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No description available.

Periodontitis is a chronic inflammatory disease initiated by gram negative anaerobic bacteria. These bacteria possess pathogen-associated molecular patterns (PAMPs) that interact with various receptors including Toll like receptors (TLRs). Bacterial DNA (bDNA) is one of the PAMPs mainly recognized by TLR9. Interaction of bDNA and its receptors leads to activation of inflammatory signaling pathways potentially resulting in periodontal bone destruction. The aim of this study was to determine the production of IL- 6 and IL-8 in response to periodontal bDNA from human osteoblastic cells (MG-63). MG- 63 cells were stimulated in duplicate for 20 hours with 100ng/μl of bDNA from various pathogens including Porhyromonas gingivalis, Esherichia coli, Streptococcus sanguinis, Aggregatibacter actinomycetemcomitans as well as heat killed whole bacteria (1:100). E.coli LPS (10ng/μl) was used as a positive control in each experiment. To block TLR9 signaling, further experiments were carried out by treating MG-63 cells with chloroquine (10ng/μl) for 2 hours at 37ºC prior to stimulations. Cytokine levels were determined using enzyme linked-immunosorbent assay. Although IL-6 and IL-8 production was increased in response to periodontal bDNA in MG-63 cells, the results were not significant compared to unstimulated controls. As expected, E.coli DNA, E.coli LPS and heat killed whole bacteria stimulated significantly increased cytokine production (p<0.05). Blocking TLR9 with chloroquine did not affect the amount of cytokine production in bDNA stimulated cells suggesting that TLR9 may not be operant in triggering IL-6 and IL-8 production from MG- 63 cells. In conlusion, periodontal bDNA did not trigger significantly increased IL-6 and IL-8 production from MG-63 cells. Considering the involvement of several inflammatory mediators in periodontal bone destruction, further studies are warranted to assess the production of other cytokines in response to periodontal bDNA in human osteoblastic cells.

Monocytic cells survive HIV replication and consequent cytopathic effects because of their decreased sensitivity to HIV-induced apoptosis. However, the mechanism underlying this resistance to apoptosis remains poorly understood. I hypothesized that exposure to microbial products, translocated from the gut, may confer anti-apoptotic properties in human monocytic cells through interaction with their corresponding Toll-like receptors (TLRs). Using HIV-Vpr(52-96) peptide as a model apoptosis-inducing agent, I demonstrated that unlike monocyte-derived macrophages, undifferentiated primary human monocytes and pro-monocytic THP-1 cells are highly susceptible to Vpr(52-96)-induced apoptosis. Interestingly, monocytes and THP-1 cells stimulated with TLR-9 agonists, CpG and E.coli DNA, induced almost complete resistance to Vpr(52-96)-induced apoptosis albiet via a TLR-9 independent signaling pathway. Moreover, CpG and E.coli DNA selectively induced the anti- apoptotic Inhibitor of Apoptosis Protein-2 (c-IAP-2) and inhibition of the c-IAP-2 gene by either specific siRNAs or synthetic second mitochondrial activator of caspases (Smac) mimetic reversed CpG-induced resistance against Vpr(52-96)-mediated apoptosis. I demonstrated that c-IAP-2 was regulated by the c-Jun N terminal kinase (JNK) and the calcium signaling pathway in particular the calmodulin-dependent protein kinase-II (CaMK-II). Furthermore, inhibition of JNK and the calcium signaling including CaMK-II by either pharmacological inhibitors or their specific siRNAs reversed CpG-induced protection against Vpr(52-96)-mediated apoptosis. I also showed that CpG-induced JNK phosphorylation through activation of calcium signaling pathway.

Monocytic cells survive HIV replication and consequent cytopathic effects because of their decreased sensitivity to HIV-induced apoptosis. However, the mechanism underlying this resistance to apoptosis remains poorly understood. I hypothesized that exposure to microbial products, translocated from the gut, may confer anti-apoptotic properties in human monocytic cells through interaction with their corresponding Toll-like receptors (TLRs). Using HIV-Vpr(52-96) peptide as a model apoptosis-inducing agent, I demonstrated that unlike monocyte-derived macrophages, undifferentiated primary human monocytes and pro-monocytic THP-1 cells are highly susceptible to Vpr(52-96)-induced apoptosis. Interestingly, monocytes and THP-1 cells stimulated with TLR-9 agonists, CpG and E.coli DNA, induced almost complete resistance to Vpr(52-96)-induced apoptosis albiet via a TLR-9 independent signaling pathway. Moreover, CpG and E.coli DNA selectively induced the anti- apoptotic Inhibitor of Apoptosis Protein-2 (c-IAP-2) and inhibition of the c-IAP-2 gene by either specific siRNAs or synthetic second mitochondrial activator of caspases (Smac) mimetic reversed CpG-induced resistance against Vpr(52-96)-mediated apoptosis. I demonstrated that c-IAP-2 was regulated by the c-Jun N terminal kinase (JNK) and the calcium signaling pathway in particular the calmodulin-dependent protein kinase-II (CaMK-II). Furthermore, inhibition of JNK and the calcium signaling including CaMK-II by either pharmacological inhibitors or their specific siRNAs reversed CpG-induced protection against Vpr(52-96)-mediated apoptosis. I also showed that CpG-induced JNK phosphorylation through activation of calcium signaling pathway.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 5 uppsatser.

