Impact of smoking on protein levels of beta-defensins in periodontal disease

Abstract Antimicrobial peptides (AMPs) are important components of the host response against invading pathogens. In addition to their direct antimicrobial activity, they can also participate in the immune system modulation. However, the role of AMPs in the etiopathogenesis of periodontal disease and the risk factors that may influence their expression in the oral cavity are not fully understood. The aim of this study was to determine the impact of smoking on beta-defensin (hBD) 1 and 2 levels analyzing samples from periodontitis patients. Fifty patients with periodontitis, 25 smokers and 25 non-smokers, and 20 periodontally healthy patients were recruited. After periodontal clinical evaluation, gingival crevicular fluid (GCF) samples were collected from healthy sites of patients without periodontal disease and from healthy and diseased sites of patients with periodontitis. Peptides quantification was performed by sandwich ELISA technique. Smokers showed reduced GCF hBD 1 levels and increased hBD 2 levels compared to non-smokers in diseased sites (p <0.05). Higher levels of hBD 1 were observed in healthy sites of patients without periodontal disease than in healthy sites of patients with periodontitis (p<0.0001). Diseased sites of non-smokers presented higher levels of hBD 2 than healthy sites (p <0.05). These results reveal that protein levels of hBDs 1 and 2 can be impaired by cigarette smoking in the presence of periodontal disease.

Resumo Peptídeos antimicrobianos (PAMs) são componentes importantes da resposta do hospedeiro contra patógenos invasores. Além de sua atividade antimicrobiana direta, eles também podem participar da modulação do sistema imunológico. No entanto, o papel dos PAMs na etiopatogenia da doença periodontal e os fatores de risco que podem influenciar a sua expressão na cavidade oral não são totalmente compreendidos. O objetivo deste estudo foi determinar o impacto do tabagismo nos níveis de beta-defensina (hBD) 1 e 2 analisando amostras de pacientes com periodontite. Cinquenta pacientes com periodontite, 25 fumantes e 25 não fumantes e 20 pacientes periodontalmente saudáveis foram recrutados. Após avaliação clínica periodontal, amostras de fluido crevicular gengival (FCG) foram coletadas de sítios saudáveis de pacientes sem doença periodontal e de sítios saudáveis e doentes de pacientes com periodontite. A quantificação dos peptídeos foi realizada pela técnica de ELISA sanduíche. Fumantes apresentaram níveis reduzidos de hBD 1 no FCG e níveis aumentados de hBD 2 em comparação com não fumantes em locais doentes (p <0,05). Níveis mais elevados de hBD 1 foram observados em sítios saudáveis de pacientes sem doença periodontal do que em sítios saudáveis de pacientes com periodontite (p<0,0001). Os sítios doentes de não fumantes apresentaram níveis mais elevados de hBD 2 do que os sítios saudáveis (p<0,05). Esses resultados revelam que os níveis das hBDs 1 e 2 podem ser prejudicados pelo tabagismo na presença de doença periodontal.

Inheritance pattern of molar-incisor hypomineralization

Abstract The aim of this study was to investigate the segregation patterns of molar incisor hypomineralization (MIH) in families, given the evidence that its etiology is influenced by genetics. Clinically, MIH may be detected in parents and/or siblings of MIH-affected children. Our study included children with at least one first permanent molar affected by MIH (proband) and their first-degree relatives (parents and siblings). The participants were examined clinically to detect MIH, according to the European Academy of Paediatric Dentistry criteria (2003). A total of 101 nuclear families (391 individuals) were studied. Proband diagnosis was followed by MIH classification of the subject, his parents and siblings, as affected, unaffected, or unknown. Segregation analysis was performed using the multivariate logistic regression model of the Statistical Analysis for Genetic Epidemiology package, and segregation models (general transmission, environmental, major gene, dominant, codominant and recessive models). The Akaike information criterion (AIC) was used to evaluate the most parsimonious model. In all, 130 affected individuals, 165 unaffected individuals, and 96 unknown individuals were studied. Severe MIH was found in 50.7% of the cases. A segregation analysis performed for MIH revealed the following different models: environmental and dominance (p = 0.05), major gene (p = 0.04), codominant (p = 0.15) and recessive models (p = 0.03). According to the AIC values, the codominant model was the most parsimonious (AIC = 308.36). Our results suggest that the codominant model could be the most likely for inheriting MIH. This result strengthens the evidence that genetic factors, such as multifactorial complex defect, influence MIH.

