Inducible Nitric Oxide Synthase Polymorphisms and Nitric Oxide Levels in Individuals with Chronic Periodontitis.

This study aimed to investigate whether the -1026(A>C)(rs2779249) and +2087(A>G)(2297518) polymorphisms in the NOS2 gene were associated with chronic periodontitis (CP) and with salivary levels of nitrite (NO2-) and/or nitrate + nitrite (NOx). A group of 113 mixed-race patients were subjected to periodontal, genetic, and biochemical evaluations (65 CP/48 periodontally healthy subjects). DNA was extracted from oral epithelial cells and used for genotyping by polymerase chain reaction (real-time). Salivary NOx concentrations were determined using an ozone-based chemiluminescence assay. Association of CP with alleles and genotypes of the -1026(A>C) polymorphism was found (X² test, p = 0.0075; 0.0308), but this was not maintained after multiple logistic regression, performed to estimate the effect of covariates and polymorphisms in CP. This analysis demonstrated, after correction for multiple comparisons, that only the female gender was significantly associated with CP. Polymorphisms analyzed as haplotypes were not associated with CP. NOx levels were significantly higher in the control group of heterozygous individuals for both polymorphisms. In conclusion, the female gender was significantly associated with CP, and higher levels of salivary NOx were found in control subjects and associated with the heterozygous state of the NOS2 polymorphisms, reinforcing the potential of NO metabolites as markers of periodontitis status.

Topical application of the lectin Artin M accelerates wound healing in rat oral mucosa by enhancing TGF-ß and VEGF production.

The lectin Artin M has been shown to accelerate the wound-healing process. The aims of this study were to evaluate the effects of Artin M on wound healing in the palatal mucosa of rats and to investigate the effects of Artin M on transforming growth factor beta (TGF-ß) and vascular endothelial growth factor (VEGF) secretion by rat gingival fibroblasts. A surgical wound was created on the palatal mucosa of 72 rats divided into three groups according to treatment: C--Control (nontreated), A--Artin M gel, and V--Vehicle. Eight animals per group were sacrificed at 3, 5, and 7 days postsurgery for histology, immunohistochemistry and determination of the levels of cytokines, and growth factors. Gingival fibroblasts were incubated with 2.5 µg/mL of Artin M for 24, 48, and 72 hours. The expression of VEGF and TGF-ß was determined by enzyme-linked immunosorbent assay. Histologically, at day 7, the Artin M group showed earlier reepithelialization, milder inflammatory infiltration, and increased collagen fiber formation, resulting in faster maturation of granular tissue than in the other groups (p < 0.05). Artin M-induced cell proliferation in vivo and promoted a greater expression of TGF-ß and VEGF in both experiments (p < 0.05). Artin M was effective in healing oral mucosa wounds in rats and was associated with increased TGF-ß and VEGF release, cell proliferation, reepithelialization, and collagen deposition and arrangement of fibers.

Variation in the CXCR1 gene (IL8RA) is not associated with susceptibility to chronic periodontitis.

BACKGROUND: The chemokine receptor 1 CXCR-1 (or IL8R-alpha) is a specific receptor for the interleukin 8 (IL-8), which is chemoattractant for neutrophils and has an important role in the inflammatory response. The polymorphism rs2234671 at position Ex2+860G>C of the CXCR1 gene causes a conservative amino acid substitution (S276T). This single nucleotide polymorphism (SNP) seemed to be functional as it was associated with decreased lung cancer risk. Previous studies of our group found association of haplotypes in the IL8 and in the CXCR2 genes with the multifactorial disease chronic periodontitis. In this study we investigated the polymorphism rs2234671 in 395 Brazilian subjects with and without chronic periodontitis. FINDINGS: Similar distribution of the allelic and genotypic frequencies were observed between the groups (p>0.05). CONCLUSIONS: The polymorphism rs2234671 in the CXCR1 gene was not associated with the susceptibility to chronic periodontitis in the studied Brazilian population.

Haplotypes in the interleukin 8 gene and their association with chronic periodontitis susceptibility.