Aggregatibacter actinomycetemcomitans is a gram-negative, facultative anaerobic coccobacillus bacterium that colonizes the human oral cavity. Regarding the bacterial consortium found into biofilms, previous studies have suggested that the occurrence of particular serotypes of A. actinomycetemcomitans may be related to other oral microorganisms. Interestingly, the relations among these bacterial species seem to be under influence of geographic and ethnical aspects. Objective: This study aimed to evaluate the frequency of A. actinomycetemcomitans serotype-specific antigens and its association with Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia and Campilobacter rectus in Brazilian subjects. Method: A wide group of subjects (n=1320) had their periodontal status determined and were screened for the subgingival presence of A. actinomycetemcomitans serotypes. Then, a total of 263 individuals A. actinomycetemcomitans positive for serotypes A, B, C and E were considered in this survey. Subsequently, in this same group of positive subjects, the presence of P. gingivalis, P. intermedia, T. forsythia and C. rectus from periodontal pockets were also evaluated by PCR. Result: Out of 263 subjects investigated, A. actinomycetemcomitans serotype A was detected in 28.5% subjects; serotype B in 15.97%; serotype C in 51.71% and serotype E in 3.80% subjects. The frequency of serotype A of A. actinomycetemcomitans was significantly higher in C. rectus positive subjects than in P. gingivalis, P. intermedia or T. forsythia positive subjects. Additionally, the frequency of serotypes B and C were significantly higher in both C. rectus and T. forsythia positive subjects in comparison to P. gingivalis and P. intermedia positive subjects. Meanwhile, the frequency of serotype E was not associated with the presence of the other periodontal pathogens investigated. Conclusion: We observed a positive association between A. actinomycetemcomitans serotypes A with C. rectus and between B and C serotypes with C. rectus and T. forsythia. / Aggregatibacter actinomycetemcomitans é uma bactéria do tipo cocobacilo anaeróbia facultativa gram-negativa que coloniza a cavidade oral humana. Levando em consideração as associações bacterianas encontradas no biofilme dentário, estudos prévios têm sugerido que a ocorrência de sorotipos específicos de A. actinomycetemcomitans pode estar relacionada à ocorrência de outros micro-organismos orais. Curiosamente, as relações entre essas espécies de bactérias parecem sofrer influência de aspectos geográficos e étnicos. Objetivo: Este estudo tem como objetivo avaliar a frequência de sorotipo-específicos de A. Actinomycetemcomitans e suas associações com Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia e Campilobacter rectus em indivíduos brasileiros. Materiais e Métodos: Um extenso grupo de indivíduos (n=1320) teve sua condição periodontal determinada e foram selecionados pela presença subgengival de algum sorotipo de A. actinomycetemcomitans. Sendo assim, um total de 263 indivíduos positivos para os sorotipos A, B, C e E foram considerados nessa pesquisa. Posteriormente, nesse mesmo grupo de indivíduos positivos, foram avaliadas, por PCR, a presença de P. gingivalis, P. intermedia, T. forsythia e C. rectus nas bolsas periodontais. Resultados: Dos 263 indivíduos investigados, o sorotipo A de A. actinomycetemcomitans foi detectado em 28,5% dos casos, o sorotipo B em 15,97%, o sorotipo C em 51,71% e o sorotipo E em 3,80% dos indivíduos. A frequência do sorotipo A de A. actinomycetencomitans foi significativamente maior em indivíduos positivos para C. rectus do que em indivíduos positivos para P. gingivalis, P. intermedia ou T. forsythia. Além disso, a frequência de sorotipos B e C foi significativamente maior tanto em indivíduos positivos para C. rectus quanto em T. forsythia quando comparados a indivíduos positivos para P. gingivalis e P. intermedia. Entretanto, a frequência do sorotipo E não foi associada com a presença de nenhum dos periodontopatógenos investigados. Conclusão: Observamos uma associação positiva entre o sorotipo A de A. actinomycetemcomitans com C. rectus e entre o sorotipo B e C com C. rectus e T. forsythia.

Abstract New rapid methods for sensitive and specific detection of pneumococci are not only needed to improve the diagnosis of pneumococcal disease but are also essential for vaccine and carriage studies. The purpose of this study was to develop sensitive PCR methods for the detection and quantification of S. pneumoniae and to study the applicability of these methods to detecting pneumococci in clinical samples. A previously described PCR method was first developed further by introducing a Europium-labelled hybridisation probe for the detection of amplification products. The hybridisation method was easy to use and improved the specificity of the PCR assay. The developed PCR assay was established as a sensitive method for detecting pneumococcal DNA when the presence of pneumococcal DNA in over 2500 middle ear fluid (MEF) samples of children with acute otitis media (AOM) was studied by using the method. Pneumococcal findings increased by 76% when using PCR detection in addition to culture, compared to using culture alone. However, the PCR-positive, culture-negative AOM events represented a less severe type of disease compared to the culture-positive events. A positive PCR finding seems to indicate the presence of viable, although often non-culturable pneumococci within the middle ear cleft. To be able to rapidly detect and quantify the initial numbers of pneumococcal genome copies in clinical samples, a real-time PCR method for the detection and quantification of pneumococcal DNA was developed. In real-time PCR, amplification and detection of amplification products occur simultaneously, which makes it possible to monitor the phase of the reaction at a particular stage or continuously. The method developed here was applied to the analysis of MEF samples and to investigating the nasopharyngeal carriage of pneumococcus. The sensitivities of bacterial culture and real-time PCR in detecting pneumococci were also compared. The real-time PCR assay was found to be rapid and sensitive and to provide information about the differences between the numbers of bacteria in samples. However, the quantitative results were shown to be dependent on the DNA extraction method applied. The real-time PCR method developed appears to be a good aid in research where an accurate and sensitive pneumococcal diagnosis is needed.

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [119]-139).