BONEFILL ® block as alternative for bone substitute: a toxicological evaluation

ABSTRACT Bone substitutes based on hydroxyapatite (HA) and Bonefill® (BO - inorganic bovine bone) associated with poly(lactic-co-glycolic acid) (PLGA) (HA/PLGA and BO/PLGA) were evaluated concerning cytotoxicity, genotoxicity and mutagenicity as potential candidates for bone repair. The materials were developed and provided by Bionnovation Biomedical Products Ltda. Eluates from these bone substitutes were prepared for toxicity evaluations using eukaryotic cell cultures. HA/PLGA was used as a comparison for Bonefill®. Cell viability was evaluated by XTT assay and surviving fraction was calculated for clonogenic survival. Additionally, tail moment was used to assess genotoxicity (comet assay). The frequencies of binucleated cells with micronucleus (FBMN), micronucleus (FMN), nucleoplasmic bridges (NPBs), and nuclear buds (NBUDs) were analysed by cytokinesis-block micronucleus assay (CBMN assay). Results showed no statistical difference in cell viability compared with negative control (NC) The eluates did not promote delayed cytotoxicity whereas the surviving fraction rate for cultured cells was similar to NC. Furthermore, no genotoxicity or mutagenicity effects were observed for cultured cells with the Bonefill/PLGA and HA/PLGA eluates. In conclusion, the negative cytotoxicity, genotoxicity and mutagenicity results indicate that these bone substitutes presented interesting preliminary results as potential biomaterials for bone repair.

Analysis of polymorphisms in Interleukin 10, NOS2A, and ESR2 genes in chronic and aggressive periodontitis

Abstract The objective of this study was to investigate the association between single nucleotide polymorphisms (SNPs) in the IL10, NOS2A, and ESR2 genes and chronic periodontitis (CP) and aggressive periodontitis (AgP). Three groups of patients underwent periodontal and radiographic evaluations: CP (n = 61), AgP (n = 50), and periodontally healthy (control group=61). Genomic DNA was extracted from oral epithelial cells and used for genotyping by real-time polymerase chain reaction using TaqMan® probes. The investigated SNPs were: -1087G > A, -819C > T and -592C > A in the IL10; +2087G > A in the NOS2A, and +1730G > A in the ESR2 gene. Differences in genotype and allele frequencies of each polymorphism and some individual characteristics were analyzed using the chi-square test and multivariate logistic regression analysis. Analysis of SNPs and haplotypes in the IL10 and SNP in the ESR2 gene did not present any significant association with AgP or CP. The +2087G allele of the NOS2A gene tended to be significantly associated with periodontal disease. Patients carrying the genotype +2087GG in the NOS2A gene were genetically protected against the development of CP (p = 0.05; OR = 0.44; 95%CI = 0.20-0.95). This result showed greater significance when patients with AgP and CP were combined (total PD) (p = 0.03; OR = 0.46; 95%CI = 0.23-0.92). In conclusion, the studied Brazilian population had a significantly higher frequency of the GG genotype for the +2087 SNP in the NOS2A gene in individuals without periodontitis, although statistical significance was not maintained after multiple logistic regression.

Detection of the single nucleotide polymorphism (rs2227307) in the human interleukin 8 gene using a different PCR-RFLP assay / Detecção do polimorfismo de base única (rs2227307) no gene interleucina 8 humano usando PCR-RFLP

Interleukin 8 (IL-8) is a chemokine that acts as a potent chemoattractant for neutrophils. Single nucleotide polymorphisms (SNPs) in the human IL8 gene have been investigated in many disease association studies. We have developed a different PCR-RFLP (Polymerase Chain Reaction - Restriction Fragment of Length Polymorphism) assay for genotyping the SNP (rs2227307) in the IL8 gene. This method was used for typing 147 white healthy Brazilian individuals, whose DNA was obtained from buccal epithelial cells and extracted with phenol: chloroform: isoamyl alcohol. Genomic DNA was amplified by PCR using a conventional thermal cycler. The PCR products (573 bp) were submitted to RFLP reactions. The RFLP fragments were analyzed in a 4% agarose gel stained with ethidium bromide. The genotype distribution observed in this study was consistent with the assumption of Hardy-Weinberg equilibrium and was similar (p=0.30) to those reported for other white populations in the SNP Database of the National Center for Biotechnology Information (NCBI). Because the PCR-RFLP method presented here was efficient, low cost, reproducible and convenient for laboratories with a limited level of technology worldwide, it should be useful for genotyping in case-control association or population genetic studies.

