CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Porphyromonas gingivalis e Aggregatibacter actinomycetemcomitans sÃo periodontopatÃgenos associados à periodontite agressiva. A fÃmbria, uma estrutura relacionada à adesÃo e à invasÃo de cÃlulas, à um dos principais fatores de virulÃncia de P. gingivalis. Baseado na sequÃncia de nucleotÃdeos, seis genÃtipos(fimA) que codificam a fÃmbria principal dessas bactÃrias foram identificados, sendo o fimA II mais comumente relacionado à destruiÃÃo periodontal. O objetivo deste trabalho foi avaliar, por meio de reaÃÃo em cadeia da polimerase em amostras de placa subgengival dos sÃtios com maior profundidade de sondagem de pacientes com periodontite agressiva, a prevalÃncia de P. gingivalis, do genÃtipo fimA II de P. gingivalis e de A. actinomycetemcomitans, assim como relacionar a presenÃa desses patÃgenos ou genÃtipo à idade e aos parÃmetros clÃnicos periodontais (Ãndice de placa, Ãndice de sangramento gengival, profundidade de sondagem e nÃvel de inserÃÃo) encontrados nesses pacientes. Foram selecionados 45 pacientes com periodontite agressiva generalizada, com idade entre 15 e 40 anos. Nessa populaÃÃo, 64,4% apresentaram P. gingivalis e 28,8% apresentaram A. actinomycetemcomitans em sua microbiota subgengival. Dos pacientes positivos para P. gingivalis, 82,6% apresentaram o genÃtipo fimA II. Ao se relacionar a presenÃa ou ausÃncia das bactÃrias ou genÃtipo aos dados clÃnicos e idade, foi observada diferenÃa estatisticamente significante entre o nÃvel clÃnico de inserÃÃo do sÃtio coletado de pacientes com presenÃa de P. gingivalis e seu genÃtipo fimA II quando comparados aos pacientes negativos para essa bactÃria e genÃtipo, sendo a perda de inserÃÃo significativamente maior em pacientes que apresentaram P. gingivalis e em paciente com seu genÃtipo fimA II. AlÃm disso, foi encontrada mÃdia de idade significativamente mais elevada em pacientes positivos para P. gingivalis que em pacientes negativos para essa bactÃria. Concluiu-se, assim, que P. gingivalis e seu genÃtipo fimA tipo II estÃo presentes em alta prevalÃncia em pacientes com periodontite agressiva, que A. actinomycetemcomitans està presente em menor proporÃÃo de indivÃduos na populaÃÃo estudada e que P. gingivalis parece ser mais comumente encontrada em bolsas mais profundas e em indivÃduos mais velhos. / Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans are periodontal pathogens associated with aggressive periodontitis. The fimbriae, a structure related to adhesion and invasion of cells, is one of the major virulence factors of P. gingivalis. Based on the nucleotide sequence, six genotypes(fimA) encoding the major fimbriae of these bacteria were identified, and the fimA II is the most commonly associated with periodontal destruction. The objective of this study was to evaluate, by polymerase chain reaction in subgingival plaque samples from sites with highest probing depth in patients with aggressive periodontitis, the prevalence of P. gingivalis, P. gingivalis genotype fimA II and A. actinomycetemcomitans, and relate the presence of these pathogens or genotype to age and clinical periodontal parameters (plaque index, gingival bleeding index, probing depth and clinical attachment level) in these patients. We selected 45 patients with generalized aggressive periodontitis, aged from 15 to 40 years. 64.4% of these patients harbored P. gingivalis and 28.8% harbored A. actinomycetemcomitans in their subgingival microbiota. In patients positive for P. gingivalis, 82.6 % presented the genotype fimA II. In relation to the presence or absence of bacteria or gene to clinical data and age, a statistically significant difference between clinical attachment level was observed in the selected sites of patients with the presence of P. gingivalis and its genotype fimA II when compared to patients negative for these bacteria and genotype, with periodontal loss significantly higher in patients harboring P. gingivalis and in patients harboring genotype fimA II. In addition, the average age in patients positives for P. gingivalis was significantly higher than in negative ones. It is therefore concluded that P. gingivalis and its genotype fimA II are present in high prevalence in patients with aggressive periodontitis, A. actinomycetemcomitans is present in a smaller proportion of individuals in the studied population and P. gingivalis seems to be more commonly found in deeper sites and older individuals.

