and collections of stimulated whole mixed saliva were performed at baseline and 4 weeks after scaling. After evaluation of salivary AST, ALT, and LDH levels, DNA was extracted from various cells in whole saliva. IL-1A+4845 G/T genotype was determined by polymerase chain reaction amplification, followed by enzyme digestion and electrophoresis. Statistical analysis was performed by the Wilcoxon signed-rank and Mann-Whitney U tests. A significant difference was set at P <0.05. RESULTS: Mean PD, CAL, and BOP values significantly decreased after scaling (mean +/- SE: 3.2 +/- 0.1 mm to 2.6 +/- 0.1 mm in PD; 3.9 +/- 0.2 mm to 3.3 +/- 0.2 mm in CAL; and 41% +/- 4% to 18% +/- 3% in BOP) (P <0.001). The values of AST, ALT, and LDH were 77.0 +/- 7.5, 43.9 +/- 5.5, and 753.4 +/- 96.5 (units per liter [U/l]) at baseline, and significantly decreased to 55.5 +/- 6.5, 30.0 +/- 5.5, and 394.7 +/- 34.0 (U/l) after scaling, respectively (P = 0.01, P = 0.006, and P <0.001). The carriage rate of the IL-1A+4845 allele 2 was 24.5%. No difference was noted in the decrease in PD, CAL, and BOP after scaling between the carriers (N = 12) and non-carriers (N = 37) of IL-1A+4845 allele 2. However, the IL-1A allele 2 non-carriers displayed a significant decrease in salivary AST and ALT levels (P <0.001), in contrast to the carriers who did not show any changes in the salivary levels of the enzymes after scaling. CONCLUSIONS: These results documented that salivary AST, ALT, and LDH levels reflect inflammation and destruction of periodontal tissue, suggesting clinically useful markers following periodontal therapy. In addition, although IL-1A+4845 alleles may not influence clinical parameters, they may influence post-scaling values of salivary AST and ALT.

Interleukin-6 receptor gene polymorphisms and periodontitis in a non-smoking Japanese population.

AIM: The indispensable role of interleukin-6 receptor (IL-6R) in regulating IL-6 responses has been clearly established. We have previously reported that IL6R polymorphisms strongly influenced the serum levels of soluble IL-6R. In this study, we investigated the association between these genetic variations and periodontitis. MATERIAL AND METHODS: Among the seven novel IL6R single-nucleotide polymorphisms (SNPs) reported, we genotyped two important sites: the +48892 A/C in exon 9 and the -183 G/A in the promoter region. The SNP in exon 9 results in Asp-->Ala substitution in the proteolytic cleavage site of IL-6Ralpha. In total, 212 periodontitis cases and 210 healthy controls were genotyped using polymerase chain reaction, restriction fragment length polymorphisms and direct sequencing methods. RESULTS: Analysis of the genotype distribution of the +48892 A/C SNP in periodontitis patients and in controls revealed a suggestive association with aggressive (p = 0.04) and chronic periodontitis (p = 0.04). In addition, the carriage rate for the A allele was significantly higher in chronic periodontitis patients [p = 0.02, odds ratio (OR) = 2.25]. No association was found in the -183 G/A SNP. The two markers were in linkage disequilibrium (LD) (|D'| = 0.53). CONCLUSION: The IL6R+48892 A/C polymorphism could act as a risk factor for periodontitis; however, further association and biological studies are needed.

Imbalance between soluble tumour necrosis factor receptors type 1 and 2 in chronic periodontitis.

BACKGROUND: Soluble types of tumour necrosis factor (TNF) receptors type 1 and 2 modulate the TNF-alpha-mediated inflammatory responses in chronic periodontitis (CP). OBJECTIVES: This study investigated the levels of TNF-alpha, soluble TNF receptor type 1 and 2 in gingival crevicular fluid (GCF) and serum of healthy subjects and CP patients. MATERIALS AND METHODS: Thirty-eight sera and 73 GCF samples were collected from 16 healthy subjects and 22 CP patients. GCF was collected from probing pocket depth (PPD)< or =3 mm sites of healthy subjects, PPD< or =3, 4-6 and > or =7 mm sites of CP patients. The levels of TNF-alpha, soluble TNF receptor type 1 and 2 in the serum and GCF were quantified by enzyme-linked immunosorbant assay. RESULTS: The total amounts of TNF-alpha, soluble TNF receptor type 1 and 2 in GCF significantly elevated with increasing PPD in both site-based (p<0.05) and subject-based (p<0.05) analyses. However, their levels progressively diverged as the pocket depths increased, with the soluble TNF receptor type 2 level being comparatively lower than type 1. On the other hand, soluble TNF receptor type 2/type 1 ratios in GCF decreased as the severity of periodontitis increased (p<0.0001). CONCLUSION: The imbalance between soluble TNF receptor type 1 and 2 levels in GCF could be related to CP severity.