Interleukin-8 (IL-8), which is responsible for the migration and activation of neutrophils, is an important inflammatory mediator involved in the initiation and amplification of acute inflammatory reactions and chronic inflammatory processes. IL-8 plays an important role in periodontitis, an inflammatory disease characterized by the loss of connective tissue and alveolar bone. The aim of this study was to investigate whether the SNPs rs2227307 (+396) and rs2227306 (+781), and the haplotypes they formed together with the previously investigated rs4073 (-251), were associated with chronic periodontitis susceptibility. Clinical periodontal exams were performed and DNA samples were collected from 493 individuals (223 with periodontitis and 270 controls). Associations between SNPs, haplotypes, and subject phenotypes were analyzed using the χ(2) test followed by multivariate logistic regression modeling. We conclude that the +396TT genotype and the haplotypes ATC/TTC and AGT/TGC were significantly associated with chronic periodontitis susceptibility in Brazilians.

Weissella oryzae DC6-facilitated green synthesis of silver nanoparticles and their antimicrobial potential.

Nanoparticles and nanomaterials are at the prominent edge of the rapidly developing field of nanotechnology. Recently, nanoparticle synthesis using biological resources has been found to be a new area with considerable prospects for development. Biological systems are the masters of ambient condition chemistry and are able to synthesize nanoparticles by utilizing metal salts. In the perspective of the current initiative to develop green technologies for the synthesis of nanoparticles, microorganisms are of considerable interest. Thus, the present study describes a bacterial strain-Weissella oryzae DC6-isolated from mountain ginseng, for the green and facile synthesis of silver nanoparticles. The particles were synthesized effectively without the need for any supplementary modification to maintain stability. The synthesized nanoparticles were evaluated by several instrumental techniques, comprising ultraviolet-visible spectrophotometry, field emission transmission electron microscopy, energy dispersive X-ray spectroscopy, elemental mapping, X-ray diffraction, and dynamic light scattering. In addition, the biosynthesized silver nanoparticles were explored for their antimicrobial activity against clinical pathogens including Vibrio parahaemolyticus, Bacillus cereus, Bacillus anthracis, Staphylococcus aureus, Escherichia coli, and Candida albicans. Furthermore, the potential of nanoparticles has been observed for biofilm inhibition against Staphylococcus aureus and Pseudomonas aeruginosa. Thus, the synthesis of silver nanoparticles by the strain W. oryzae DC6 may serve as a simple, green, cost-effective, consistent, and harmless method to produce antimicrobial silver nanoparticles.

Pseudomonas deceptionensis DC5-mediated synthesis of extracellular silver nanoparticles.

The biological synthesis of metal nanoparticles is of great interest in the field of nanotechnology. The present work highlights the extracellular biological synthesis of silver nanoparticles using Pseudomonas deceptionensis DC5. The particles were synthesized in the culture supernatant within 48 h of incubation. Extracellular synthesis of silver nanoparticles in the culture supernatant was confirmed by ultraviolet-visible spectroscopy, which showed the absorption peak at 428 nm, and also under field emission transmission electron microscopy which displayed the spherical shape. In addition, the particles were characterized by X-ray diffraction spectroscopy, which corresponds to the crystalline nature of nanoparticles, and energy-dispersive X-ray analysis which exhibited the intense peak at 3 keV, resembling the silver nanoparticles. Further, the synthesized nanoparticles were examined by elemental mapping which displayed the dominance of the silver element in the synthesized product, and dynamic light scattering which showed the distribution of silver nanoparticles with respect to intensity, volume, and number of particles. Moreover, the silver nanoparticles have been found to be quite active in antimicrobial activity and biofilm inhibition activity against pathogenic microorganisms. Thus, the present work emphasized the prospect of using the P. deceptionensis DC5 to achieve the extracellular synthesis of silver nanoparticles in a facile and environmental manner.

Pathogen levels and clinical response to periodontal treatment in patients with Interleukin 8 haplotypes.

The aim of this study was to investigate the effect of non-surgical treatment of periodontitis on the levels of periodontopathogens and clinical parameters in patients with different genetic backgrounds produced by polymorphisms in the Interleukin ( IL8) gene. Thirty patients grouped according to IL8 ATC/TTC or AGT/TTC haplotypes were submitted to non-surgical periodontal treatment. Levels of Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola were determined in 240 subgingival plaque samples by qPCR. The association between IL8 haplotypes and the levels of periodontopathogens and clinical parameters was investigated by multilevel analysis accounting for the clustering of diseased sites analyzed within patients. It was observed that neither levels of periodontopathogens nor non-surgical treatment was associated with the IL8 haplotype. The clinical parameters after periodontal treatment were similar in diseased and healthy sites, independently of the IL8 haplotype. Nonetheless, in the same period, diseased sites of AGT/TTC patients harbored higher levels of P. gingivalis, T. denticola, T. forsythia, and red complex than those of ATC/TTC patients. However, the non-surgical periodontal therapy decreased the levels of these periodontopathogens and of the tested clinical parameters of diseased sites in both groups. Non-surgical therapy is equally effective in improving clinical parameters and decreasing the levels of periodontopathogens, independent of the genotype groups produced by the IL8 haplotype.