O ligamento periodontal e o tecido gengival são formados por tecido conjuntivo frouxo, sendo constituídos por diversas células, dentre as quais os fibroblastos são as mais numerosas. Ao serem submetidas a agressões diversas, estas células respondem com a liberação de substâncias, tais como citocinas e quimiocinas, que participam de maneira ativa no processo inflamatório, e com a produção de componentes da matriz extracelular fundamentais para o reparo, como por exemplo, o colágeno. Assim sendo, este trabalho teve como objetivo: Avaliar e comparar a expressão e a produção de prócolágeno tipo I, IL6, MIP1 e SDF1 por fibroblastos humanos, cultivados de ligamento periodontal e de tecido gengival, estimulados por lipopolissacarídeo (LPS) de Porphyromonas gingivalis. Foram coletados ligamentos periodontais de terceiros molares não irrompidos e biópsias de gengiva de um mesmo indivíduo (n=3). Estes tecidos foram picotados e mantidos em meio de cultura adequado para fibroblastos, que foram utilizados na quarta passagem. Após adesão dos fibroblastos a placas de 24 poços, o meio de cultura contendo 0,1 10 g/mL de LPS de P. gingivalis foi adicionado às placas em duplicata. O sobrenadante e as células foram coletados após 1, 6 e 24 horas e analisados por ELISA e PCR em tempo real, respectivamente. A análise estatística foi realizada por meio do programa GraphPad Prism, aplicandose o teste ANOVA a 1 critério com nível de significância de 5%. A expressão de prócolágeno tipo I mostrouse - ligeiramente diferente entre fibroblastos de ligamento periodontal e de gengiva. A produção de IL6, MIP1 e SDF1 foi significativamente maior em fibroblastos gengivais. A citocina IL6 foi produzida de maneira tempodependente com LPS de P gingivalis, principalmente por fibroblastos gengivais. Para MIP1, os fibroblastos gengivais mostraram maior produção com a menor concentração de estímulo (0,1g/ml). Para SDF1, foi detectada produção constitutiva que foi inibida com o aumento da concentração de LPS ao longo do tempo nestas mesmas células. Já para fibroblastos de ligamento periodontal, não foi observado um padrão homogêneo e linear, apesar de a produção basal de SDF1 também existir, porém em níveis bem mais discretos, como aquele observado para a produção de MIP1. A capacidade dos fibroblastos modificarem o padrão de produção dessas citocinas frente ao estímulo com LPS de P. gingivalis reforça a importância dessas células no contexto da resposta imune do indivíduo frente à doença periodontal. / The fibroblast is considered an important cell in periodontitis because it is the predominant cell type in the periodontal connective tissue. When challenged by different agents, fibroblasts respond through the release of substances, such as cytokines and chemokines that participate in an active way in the inflammatory process as well as the production of basic components of the extracellular matrix for repair, like collagen. The aim of this study was to: to evaluate and to compare the expression and production of type I procollagen, IL6, MIP1 and SDF1 by cultured human periodontal ligament and gingival fibroblasts challenged with lipopolyssacharide from Porphyromonas gingivalis. Human periodontal ligament and gingival fibroblasts were cultured from biopsies of the same donor and were used on the fourth passage. After confluence in 24well plates, the culture medium alone (control) or with 0,1 10 ug/mL of LPS from P. gingivalis were added and after 1, 6 and 24 hours, the supernatant and the cells were collected and analysed by ELISA and Real time PCR, respectively. Data were analysed by GraphPad Prism Program (1 way ANOVA test) and a significance level of 5% was adopted. Procollagen type I expression by Real Time PCR differ between periodontal ligament and gingival fibroblasts. In vitro experiments revealed that IL6, MIP 1 and SDF1 production were significantly greater in gingival fibroblasts when compared to periodontal ligament. In addition, IL6 was upregulated in a timedependent manner, mainly by the gingival fibroblasts. On one hand, MIP1 was stimulated with a low concentration (0,1ugml) of LPS by gingival fibroblasts. On the other hand, SDF1 was constitutively secreted by the same cells but its production was inhibited when challenged by a higher concentration of LPS from P gingivalis. In general, periodontal ligament fibroblasts did not show a pattern of production of these cytokines under the challenge with LPS, despite of the basal production of SDF1 in lower levels than gingival cells and the low production of MIP1 over time. The differential ability of the gingival and periodontal ligament fibroblasts to secrete these cytokines emphasizes their crucial role in the inflammatory microenvironment and in the host immune response to periodontal disease.

