Microbiological and Clinical Effects of Sitafloxacin and Azithromycin in Periodontitis Patients Receiving Supportive Periodontal Therapy.

Sitafloxacin (STFX) is a newly developed quinolone that has robust antimicrobial activity against periodontopathic bacteria. We previously reported that oral administration of STFX during supportive periodontal therapy was as effective as conventional mechanical debridement under local anesthesia microbiologically and clinically for 3 months. The aim of the present study was to examine the short-term and long-term microbiological and clinical effects of systemic STFX and azithromycin (AZM) on active periodontal pockets during supportive periodontal therapy. Fifty-one patients receiving supportive periodontal therapy were randomly allocated to the STFX group (200 mg/day of STFX for 5 days) or the AZM group (500 mg/day of AZM for 3 days). The microbiological and clinical parameters were examined until 12 months after the systemic administration of each drug. The concentration of each drug in periodontal pockets and the antimicrobial susceptibility of clinical isolates were also analyzed. The proportions of red complex bacteria, i.e., Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia, which are the representative periodontopathic bacteria, were significantly reduced at 1 month and remained lower at 12 months than those at baseline in both the STFX and AZM groups. Clinical parameters were significantly improved over the 12-month period in both groups. An increase in the MIC of AZM against clinical isolates was observed in the AZM group. These results indicate that monotherapy with systemic STFX and AZM might be an alternative treatment during supportive periodontal therapy in patients for whom invasive mechanical treatment is inappropriate. (This study has been registered with the University Hospital Medical Information Network-Clinical Trials Registry [UMIN-CTR] under registration number UMIN000007834.).

Profiling biomarkers in gingival crevicular fluid using multiplex bead immunoassay.

OBJECTIVE: Biomarkers in gingival crevicular fluid (GCF) have been investigated; however, measurements were limited by the small sample volume available. The aim of this study was to determine the levels of 40 different cytokines and chemokines in GCF samples. DESIGN: Eleven patients with generalised chronic periodontitis participating in a supportive periodontal therapy programme with remaining probing pocket depths (PDs) of >5mm were enrolled. One healthy and two diseased sites were sampled in each subject. Forty biomarkers in GCF were examined using a multiplex bead immunoassay. Porphyromonas gingivalis from the diseased sites was quantified by real-time polymerase chain reaction. RESULTS: Twenty-six biomarkers were detected in the GCF samples using the multiplex bead immunoassay. The levels of nine biomarkers were significantly different between the diseased and healthy sites after adjustment with Bonferroni's correction. The level of 26 biomarkers in diseased sites was compared between bleeding on probing (BOP)-positive and BOP-negative sites. Interleukin (IL)-1ß and interferon-inducible protein (IP)-10 levels were significantly higher in BOP-positive diseased sites than BOP-negative diseased sites after adjustment for multiple comparisons (IL-1ß, p=0.0007, IP-10; p=0.0009). In addition, the levels of IL-1ß in GCF were found to be strongly correlated with the P. gingivalis ratio (r=0.646, p=0.0012). CONCLUSION: IL-1ß levels in GCF correlate with the PDs, BOP and the presence of P. gingivalis in subgingival plaque. Multiplex bead assays can be useful in GCF studies. These findings can help in identifying new diagnostic methods in the diagnosis of periodontal disease.

Effects of systemic sitafloxacin on periodontal infection control in elderly patients.

OBJECTIVE: To evaluate the microbiological and clinical effects of the systemic administration of sitafloxacin (STFX) on periodontal pockets in elderly patients receiving supportive periodontal therapy (SPT). BACKGROUND: Periodontitis is a risk factor for atherosclerosis. Better periodontal health contributes to reduce atherosclerosis-related diseases in elderly population. MATERIALS AND METHODS: Forty-four patients undergoing SPT were randomly assigned to two groups: a test group took 100 mg/day of STFX for five consecutive days, or a control group received scaling and root planing (SRP) under local anaesthesia. Microbiological and clinical parameters were examined at baseline and at 1 and 3 months after therapy. RESULTS: The presence of Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia was significantly reduced at 1 month after treatment in both groups. The median reductions of the bacteria between the baseline and 1 month were 3.08 and 2.54% in the STFX- and SRP-treated groups, respectively. Both treatments significantly decreased the probing depth at 1 and 3 months compared to the baseline. CONCLUSION: The systemic administration of STFX is effective at improving periodontal health during SPT and could be an alternative to SRP for elderly patients who cannot undergo anaesthesia or are at risk of tissue injury.

Immunoregulatory gene polymorphisms in Japanese women with preterm births and periodontitis.

Many studies have reported an association between periodontal disease and preterm birth, although this remains controversial. Cytokines and antibodies produced to give resistance to infection can enter the bloodstream and cause preterm labor. We analyzed maternal genetic polymorphisms in various immunoregulatory genes that could affect both preterm birth and periodontitis. A total of 1099 women referred to the Department of Obstetrics and Gynecology, Niigata University Medical and Dental Hospital were candidates for participation, 424 of whom refused, and 553 were excluded. The final number of subjects was 122 (51 with preterm birth, 71 with term birth). Genomic DNA was isolated from venous blood, and 22 polymorphisms were determined: IL-1A, IL-1B, IL-1RN, IL-2, IL-4, IL-6, IL-10, TNFA, TNFRI, TNFRII, FcγRIIA, FcγRIIB, FcγRIIIA, FcγRIIIB, and FcαR. Within five days of labor, periodontal parameters were evaluated, and bacteria from subgingival plaque were detected using real-time PCR. There was no difference in the prevalence and degree of periodontitis between term and preterm births. Chi-squared tests showed that an age <33 years and FcαR(+56)T/C alleles were associated with preterm birth. Multiple logistic regression analysis represented a model with significant fitness in which four variables were associated with preterm birth: maternal age, number of Aggregatibacter actinomycetemcomitans, IL-6(-572)G/C, and FcαR(+56)T/C. In conclusion, there was no association between preterm birth and periodontitis in this study. A. actinomycetemcomitans, IL-6, and FcαR were suggested to be associated with preterm birth. Multiple logistic regression models with both genetic and environmental factors would be useful for evaluating susceptibility to preterm birth.