Platelet-rich plasma combined with a porous hydroxyapatite graft for the treatment of intrabony periodontal defects in humans: a comparative controlled clinical study.

BACKGROUND: The aim of the present controlled clinical study was to compare platelet-rich plasma (PRP) combined with a biodegradable ceramic, porous hydroxyapatite (HA) with a mixture of HA and saline in the treatment of human intrabony defects. METHODS: Seventy interproximal intrabony osseous defects in 70 healthy, non-smoking subjects diagnosed with chronic periodontitis were included in this study. Thirty-five subjects each were randomly assigned to either the test group (PRP and HA) or control group (HA with saline). Clinical and radiographic measurements were determined at baseline and the 12-month evaluation. RESULTS: When compared to baseline, the 12-month results indicated that, while both treatment modalities resulted in significant changes in all clinical parameters (gingival index, bleeding on probing, probing depth, clinical attachment level, and intrabony defect fill; P <0.001), the test group exhibited statistically significant changes compared to the control sites in probing depth reduction: 4.7 +/- 1.6 mm versus 3.7 +/- 2.0 mm (P <0.05); clinical attachment gain: 3.4 +/- 1.7 mm versus 2.0 +/- 1.2 mm (P <0.001); and vertical relative attachment gain: 70.3% +/- 23.4% versus 45.5% +/- 29.4% (P <0.001). CONCLUSION: Treatment with a combination of PRP and HA compared to HA with saline led to a significantly more favorable clinical improvement in intrabony periodontal defects.

Matrix metalloproteinase-1 and -3 gene promoter polymorphisms in Japanese patients with periodontitis.

BACKGROUND/AIMS: Matrix metalloproteinase (MMP)-1 and MMP-3 have important roles in the connective tissue remodelling and destruction processes in periodontitis. MMP-1 1G/2G (-1607) and MMP-3 5A/6A (-1171) polymorphisms have been identified and appear to influence the transcription of the genes. The aim of this study was to investigate whether these gene promoter polymorphisms were associated with the susceptibility to periodontitis. MATERIAL AND METHODS: Genomic DNA was obtained from 37 generalised aggressive, 205 slight-to-severe generalised chronic-periodontitis patients and 142 healthy subjects. All subjects were non-smoking Japanese. We genotyped by using TaqMan PCR assay. The statistics were analysed by chi2-test. RESULTS: We found no significant differences in genotype distributions, allele frequencies, carriage rates and haplotype frequencies in the MMP-1 and the MMP-3 gene promoter polymorphisms among all groups. The distributions of MMP-1 and MMP-3 genotypes in our study were different from those of previously reported in Caucasians or Brazilians, but consistent with previously reported in Japanese. CONCLUSION: Our data did not support the hypothesis that MMP-1 and/or MMP-3 gene promoter polymorphisms influenced the susceptibility to periodontitis in Japanese patients, indicating MMP-1 and MMP-3 expressions were regulated by complex processes such as cytokine network in periodontal disease rather than gene polymorphisms.

Association of tumor necrosis factor receptor type 2 +587 gene polymorphism with severe chronic periodontitis.

BACKGROUND: Genetic polymorphisms for cytokines and their receptors have been proposed as potential markers for periodontal disease. Tumor necrosis factor receptor 2 (TNFR2) is one of the cell surface receptors for TNF-alpha. Recent studies have suggested that TNFR2 gene polymorphism is involved in autoimmune and other diseases. OBJECTIVES: The aim of the present study is to evaluate whether TNFR2(+587T/G) gene polymorphism is associated with chronic periodontitis (CP). METHODS: One hundred and ninety-six unrelated subjects (age 40-65 years) with different levels of CP were identified according to established criteria, including measurements of probing pocket depth (PPD), clinical attachment level (CAL), and alveolar bone loss (BL). All subjects were of Japanese descent and non-smokers. Single nucleotide polymorphism at position +587(T/G) in the TNFR2 gene was detected by a polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) method. RESULTS: The frequency and the positivity of the +587G allele were significantly higher in severe CP patients than in controls (p=0.0097; odds ratio=2.61, p=0.0075; odds ratio=3.06). In addition, mean values of PPD, CAL, and BL were significantly higher in the +587G allele positive than in the negative subjects (p=0.035, 0.022, and 0.018, respectively). CONCLUSIONS: These findings suggest that the TNFR2(+587G) polymorphic allele could be associated with severe CP in Japanese.