A Interleucina 8 é uma quimiocina com potente ação quimioatrativa para neutrófilos. Polimorfismos de base única (SNPs) no gene humano IL8 têm sido investigados em vários estudos de associação com doenças. Um método diferente de PCR-RFLP para genotipagem do SNP (rs2227307) do gene IL8 foi desenvolvido pelo nosso grupo. Esse método foi utilizado para genotipar 147 indivíduos brasileiros brancos saudáveis que tiveram seu DNA obtido de células da mucosa oral e extraído com fenol:clorofórmio:álcool isoamílico. DNA genômico foi amplificado por PCR usando um termociclador convencional, e a seguir os produtos da PCR (573 pb) foram submetidos a reações de RFLP. Os produtos de RFLP foram analisados em gel de agarose 4% impregnado com brometo de etídio. A distribuição do genótipo observado neste estudo foi consistente com o equilíbrio de Hardy-Weinberg e foi similar (p=0,30) ao reportado em outras populações brancas no banco de dados de SNPs do National Center for Biotechnology Information (NCBI). Este novo método de PCR-RFLP apresentado neste estudo foi eficiente, de baixo custo, reprodutível e conveniente para laboratórios com tecnologia limitada, podendo ser útil para utilização em estudos de genética de populações ou de associação com doenças (tipo caso-controle).

Doença periodontal em indivíduos com Síndrome de Down: enfoque genético / Periodontal disease in individuals with Down Syndrome: genetic focus

Conceitos fundamentais da etiologia, herança e características clínicas da Síndrome de Down são utilizados nesta revisão como base para apresentação de estudos que enfocam a doença periodontal em indivíduos com Síndrome de Down, já que praticamente 100% dos mesmos desenvolvem a doença na vida adulta. Acredita-se que, juntamente com os fatores ambientais e culturais relacionados à higienização e deficiência de coordenação motora, as características imunológicas que se encontram alteradas em indivíduos portadores de Síndrome de Down, como a quimiotaxia deficiente dos neutrófilos e o número reduzido de linfócitos T maduros, possam contribuir para a maior prevalência e severidade de acometimento periodontal nos pacientes portadores de Síndrome de Down. Além disso, o padrão de destruição periodontal observado em indivíduos com Síndrome de Down é compatível com o da periodontite agressiva, com predominância de periodontopatógenos como Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis e Tannerella forsythensis, encontrados precocemente em crianças com Síndrome de Down (2 anos de idade) e adultos jovens (até 25 anos). É possível notar uma ligação entre o desenvolvimento de técnicas moleculares e a evolução do conhecimento sobre a Síndrome de Down, por exemplo: a identificação da presença da síndrome pela trissomia somente de uma parte do cromossomo 21 (distal do braço longo); a identificação dos genes presentes nessa região e o padrão de superexpressão (ou não) desses genes. Ainda, nesta revisão, são apresentadas perspectivas para o futuro sobre a melhor compreensão da Síndrome de Down, dentro do contexto genético, o que refletirá em tratamentos clínicos mais individualizados e eficientes, que proporcionarão melhor qualidade de vida para esses pacientes.

Fundamental concepts of etiology, inheritance and clinical characteristics of Down syndrome are used in this review as a basis for submission of studies that focus on periodontal disease in individuals with Down syndrome, since almost 100% of them develop the disease in adult life. It is believed that in association with environmental and cultural factors related to hygiene and disabilities of coordination, the immunological characteristics that are found altered in individuals with Down syndrome, such as deficient neutrophil chemotaxis and reduced number of mature T lymphocytes, may contribute to the greater prevalence and severity of periodontal involvement in patients with Down syndrome. Moreover, the pattern of periodontal destruction observed in individuals with Down syndrome is consistent with aggressive periodontitis, with a predominance of periodontopathogens such as Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Tannerella forsythensis during childhood and adolescence of Down's syndrome patients. It is possible to note a relationship between the development of molecular techniques and the evolution of knowledge about Down syndrome, for example: identification of the trisomy syndrome by observing only part of chromosome 21 (distal long arm); identification of genes in this trisomic region and the pattern of superexpression (or not) of these genes. Moreover, in this review future perspectives are presented with regard to better understanding Down syndrome in the genetic context, which will reflect in more individualized and effective clinical treatments that will provide these patients with a better quality of life.