Tesis presentada para optar al Grado de Doctor en Farmacología / La periodontitis es una enfermedad infecciosa con un componente inflamatorio crónico que se produce como resultado del desbalance entre los microorganismos que conforman la biopelícula subgingival y la respuesta inmune del hospedero. La colonización de microorganismos patógenos y la respuesta inmunológica desencadenada que conducen a la destrucción de los tejidos de inserción de los dientes, como son el hueso alveolar, el ligamento periodontal y el cemento radicular, resultan finalmente en la pérdida de dientes. La presencia de bacterias anaerobias Gram-negativo específicas en la biopelícula subgingival es relevante en la etiología y progresión de la periodontitis. Dentro de ellas Porphyromonas gingivalis (P. gingivalis) es considerada un patógeno “clave” en el inicio de la enfermedad periodontal. A pesar de presentarse en una baja abundancia en sitios enfermos comparado con la microbiota oral total, esta bacteria es capaz de inducir cambios en el microbioma oral y causar disbiosis, conduciendo al desarrollo de la periodontitis. P. gingivalis además, es la bacteria más frecuentemente aislada en pacientes con periodontitis crónica (P. crónica) y ha sido relacionada con la progresión de esta enfermedad. No obstante, no está claro por qué su presencia en individuos sanos no se asocia con la enfermedad y los mecanismos que utiliza para el desarrollo y/o progresión de la periodontitis tampoco han sido totalmente dilucidados. Entre los factores de virulencia de P. gingivalis, el LPS destaca por su variabilidad estructural la cual le otorga la capacidad de dirigir e incluso evadir la respuesta inmunológica del hospedero, así como la actividad antimicrobiana de los péptidos endógenos, como es el caso de las defensinas (hBDs). P. gingivalis es capaz de reducir su interacción electrostática con péptidos catiónicos como Polimixina B (PMB), incrementando su resistencia por una de-fosforilación mediada por una fosfatasa en la posición 4´ del lípido A (gen PGN_0524). Junto con modificar su lípido A, P. gingivalis sintetiza dos tipos de LPS (A-LPS y O-LPS) que difieren en la región del Antígeno O polimérico (APS y AgO, respectivamente). Varios estudios sugieren que el lípido A es relevante en la resistencia a PMB, sin embargo su relación con la resistencia a otros péptidos catiónicos presentes en la cavidad oral (hBDs) no ha sido estudiada, así como tampoco se conoce el rol que pudieran tener otras estructuras del LPS, como el APS y AgO en dicha resistencia. Por lo anteriormente expuesto, en el presente trabajo se propuso determinar la susceptibilidad de aislados clínicos de P. gingivalis, provenientes de individuos clínicamente diagnosticados con periodontitis crónica (P. crónica) e individuos periodontalmente sanos, en presencia de PMB y evaluar la participación de la fosfatasa 4´ del lípido A de P. gingivalis en la resistencia a PMB y las hBDs-1 a -4. Además, se evaluó el rol de otras regiones del LPS como el AgO y/o APS en la resistencia a péptidos catiónicos, utilizando como modelo la PMB. Nuestros resultados muestran que aislados de P. gingivalis de pacientes con P. crónica presentan mayor resistencia a PMB respecto a aquellos obtenidos de individuos sanos. Además, aislados de individuos sanos son significativamente menos susceptibles a PMB respecto de la cepa de referencia ATCC 33277. Si bien una mutante que carece del gen de la fosfatasa en la posición 4´ del lípido A es totalmente susceptible a PMB, la presencia de este gen en los aislados no se correlaciona con la resistencia diferencial observada, ya que todos los aislados presentaban el gen. Más aún, en el análisis estructural del lípido A de los aislados se demostró la presencia de estructuras fosforiladas en la posición 4´, tanto en aislados susceptibles como resistentes a PMB, indicando que la composición del lípido A de P. gingivalis no es el único factor determinante en la resistencia de los aislados clínicos a PMB, y muy probablemente