Helium leak test for sterility assurance of a sealed bag. II: Establishing a test method for the manufacturing process.

To establish a simpler and more reliable method for retaining the aseptic condition of freeze-dried bulk product of a drug substance, a helium leak test method was developed. The bulk product is for the new kit system for infusion of our antibiotic product. In manufacturing the kit system, the bulk product needs to be transported outside of the aseptic area. We had to use a proper container to enclose the bulk product under aseptic conditions and establish an appropriate method for sterility assurance of the container. We decided to use a flexible aluminum laminate bag as a container and to seal it in a polyethylene bag. To detect tears or pinholes in the bag, a helium leak test was considered. As a tear model, a pinhole of known diameter was made in the aluminum laminate bag which was then filled with helium and sealed in a polyethylene bag. Helium leaking from the pinholes was measured with a helium leak detector, and leakage from a pinhole of more than 50 microm in the aluminum laminate bag could be detected. The amount of leakage was strongly affected by the pinhole diameter, and we developed a scientific approach for measuring leakage using the Poiseiulle Equation. The detection sensitivity of our method was enough to retain an aseptic condition inside the aluminum laminate bag, confirmed by the results of the process simulation test using our helium leak test. We concluded that our helium leak test was useful for sterility assurance of the bulk product sealed in the aluminum laminate bag in the manufacturing process of our kit system for infusion of our antibiotic product.

Association of interleukin-1 receptor antagonist gene polymorphisms with early onset periodontitis in Japanese.

BACKGROUND/AIMS: Early onset periodontitis (EOP), newly 'aggressive periodontitis', is considered to have genetic basis, which have not been clearly defined. The interleukin-1 (IL-1) gene cluster polymorphism as one of genetic factors may influence the expression of several chronic inflammatory diseases. The aim of this study is to investigate the frequency of single nucleotide polymorphisms (SNPs) in the genes encoding IL-1alpha, IL-1beta and a variable number of tandem repeat (VNTR) polymorphisms in the IL-1 receptor antagonist gene (IL-1RN) in 47 generalized EOP (G-EOP) patients and 97 periodontally healthy controls. MATERIAL AND METHODS: All subjects were of Japanese descent and systemically healthy. They were identified according to established clinical criteria. SNPs in the IL-1alpha (+ 4845) and IL-1beta (- 511, + 3954) genes were analyzed by amplifying the polymorphic region using polymerase chain reaction (PCR), followed by restriction-enzyme digestion and agarose gel electrophoresis. IL-1RN (VNTR) polymorphisms were then detected by PCR amplification and fragment size analysis. RESULTS: There was no significant difference in the IL-alpha (+ 4845) and IL-1beta (- 511, + 3954) genotypes and allele frequencies between G-EOP patients and healthy controls. However, the frequency of IL-1RN (VNTR) polymorphic alleles was found to be significantly increased in G-EOP patients (chi2 test, P = 0.007; odds ratio = 3.40). Additionally, the carriage rate of IL-1RN (VNTR) polymorphisms was significantly higher in G-EOP patients than in healthy controls (chi2 test, P = 0.005; odds ratio = 3.81). CONCLUSION: These findings suggest that IL-1RN (VNTR) polymorphisms are associated with G-EOP in Japanese.

Helium leak test for sterility assurance of a sealed bag. I: Relationship of helium leak and pinhole diameter.

Described herein is an attempt to establish a simpler, more reliable method to maintain the aseptic condition of bulk materials of drug substances. The method would be used in the manufacturing process of our "double-bag" kit system for infusion, which has two compartments, one for the infusion liquid and another for the drug product. To manufacture the kit system, we used a flexible inner container to enclose the bulk under aseptic conditions and a method for ensuring sterility of the container. We used an aluminum laminate bag as the inner container, which was then enclosed in a polyethylene bag. To detect tears or pinholes in the bag, a helium leak test was evaluated. First, a simple experimental model of helium leakage from bags was established. In the model, a pinhole was made in a film disk of the aluminum laminate or polyethylene material used for the inner and outer bags. A helium leak detector was used to measure the escape of helium through the pinhole, and the leak could be detected from a pinhole from 10 microns in diameter. As the bulk product was doubly sealed in an aluminum bag and a polyethylene bag in the manufacturing process to maintain an aseptic condition, we also checked for helium leak from pinholes of film disks after connecting two film disks. The results showed that helium leak was detectable when the pinhole diameters of both film disks were more than 20 microns. Clearly, helium leak is strongly affected by pinhole diameter in both experimental models. We have calculated, for the pinhole geometries studied, helium leak rates by using the Poiseuille Equation. Calculated values were in agreement with experimental values.