The association of Aggregatibacter actinomycetemcomitans with preeclampsia in a subset of Japanese pregnant women.

AIM: To determine whether periodontitis and three prominent members of the periodontal flora are associated with the development of preeclampsia (hypertension plus proteinuria) Materials and Methods: The samples were composed of 127 systemically healthy women. Within 5 days after labour, clinical periodontal parameters and Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Prevotella intermedia in subgingival plaque were evaluated. Maternal serum IgG antibody specific for each bacteria was determined by enzyme-linked immunosorbent assay. Multivariate logistic regression analysis was used to control for confounders (maternal age, body mass index before pregnancy, parity, and smoking). RESULTS: Eighteen women were affected with preeclampsia. The number of A.actinomycetemcomitans was shown to be significantly associated with preeclampsia in the logistic regression model (odds ratio; 1.7, 95% confidence interval; 1.1­2.7). There were statistically significant differences between the preeclamptic and control groups in body mass index before pregnancy, pre-term birth and low birthweight (respectively, p = 0.014, p = 0.010 and p < 0.0001). We found no statistically significant association between preeclampsia and periodontal clinical parameters or the presence of periodontitis. CONCLUSION: In systemically healthy pregnant women, our findings suggested that the levels of maternal subgingival A. actinomycetemcomitans DNA were elevated in preeclamptic women.

Salivary biomarkers for predicting the progression of chronic periodontitis.

OBJECTIVE: Predicting the progression of periodontitis would allow for targeted supportive periodontal therapy. The purpose of this study was to determine the usefulness of salivary biomarkers for predicting the progression of periodontitis. DESIGN: Eighty-five chronic periodontitis patients were enrolled in an 18-month longitudinal study. Amongst them, 57 experienced progression of periodontitis, indicated at the end of the 18 months by at least one site with >3mm loss of attachment compared with baseline. We determined the levels of aspartate aminotransferase, alanine aminotransferase (ALT), lactate dehydrogenase, alkaline phosphatase and free haemoglobin as biomarkers, as well as the counts of Porphyromonas gingivalis, Prevotella intermedia and Tannerella forsythia, which represented the periodontal bacteria, in the stimulated saliva. The Mann-Whitney U test was used to compare patients with and without progression. After categorising the diagnostic values, the chi-square test was applied. RESULTS: Counts and ratios (ratio to total bacteria) of P. gingivalis and P. intermedia were found to be significant predictors of the progression of periodontitis. To increase prediction accuracy, combination analyses were performed. The combination of ALT level and the P. gingivalis ratio showed the highest likelihood (p<0.001, sensitivity 0.40, specificity 0.96, likelihood 11.30). CONCLUSION: Our findings suggest that salivary ALT level and the P. gingivalis ratio may be potential indicators for the progression of periodontitis. Such a salivary test could be a useful diagnostic tool for predicting periodontal disease progression.

Peroxisome proliferator-activated receptor gamma polymorphism and periodontitis in pregnant Japanese women.

BACKGROUND: Recent studies suggest an association between maternal periodontitis and preterm birth, although the association remains controversial. It was suggested that mechanisms such as a genetic predisposition for a hyperinflammatory response cause periodontitis and preterm births. Peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear hormone receptor and ligand-dependent transcription factor. PPARgamma inhibits the transcriptional activity of the genes that produce proinflammatory mediators and repress periodontitis. Recently, a common polymorphism, proline(PRO)-to-alanine(ALA) mutation at codon12 in exonB (Pro12Ala: rs 1801282) PPARgamma, was reported to reduce the ability to transactivate responsive promoters. In this study, we tested whether the PPARgammaPro12Ala polymorphism was associated with maternal periodontitis and/or preterm birth. METHODS: Genomic DNA was isolated from the venous blood of pregnant Japanese women (term birth: n = 72; preterm birth: n = 58). The PPARgammaPro12Ala genotype was determined by polymerase chain reaction (PCR)-restriction fragment length polymorphism. Within 5 days after labor, clinical periodontal parameters were evaluated, and periodontopathic bacteria from the subgingival plaque were detected by species-specific PCR. RESULTS: The mean clinical attachment level (P = 0.012), mean probing depth (P = 0.031), mean gingival index (P = 0.037), and percentages of sites with bleeding on probing (P = 0.041) in women with the PPARgammaPro12Ala genotype were significantly higher than in women with the PPARgammaPro12Pro genotype. However, there was no association between preterm birth and periodontitis. CONCLUSION: We suggest that the PPARgammaPro12Ala polymorphism may represent a genetic susceptibility factor for the clinical measurements of periodontitis in a limited number of pregnant Japanese women, but it probably cannot influence the relationship between periodontitis and preterm birth.

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.

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.

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.

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.