Porphyromonas gingivalis, a gram-negative anaerobic, pathogenic bacterium is a major etiological agent in the initiation and progression of periodontal disease. Due to the ever-changing environment of the oral cavity, inhabitants like Porphyromonas gingivalis must possess the ability to adapt to changes in environmental conditions like pH, temperature, oxygen tension, and metal concentration. P. gingivalis should therefore have an efficient regulatory system in order to adapt and survive in the oral cavity. This response adaptation occurs at the transcriptional level, which involves alternative sigma factors. Extracytoplasmic function sigma (ECF-s) factors are the largest group of alternative sigma factors that play a role in the bacterial response to environmental stress conditions. Here we analyze the s-70 factor gene, PG0214, an extracytoplasmic function sigma factor encoded in the P. gingivalis genome, and examine its role in the bacterial response to environmental stress and virulence. Our findings indicate that the PG0214 gene is important in regulating major functional gene groups and pathways in the P. gingivalis genome. Strains deficient in the PG0214 gene were analyzed and shown to have decreased protease activity, as well as reduced survivability and invasion rates in eukaryotic host cells when compared against wild-type W83 and ATCC 33277 strains. Collectively our studies demonstrate that the PG0214 gene is a positive regulator of gene expression for the survival and virulence of P. gingivalis in the presence of oxidative- and iron-stress, although further study is needed to fully characterize the gene and determine its specific function.

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.

Matrix metalloproteinases (MMP) are zinc- or calcium-dependent proteinases involved in the normal maintenance of the extracellular matrix. When elevated, MMPs degrade matrix components contributing to tissue destruction in infected periodontal sites. The objectives of this study were two-fold: first to assess the ability of Porphyromonas gingivalis hemagglutinin B (HagB) to induce MMP responses in human myeloid dendritic cells and second, to assess the effect of host defense peptide human β defensin 3 (HBD3) to regulate and attenuate the MMP response of HagB treated dendritic cells. HBD3 (0.2, 2.0, or 20.0 µM) was given to primary dendritic cells pre-, co-, or post-treatment to HagB (0.02 or 0.2 µM). At 16 hours, MMP concentrations were determined. There were no significant differences in concentrations for all 3 replications for MMP-2 and -13. There were few significant differences in some of the replications for MMP-3, -7, and -9. There were more pronounced differences in MMP-1, -10, and -12 expression, which were significantly influenced by both the concentration of HBD3 and the timing of administration. Chemokine and cytokine responses were inversely related to MMP production. While MMP responses decreased in a dose related manner, chemokine responses were increased. Concentrations of MIP-1α were high and there were no differences in response to 0.02 and 0.2 M HagB with or without 20.0 M HBD3. However, the MIP-1β and TNFα response to 0.2 M HagB were only attenuated. HagB induces the production of MMPs in dendritic cells and treatment of dendritic cells with HBD3 can alter the profile of HagB-induced MMPs. Such a finding may have importance in the pathogenesis of periodontal disease.

Abstract not available

published_or_final_version / Medicine / Master / Master of Medical Sciences

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Thesis (M. Med. Sc.)--University of Hong Kong, 200.

Diss. (sammanfattning) Umeå : Umeå universitet, 2008. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 52-68).

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 115-123).