Interleukin 4 haplotypes of susceptibility to chronic periodontitis are associated with IL-4 protein levels but not with clinical outcomes of periodontal therapy.

Different IL4 haplotypes were associated to susceptibility to/or protection against chronic periodontitis (CP). The aim of this study was to investigate if individuals carrying different haplotypes would present differences in clinical periodontal parameters and in the IL-4 levels at baseline, 45 and 90 days after non-surgical periodontal therapy. 62 patients were subdivided: genetically protected without CP (PH), genetically protected with CP (PCP), genetically susceptible with CP (SCP), genetically susceptible without CP (healthy) (SH). Clinical examination and gingival crevicular fluid (GCF) collection were performed for all patients, and IL-4 levels were measured by ELISA. At baseline, higher values for plaque index (PI, p = 0.013), gingival index (GI, p = 0.005) were observed for the SCP group in comparison to the PCP group but not after the completion of periodontal therapy. 45 and 90 days after the non-surgical therapy, PCP demonstrated significantly higher IL-4 levels than the SCP (p = 0.000002). Correlation analysis showed different results between clinical parameters and IL-4 production or GCF volume for groups with different genetic loads. The IL4 gene which was previously associated with susceptibility to CP was related with differences in the IL-4 protein levels in the GCF. However, independent of genetic carriage, individuals responded similarly to this therapy.

Haplotypes of susceptibility to chronic periodontitis in the Interleukin 8 gene do not influence protein level in the gingival crevicular fluid.

OBJECTIVE: Previously, we identified that the ATC/TTC haplotype formed by polymorphisms in the Interleukin-(IL)8 gene conferred susceptibility to chronic periodontitis (CP). The aim of the study was to investigate whether the IL8 haplotype ATC/TTC was associated with the volume of gingival crevicular fluid (GCF), the concentration of interleukin IL-8 in the GCF, as well as periodontal conditions in patients with CP in comparison to controls without CP. METHODS: Seventy-nine individuals (CP: n=41, controls: n=38) were grouped according to the presence (susceptible for CP) or absence (not susceptible for CP) of the IL8 ATC/TTC haplotype. After periodontal clinical evaluation, they were subdivided by the presence or absence of CP. GCF was collected from each patient and the IL-8 levels were determined by ELISA. The GCF volume of each subject was measured by means of a calibrated electronic device. Comparisons of means between carriers and non-carriers of the ATC/TTC haplotype were evaluated using the Mann-Whitney test. Linear regression and stepwise linear regression analysis were used to analyse the association of the GCF volume with potential covariates and their contribution for the phenotype. RESULTS: We did not find significant differences of both periodontal conditions and IL-8 concentration in the GCF of patients with the presence or absence of the IL8 ATC/TTC haplotype. However, the GCF volume was significantly higher amongst the patients affected by CP that are absent for the IL8 ATC/TTC haplotype. In addition, linear regression analysis showed a statistically significant association between GCF volume and CP, IL8 haplotype ATC/TTC and IL-8 concentration. CONCLUSIONS: The IL8 haplotype of susceptibility to CP was neither associated with IL-8 cytokine levels nor with clinical periodontal parameters. Also, CP, IL8 haplotype and IL-8 concentration showed a positive association with the GCF volume levels in the studied patients.

Polymorphisms and haplotypes in the interleukin-4 gene are associated with chronic periodontitis in a Brazilian population.