frente a otros péptidos catiónicos, puesto que la mutante fue capaz de crecer incluso a las concentraciones más altas de las hBDs-1 a -4. Aislados de individuos sanos carecen de moléculas de AgO de alto peso molecular y APS, sugiriendo que P. gingivalis presente en individuos periodontalmente sanos no produce A-LPS o lo pierde. Finalmente, aislados de pacientes con P. crónica carecen de moléculas de APS de bajo peso molecular, sugiriendo que moléculas de AgO y APS de alto peso molecular son relevantes en la resistencia diferencial de P. gingivalis a los péptidos catiónicos PMB y hBDs-1 a -4. / Periodontitis is an infectious disease with a chronic inflammatory component that occurs as result from imbalance between microorganisms forming biofilm and subgingival host immune response. Pathogenic microorganisms colonization and the immune response elicited lead to destruction of teeth insertion tissue such as: alveolar bone, periodontal ligament and cementum, finally resulting in tooth loss. The presence of specific Gram-negative anaerobic bacteria in subgingival biofilm is relevant for the etiology and progression of periodontitis. Among them, Porphyromonas gingivalis (P. gingivalis) is a "key" pathogen for the onset of periodontal disease. Although present in diseased sites in low abundance compared to the total oral microbiota, this bacterium is able to induce changes in the oral microbiome and cause dysbiosis, leading to development of periodontitis. P. gingivalis is the bacteria most frequently isolated in patients with chronic periodontitis and has been linked to progression of disease. However, it is unclear how their presence in healthy individuals is not associated with disease and the mechanisms for the development and/or progression of periodontitis have not been completely understood. Among P. gingivalis virulence factors the LPS noted for its structural changes conferring the ability to orchestrate and subvert the host immune response, and to resist endogenous peptides antimicrobial activity, such as defensins (hBDs). P. gingivalis reduces its electrostatic interaction with cationic peptides as Polymyxin B (PMB) and increases resistance by phosphatase lipid A 4´dephosphorylation (PGN_0524 gene). Besides modify their lipid A, P. gingivalis synthesizes two different types of LPS (A-LPS and O-LPS) differing at region of polymeric O antigen (OAg and APS, respectively). Several studies suggest that relevance for lipid A in PMB resistance, but its relationship with other cationic peptides resistance, relevant in the oral cavity (hBDs) has not been studied, and the possible role for the other known structures in LPS, such as APS and OAg in resistance. By the above, the present work proposed to determine the susceptibility of P. gingivalis clinical isolates, from chronic periodontitis (P. chronic) patients and periodontally healthy individuals to PMB and evaluate participation of lipid A 4' phosphatase in resistance to PMB and hBDs-1 to -4. In addition, the role of other regions of LPS as OAg and/or APS in resistance to cationic peptides was evaluated using as a model the PMB. Our results showed that isolates of P. gingivalis from P. chronic patients are more resistant to PMB respect from healthy individuals. Furthermore, healthy isolates are less resistant to PMB respect to the reference strain ATCC 33277. While, a mutant lacking phosphatase gene in the lipid A 4' position is totally susceptible to PMB, the presence of this gene in isolates does not correlate with the differential resistant observed since all isolates had the gene. Moreover, the lipid A structural analysis of the isolates shown 4' phosphorylated structures in both, susceptible and resistant PMB isolates, suggesting that P. gingivalis lipid A composition is not the only determining factor in clinical isolates PMB resistance, and probably to other cationic peptides, since the mutant was able to grow even to highest hBDs-1 to -4 concentrations. In healthy subject isolates lacked high molecular weight OAg and APS molecules, suggesting that P. gingivalis in healthy individuals do not produce or lose A-LPS. Finally, P. chronic isolates lacked APS molecules, suggesting that OAg and APS molecules are relevant in P. gingivalis resistance to cationic peptides PMB and hBDs -1 to -4 / Fondecyt