BACKGROUND: Some haplotypes in the interleukin-4 (IL4) gene were reported to influence IL-4 cytokine production and were associated with inflammatory diseases. Association studies focusing on IL4 gene polymorphisms and periodontal disease provided conflicting results. The aim of this study is to investigate whether IL4 gene polymorphisms and haplotypes were related to chronic periodontitis in a Brazilian population. METHODS: The polymorphisms -590(C/T) and +33(C/T) in the IL4 gene were identified by polymerase chain reaction (PCR) and restriction fragment-length polymorphism methods; the variable number of tandem repeats (VNTR) was identified by PCR. To assess the differences between the periodontitis group (n = 125) and control group (n = 125), the chi(2) test was used to assess genotype and allele distributions of individual polymorphisms. For haplotypes reconstructed by an expectation-maximization algorithm, the CLUMP program and Fisher exact test were used. Multivariate logistic regression modeling was used to assess the association of age, gender, smoking status, and polymorphism/haplotype with periodontitis. RESULTS: The -590(T), +33(C), and insertion (I) of 70-base pair (bp) alleles and genotypes were more prevalent in the periodontitis group, even after adjusting for covariates. The -590, +33, and insertion (TCI) haplotype was associated with a susceptibility to periodontitis (adjusted odds ratio [OR(adjusted)] = 2.68; 95% confidence interval [CI] = 1.50 to 4.80) as was the genotype TCI/CCI (OR(adjusted) = 5.27; 95% CI = 2.28 to 12.18), whereas the TTD (OR(adjusted) = 0.48; 95% CI = 0.26 to 0.91), CTI (OR(adjusted) = 0.28, 95% CI = 0.11 to 0.70), and TTD/CTI (OR(adjusted) = 0.29; 95% CI = 0.08 to 1.13) genotypes were a associated with protection against the development of chronic periodontitis. CONCLUSION: Significant associations between alleles, genotypes, and haplotypes of polymorphisms in the IL4 gene and chronic periodontitis were verified in Brazilian individuals.

Association of haplotypes in the CXCR2 gene with periodontitis in a Brazilian population.

CXCR-2 is a receptor of interleukin-8, which is involved in acute and chronic inflammatory processes. Polymorphisms in the CXCR2 gene have been associated with chronic inflammatory conditions. The aim of this study was to investigate whether the +785(C/T), +1208(T/C), and +1440(G/A) single-nucleotide polymorphisms (SNPs) in the CXCR2 gene, as well as their haplotypes, are associated with susceptibility to periodontitis in Brazilians. DNA was extracted from the buccal epithelial cells of 487 individuals (control = 215; periodontitis = 272). The SNPs were investigated using the sequence-specific primer-polymerase chain reaction method. Associations between the polymorphisms and subject phenotypes were analyzed using the chi-squared statistical test, followed by univariate and multivariate logistic regression modeling. Haplotypes were reconstructed using the expectation-maximization algorithm, and differences in haplotype distribution between the groups were analyzed to estimate genetic susceptibility for periodontitis development. Univariate and multivariate analysis revealed that age, skin color, and smoking status were associated with periodontitis. The +1440 GG genotype was shown to be protective against periodontitis in both univariate and multivariate analysis (odds ratio [OR](adjusted) = 0.42; 95% confidence interval [CI] = 0.19, 0.96). A similar relevant result for the +1440 GG was obtained in an alternative analysis considering a subgroup containing only white nonsmokers (OR = 0.37; 95% CI = 0.15, 0.92). White nonsmokers with the CTG/TCG haplotype appeared to be genetically protected against the development of periodontitis (OR = 0.29; 95% CI = 0.09, 0.89), while those carrying the CTG/TCA haplotype were more susceptible to the development of periodontitis (OR = 2.08; 95% CI = 1.24, 3.51). In conclusion, the +1440 SNP and some haplotypes are associated with periodontitis in Brazilian individuals.

Lack of association of a functional polymorphism in the interleukin 8 gene with susceptibility to periodontitis.

The important inflammatory mediator interleukin-8 (IL-8) is responsible for the migration and activation of neutrophils. The IL8 gene contains a functional single-nucleotide polymorphism (SNP) (rs4073) in its promoter region that may influence the expression of IL-8, and which has been associated with inflammatory diseases. The purpose of this study was to investigate the association of the SNP (rs4073) in the IL8 gene with susceptibility to periodontitis. DNA was extracted from the buccal epithelial cells of 500 individuals (control n = 224 and periodontitis n = 276). Individuals were genotyped for the SNP (rs4073) using sequence-specific primer polymerase chain reaction. Associations between the SNP (rs4073) and subject phenotypes were analyzed using the chi-squared test, followed by univariate and multivariate logistic regression modeling. The genotype distributions in both groups were consistent with Hardy-Weinberg equilibrium. Univariate and multivariate analysis showed that age, skin color, and smoking status were associated with periodontitis. No significant differences were found for sex and frequencies of alleles and genotypes between the control and periodontitis groups in the univariate analysis. These findings were replicated in the multivariate analysis. The SNP (rs4073) in the IL8 gene is not associated with susceptibility to periodontitis in Brazilian individuals, even after controlling for covariates.