Orientador: Ana Cláudia Pavarina / Resumo: O objetivo deste estudo foi avaliar a eficácia da terapia fotodinâmica antimicrobiana associada (aPDT) ao metronidazol (MTZ) em biofilmes periodontopatogênicos. Para tal finalidade, foram realizadas as seguintes etapas: (1) determinação do tempo de adesão (24 e 48 horas) e formação de biofilme mono e duo-espécie (3, 5 e 7 dias) de Fusobacterium nucleatum (NCTC 11326) e Porphyromonas gingivalis (ATCC 33277); (2) aplicação da aPDT mediada por PDZ associada ao MTZ em biofilmes mono-espécie de F. nucleatum e P. gingivalis. Foram avaliadas diferentes concentrações do PDZ (50, 75 e 100 mg/L) e dose de luz de 50 J/cm2 (660nm). Após a aplicação da aPDT, os biofilmes foram incubados com diferentes concentrações do MTZ (MIC, 50x MIC e 100x MIC) por 24 horas. Os grupos controles positivos (L-F-) não receberam fotossensibilizador e não foram iluminados. A viabilidade dos microrganismos após os tratamentos foi avaliada por meio da contagem de UFC/ml. Os resultados demonstraram que o período de adesão de 24 horas, seguido de 5 dias de formação de biofilme foi satisfatório para a obtenção de biofilmes maduros mono-espécie. Para F. nucleatum, os resultados demonstraram que aPDT 75 mg/mL associado com MTZ 100x MIC e aPDT 100 mg/L associado com MTZ nas concentrações de 50x MIC e 100x MIC reduziu significativamente o número de UFC/mL, 2,99; 2,9 e 3,94 Log10 respectivamente. Para P. gingivalis, a redução mais significativa de UFC/mL foi obtida quando a associação de aPDT 100 mg/L e MTZ 100x MIC ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The aim of this study was to evaluate the efficacy of metronidazole (MTZ) associated antimicrobial photodynamic therapy (aPDT) on periodontopathogenic biofilms. For this purpose, the following steps were performed: (1) determination of adhesion period (24 and 48 hours) and single and duo species biofilm formation (3, 5 and 7 days) of Fusobacterium nucleatum (NCTC 11326) and Porphyromonas gingivalis (ATCC 33277); (2) Photodithazine ® (PDZ)- mediated aPDT in association with MTZ in single-specie biofilms of F. nucleatum and P. gingivalis. Different concentrations of PDZ (50, 75 e 100 mg/L) and light dose of 50 J / cm2 (660nm) were evaluated. After application of aPDT, the biofilms were incubated with different concentrations of MTZ (MIC, 50x MIC and 100x MIC) for 24 hours. Positive control groups (L-F-) received no photosensitizer and were also not illuminated. The viability of the microorganisms after the treatments was evaluated by counting CFU/ml. The results demonstrated that the 24 hours adhesion period followed by 5 days of biofilm formation was satisfactory for obtaining a mature biofilm in single-specie. For F. nucleatum, the results demonstrated that 75 mg/L aPDT associated with MTZ 100x and 100 mg/mL aPDT associated with MTZ at 50x MIC and 100x MIC concentrations significantly reduced the number of CFU/mL, 2.99; 2.9 and 3.94 Log10 respectively. For P. gingivalis, the greatest reduction of CFU/mL was obtained when the association of aPDT 100 mg/L and MTZ 100x MIC was p... (Complete abstract click electronic access below) / Doutor

Thesis (Ph.D.)--Boston University, Henry M. Goldman School of Dental Medicine. / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / Porphyromonas gingivalis , a Gram-negative anaerobic pathogen of periodontal diseases, requires iron in the form of heme (a term used to denote either the ferrous or ferric form of iron protoporphyrin IX) for growth. P. gingivalis is capable of utilizing a broad range of heme-containing compounds such as hemoglobin, hemoglobin-bound haptoglobin, hemin-bound hemopexin and hemin-saturated serum. Heme and hemoglobin utilization in P. gingivalis requires the participation of an outer membrane protein HmuR (heme utilization receptor), as well as cysteine proteinase gingipains (Lysine-specific gingipain Kgp and Arginine specific gingipains Rgps). However, the specific mechanisms utilized for heme acquisition are poorly understood. In this study, the role of HmuR in heme utilization was characterized in both E. coli and P. gingivalis . Molecular interaction between HmuR and hemin/hemoproteins was also characterized by construction and analysis of HmuR site-directed mutants. Our results support the direct role of HmuR in heme utilization. Hemoprotein utilization in P. gingivalis requires the participation of HmuR conserved residues. The HmuR residues 95 and 434, as well as the NPDL motif, seem to be involved in whole cell binding of hemoproteins; while the YRAP motif does not. All these residues seem essential for serum hemoprotein utilization. Analyses of HmuR by homology modeling provided a structural basis for functional analysis and supported the results from mutagenesis studies. In addition, expression of the hmuR, kgp and rgpA genes in response to different heme sources was also examined. We found that expression of the hmuR gene was negatively regulated by heme, while expression of the kgp and rgpA genes seemed to be regulated by growth phase. These different regulatory mechanisms, as well as the coordinate expression between HmuR and gingipains, indicate a complementary regulation mechanism for optimal heme utilization in P. gingivalis. / 2031-01-01

Mounting evidence has pointed to associations between Alzheimer's disease (AD) and dysbiotic microbiota in the oral cavity and GI tract. However, no studies have compared the role of specific oral and GI-tract pathogens in the development of AD or the roles inflammation severity and sex may play. We compared the effect of chronic exposure to two doses of lipopolysaccharides (LPS) from a periodontal pathogen, Porphyromonas gingivalis, and a GI-tract pathogen, Escherichia coli, on neuroinflammation, cognition, and AD-like pathology. P. gingivalis-LPS, E. coli-LPS, or endotoxin-free PBS was continuously subcutaneously administered to adult male and female C57BL/6 mice for 28 days via osmotic pumps at 250 µg/kg/day (low-dose) or 500 µg/kg/day (high-dose). After 21 days, spatial learning and reference memory were assessed using the Morris Water Maze (MWM) and Y-Maze. After 28 days of LPS treatment, levels of proinflammatory cytokines, TNF-α and IL-β, in serum and brain tissue were measured by ELISA. Markers of AD pathology and inflammation were detected in situ using immunohistochemistry (IHC) in paraffin-embedded brain sections. Western blot (WB) analysis was used to compare the expression of Toll-Like Receptors (TLR) 2 and 4 in brain, bone, liver, and kidney tissues. Sustained exposure to high-dose, but not low-dose, P. gingivalis-LPS or E. coli-LPS significantly impaired cognition in male mice only. P. gingivalis-LPS triggered worse cognitive impairment and neuroinflammation, whereas E. coli-LPS caused more pronounced serum inflammation. IHC analysis revealed that P. gingivalis-LPS-treated male mice had increased amyloid precursor protein and hyperphosphorylated tau protein in both the hippocampus and neocortex, while E. coli-LPS treatment had a less prominent effect. P. gingivalis-LPS also induced a greater degree of astrogliosis. The mechanisms underlying these observations may be related to differential TLR2 and TLR4 expression, as WB analysis showed TLR2 was predominantly expressed in the brain while TLR4 was constitutively expressed in both the brain and peripheral tissues. Overall, P. gingivalis-LPS treatment triggered more prominent neuroinflammation, cognitive impairment, and AD-like pathological brain changes than E. coli-LPS treatment. Moreover, these phenotypes were dose-dependent and sex-dependent. In conclusion, chronic periodontitis may be a significant risk factor for AD, and this relationship warrants further investigation.