A novel PCR-RFLP assay for the detection of the single nucleotide polymorphism at position +1440 in the human CXCR2 gene.

We designed a novel PCR-RFLP assay to genotype for the CXCR2 +1440 (G/A) single nucleotide polymorphism, which provides a simple, low-cost, practical, and reproducible method. Allele frequencies in healthy Brazilian individuals were found to be 0.65% for allele A and 0.35% for allele G.

An in vitro bioassay for xenobiotics using the SXR-driven human CYP3A4/lacZ reporter gene.

The dose and time effect of nine xenobiotics, including 17beta-estradiol, corticosterone, dexamethasone, progesterone, nifedipine, bisphenol A, rifampicin, methamphetamine, and nicotine were investigated, in vitro, using human steroid and xenobiotics receptor (SXR)-binding sites on the human CYP3A4 promoter, which can enhance the linked lacZ reporter gene transcription. To test this, liver-specific SAP (human serum amyloid P component)-SXR (SAP/SXR) and human CYP3A4 promoter-regulated lacZ (hCYP3A4/lacZ) constructs were transiently transfected into HepG2 and NIH3T3 cells to compare the xenobiotic responsiveness between human and nonhuman cell lines. In the HepG2 cells, rifampicin, followed by corticosterone, nicotine, methamphetamine, and dexamethasone, exhibited enhanced levels of the lacZ transcript, whereas those of bisphenol A and nifedipine were found to be reduced. No significant responses were observed with 17beta-estradiol or progesterone. In addition, 17beta-estradiol and progesterone did not change the levels of the lacZ transcripts in the HepG2 cells, but did induce significant increases in the transcripts of the NIH3T3 cells. Treatment with corticosterone and dexamethasone, which were highly expressed in the HepG2 cells, did not affect the levels of the lacZ transcript in NIH3T3 cells. These results show that lacZ transcripts can be measured, rapidly and reproducibly, using reverse transcriptase-polymerase chain reaction (RT-PCR) based on the expression of the hCYP3A4/lacZ reporter gene, and was mediated by the SXR. Thus, this in vitro reporter gene bioassay is useful for measuring xenobiotic activities, and is a means to a better relevant bioassay, using human cells, human genes and human promoters, in order to get a closer look at actual human exposure.

Influência dos fatores genéticos na etiopatogênese da doença periodontal / Influences of genetic factors in the etipothogenesis of periodontal disease

A doença periodontal (DP) tem caráter multifatorial pois a infecção por microrganismos periodontopatogênicos que leva à inflamação e à destruição do periodonto é modulada pela resposta imune do hospedeiro, a qual é influenciada por hábitos como o fumo. Fatores relacionados ao hospedeiro no contexto da DP têm sido bastante estudados nos últimos anos, principalmente no que se refere à imunogenética, fato que motivou esta revisão da literatura, que teve como objetivo comentar a influência que fatores genéticos podem desempenhar na etiopatogênese da DP, dando ênfase aos polimorfismos genéticos. O levantamento bibliográfico foi realizado nas bases de dados Bireme e PubMed utilizando-se os termos “Periodontite, Genética e Polimorfismos”. Estudos sobre genética humana foram selecionados de forma a apresentar relação positiva ou negativa de fatores genéticos importantes, como polimorfismos, na DP. Concluiu-se pelos crescentes trabalhos na área que, apesar de ser inegável a influência genética na DP, ainda são necessários muitos outros estudos para melhor compreender esse mecanismo.

Periodontal diseases (PD) encompass multifactorial diseases due to the infection caused by periodontopathogenic microorganisms resulting in inflammation and periodontal destruction, are modulated by the host response. The immune response is influenced by host habits such as smoking. Important host factors regarding PD have been investigated in the last years, especially in immunogenetics. The aim of this literature review was to comment about genetic factors that might influence periodontal diseases. A search of Bireme and PubMed data bases was performed using terms like “Periodontitis, Genetics and Polymorphisms”. Important studies focusing on human genetics in individuals with periodontitis were selected to demonstrate positive and negative relationship between genetics factors, like polymorphisms, and PD. It can be concluded, considering the crescent number of studies in this area, that PD is influenced by genetic factors, although more researches are necessary to better understand which mechanisms are involved in this process.