No description available.

Les parodontites sont des maladies inflammatoires de nature infectieuse affectant les tissus de soutien de la dent. La présence de bactéries parodontopathogènes dans le sillon gingival représente le facteur étiologique primaire responsable du déclenchement de la parodontite. La réponse immunitaire de l'hôte face à l'agression par ces parodontopathogènes détermine l'évolution de la maladie vers la destruction tissulaire ou la guérison. En effet, la stimulation des cellules immunitaires et mucosales par les bactéries et leurs facteurs de virulence engendre une forte production de médiateurs inflammatoires et de métalloprotéinases matricielles. Ce phénomène a pour conséquence d'activer diverses voies de dégradation tissulaire et osseuse. Le système du complément est l'un des éléments importants de la réaction immunitaire puisqu'il permet l'élimination de microorganismes pathogènes. Pour éviter un effet néfaste du système du complément, les cellules de mammifères expriment à leur surface des protéines régulatrices du complément (CRPs) qui neutralisent les composantes du complément. Le but de cette étude était d'investiguer la capacité des bactéries parodontopathogènes à déjouer le système de défense de l'hôte et/ou de contribuer à l'inflammation et à la destruction tissulaire, en interagissant avec les CRPs. D'une part, nous avons démontré que Porphyromonas gingivalis, une bactérie anaérobie stricte à Gram négatif fortement associée à la parodontite chronique, peut induire le largage de la protéine CD46 de la surface des cellules épithéliales buccales. Une fois détachée des cellules, cette protéine est dégradée par les enzymes protéolytiques sécrétées par P. gingivalis. D'autre part, Fasobacterhim nucleatum, une bactérie cohabitant avec P. gingivalis, a montré une capacité à lier à sa surface la protéine CD46 soluble. F. nucleatum couvert de la protéine CD46 s'est avéré capable d'induire une sécrétion de cytokines proinflammatoires par les cellules épithéliales. Enfin, une stimulation des cellules épithéliales par le lipopolysaccharide des parodontopathogènes a révélé une surexpression des gènes codant pour les CRPs, dont CD46, CD55 et CD59. L'ensemble de ces résultats ont mené à une meilleure compréhension des interactions des interactions des bactéries parodontopathogènes avec le système du complément et des mécanismes contribuant à l'inflammation et à la destruction tissulaire.

Dans ce projet, la capacité des polyphénols du thé vert à moduler l’expression de certains gènes chez Porphyromonas gingivalis, le principal agent étiologique de la parodontite chronique, a été évaluée. Une analyse par PCR quantitative a démontré qu’à des concentrations sous-inhibitrices, l’extrait de thé vert ainsi que l’épigallocatéchine-3-gallate (EGCG) réduisent à différents degrés le niveau d’expression de gènes codant pour d’importants facteurs de virulence chez P. gingivalis. Ces facteurs participent notamment à la colonisation, l’acquisition des nutriments et la destruction tissulaire. De plus, les deux composés ont augmenté le niveau d’expression du gène codant pour la protéine de résistance au stress HtrA chez P. gingivalis. Les résultats de cette étude suggèrent que le thé vert et l’EGCG pourraient contribuer à réduire la virulence de P. gingivalis, supportant ainsi une potentielle utilisation pour la prévention et le traitement de la parodontite.

As doenças periodontais são infecções locais que apresentam morbidade e têm sido relacionadas com outras doenças ou complicações sistêmicas. Pacientes tabagistas apresentam taxas elevadas e maior predisposição às doenças periodontais severas e avançadas. A administração sistêmica de antibióticos, particularmente metronidazol (M) e amoxicilina (A), associados ao tratamento periodontal mecânico (raspagem e alisamento radiculares - RAR) tem sido amplamente estudada frente às doenças periodontais crônicas. Porém, pouco tem sido esclarecido referente aos efeitos desses tratamentos sobre Porphyromonas gingivalis e seus genótipos. Os objetivos deste trabalho foram: avaliar os efeitos clínicos (profundidade a sondagem - PS, nível de inserção clínica - NIC, índice de placa - IP, sangramento gengival - SG, sangramento à sondagem SS, e supuração - SUP) e microbiológicos (referente à presença de P. gingivalis e seus genótipos fimA II, fimA IV e ragB) após 180 dias do tratamento periodontal mecânico (RAR) associado à administração sistêmica de antibióticos (RAR+M+A), comparados ao tratamento periodontal mecânico (RAR), utilizando-se o PCR convencional (primers específicos para 16S rRNA); e relacionar a presença de P. gingivalis e seus genótipos fimA II, fimA IV e ragB com a profundidade de sondagem (PS) em pacientes fumantes com periodontite crônica. Foram avaliadas 167 amostras oriundas de sítios de 20 sujeitos tratados com RAR (n=11) e RAR + M + A (n=9), no exame inicial e 180 dias após a terapia. Parâmetros clínicos (PS, NIC, IP, SG, SS e SUP) e coletas microbiológicas foram mensuradas em ambos tempos. A detecção da freqüência de P. gingivalis foi realizada por PCR convencional, bem como para os genótipos fimA II, fimA IV e ragB. Não houve diferença estatisticamente significante entre o tratamento periodontal mecânico associado à administração sistêmica de antibióticos (RAR+M+A) e o tratamento periodontal mecânico (RAR), nos pacientes fumantes, em relação à detecção de P. gingivalis. O grupo RAR apresentou-se mais efetivo (estatisticamente significante) na redução de prevalência do genótipo ragB, em comparação ao grupo RAR+M+A, após 180 dias do tratamento. Não foi observada relação estatisticamente significante entre a prevalência de P. gingivalis e dos genótipos fimA II, fimA IV e ragB com a PS no exame inicial. / Periodontal diseases are local infections that present morbidity and have been relation with others systemic diseases or complications. Smoking patients present high levels and bigger predisposition to the severe and advanced periodontal diseases. The systemic administration of antibiotics, mainly metronidazole (M) and amoxicillin (A), associated to the mechanical periodontal treatment (scaling and root planing - SRP) has been largely researched referring to the chronic periodontal diseases. However, not much has been cleared up referring to the effects of those treatments about Porphyromonas gingivalis and its genotypes. The purpose of this research was to evaluate the clinical effects (depth probing - DP, clinical attachment level - CAL, plaque index - PI, gingival bleeding - GB, bleeding on probing - BOP, and supuration - SUP) and microbiological (referring to the presence of P. gingivalis and its genotypes fimA II, fimA IV and ragB) after 180 days of the mechanical periodontal treatment (SRP) associated to the systemic administration of antibiotic (SRP+M+A), compared to the mechanical periodontal treatment (SRP) in smoking patients with chronic periodontitis, by using the conventional PCR (specific primers to the 16S rRNA); to associate the P. gingivalis presence and its genotypes fimA II, fimA IV and ragB with depth probing (DP) of the researched population. 167 samples from the sites of 20 subjects treated with SRP (n=11) and SRP+M+A (n=9) have been evaluated, at the baseline and 180 days after the therapy. Clinical parameters (DP, CAL, PI, GB, BOP and SUP) and microbiological samples were evaluated in both moments. The detection of P. gingivalis frequence was made by using conventional PCR, and also to the genotypes fimA II, fimA IV and ragB. There was no statistically significative difference between the mechanical periodontal treatment associated to the systemic administration of antibiotics (SRP+M+A) and the mechanical periodontal treatment (SRP), in the smoking patients, related to the detection of the P. gingivalis. The SRP group showed more effective (statiscally significative) on the reduction of the genotype ragB prevalence, comparing to the SRP+M+A group, after 180 days of therapy. No statistically significative relation between the prevalence of P. gingivalis and its genotypes fimA II, fimA IV e ragB with DP at the baseline have been observed.