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We demonstrated that infection of HeLa cells, which are non-responsive to flagellin, with wild type Salmonella enterica serotype Typhimurium (S. typhimurium) activated chemokine expression at higher level than S. typhimurium lacking sipAsopABDE2, indicating that the corresponding effector proteins (SipA, SopA, SopB, SopD and SopE2) are required to induce chemokines independent of flagellin. The S. typhimurium sipAsopABDE2 mutant complemented with sipA activated IL-8 expression at significantly higher level than a S. typhimurium sipAsopABDE2 mutant. However, extracellular addition of recombinant SipA failed to induce IL-8. Phosphorylation analyses demonstrated that S. typhimurium carrying a chromosomal copy of sipA (sopABDE2 mutant) induced phosphorylation of CREB1, JUN and p38MAPK, which are proteins involved in IL-8 expression. The contribution of effector proteins to S. typhimurium-induced intracellular Ca2+ mobilization and its role in IL-8 expression and bacterial internalization were also investigated. Our results demonstrated that wild type S. typhimurium significantly increased the amplitude of intracellular Ca2+ beginning 30 sec after infection. However, further analyses of intracellular Ca2+ changes in HeLa cells infected with S. typhimurium mutants indicated no correlation between increased intracellular Ca2+ and IL-8 expression or bacterial internalization. To analyze specific cell populations targeted by wild type S. typhimurium or S. typhimurium carrying a chromosomal copy of sipA (sopABDE2 mutant), laser capture microdissection was performed. Our data indicated that in wild type S. typhimurium-infected bovine Peyer’s patches, high levels of IL-8 were expressed in enterocytes of crypts, whereas Gro-α was expressed in enterocytes of both crypts and absorptive villi. A strain carrying a chromosomal copy of sipA colonized the same cell population as wild type, but induced IL8 and Gro-α in enterocytes of both crypts and absorptive villi. In conclusion, we demonstrated that in vitro S. typhimurium effector proteins induce chemokine expression independent of Ca2+ changes through phosphorylation of proteins related to IL-8 pathway. In vivo, we found higher levels of IL-8 expression in enterocytes of crypts than enterocytes of absorptive villi, although both cell populations contributed to Gro-α expression. These data extend the knowledge of the molecular mechanism by which S. typhimurium induces inflammatory genes by identifying pathogen and host molecules involved in inflammation.

Thesis (M.S.)--Case Western Reserve University, 2010. / [School of Medicine] Department of Pathology. Includes bibliographical references.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 6 uppsatser.

Thesis (M.S.)--University of Wisconsin--Madison. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves [70-72]).

Thesis (doctoral)--Freie Universität Berlin, 1980.

Orientadores: Ana Paula de Souza Pardo, Darcy de Oliveira Tosello / Tese (doutorado) Universidade Estadual de Campinas, Faculdade de Odontologia / Made available in DSpace on 2018-08-15T22:45:20Z (GMT). No. of bitstreams: 1 Andia_DeniseCarleto_D.pdf: 1152873 bytes, checksum: 26e93b7e638606b9fcb023a1ebbea875 (MD5) Previous issue date: 2010 / Resumo: A Doença Periodontal ou Periodontite é uma afecção complexa e multifatorial, resultante da interação dos mecanismos de defesa do hospedeiro com as espécies bacterianas da placa. Estudos em animais e humanos indicam que fatores genéticos e epigenéticos podem modificar a resposta inflamatória e imune, afetando a experiência da periodontite. Constitutivamente expressa por células epiteliais, a quimiocina Interleucina-8 (IL-8) participa ativamente da resposta inflamatória do hospedeiro, frente ao desafio bacteriano, por sua habilidade em mediar e ativar a migração de neutrófilos. A proposição deste estudo foi investigar aspectos epigenéticos (metilação), genéticos (polimorfismo) e os níveis de transcritos gênicos no promotor do gene IL8 em pacientes com periodontite crônica e agressiva. O primeiro capítulo relata a investigação sobre o single-nucleotide polymorphism (SNP) rs4073 do gene IL8 e sua relação com a periodontite crônica, observando também os níveis de mRNA IL-8 nos tecidos controles e nos acometidos pela doença. O SNP rs4370 foi detectado e analisado por PCR-RFLP em 289 amostras de DNA genômico de pacientes controles (108) e com periodontite crônica generalizada (181). A expressão relativa do mRNA da IL-8 (12 pacientes controles x 12 periodontite) foi investigada utilizando PCR quantitativa para a detecção dos níveis de transcritos gênicos. As análises dos resultados foram ajustadas pelo modelo de regressão logística multivariada e uma associação estatisticamente significante da periodontite com o genótipo TA (p=4.78x10-3) e com a idade (p=4.32x10-7) foi encontrada. Observou-se também um aumento da frequência do alelo A no grupo doente. Além disso, os níveis mais altos de produção de mRNA da IL-8 foram encontrados no grupo periodontite, principalmente nos indivíduos que apresentaram o genótipo TA. Esta diferença entre os dois grupos foi estatisticamente significante (p=0.03). Concluiu-se que o SNP rs4073 pode estar associado à periodontite crônica generalizada, em indivíduos brasileiros não fumantes, já que se observou um aumento na produção dos transcritos de mRNA da IL-8, na presença da inflamação periodontal. Esta significância foi estatisticamente significante mesmo após as correções para fatores como idade, gênero e etnia. O segundo capítulo investigou o mesmo SNP em famílias acometidas pela periodontite agressiva. PCR-RFLP foram utilizadas para a análise do genótipo da IL-8 em 13 famílias e para a investigação da frequência e do padrão de transmissão do alelo A, considerado o alelo de risco. A análise estatística foi realizada utilizando-se o teste de desequilíbrio de transmissão (TDT). Não houve associação do polimorfismo com a doença, com uma transmissão equilibrada entre os alelos (T: Z=1,213, p=0,2252; A: Z= - 1,213, p=0,2252) e frequência similar entre eles (T:52% e A:48%). Concluiu-se que o padrão de transmissão do alelo A e a combinação dele em genótipos são equilibrados, tanto em indivíduos com periodontite agressiva generalizada quanto nos controles. O terceiro capítulo relata a investigação do padrão de metilação no promotor do gene IL8 das células do epitélio oral em pacientes com periodonto saudável (controles) e nos acometidos pela periodontite agressiva. Para tanto, DNA genômico de células epiteliais orais de 37 indivíduos apresentando periodontite agressiva generalizada e de 37 pacientes controles foram purificados, modificados pelo bissulfito de sódio e submetido à técnica de PCR-MSP. A diferença no padrão de metilação entre os grupos foi estatisticamente significante (p=0.016; ?2 test), sendo que a condição não metilada foi encontrada em 62% dos indivíduos controles, enquanto que esta frequência esteve presente em 86.5% no grupo com periodontite. Concluiu-se, portanto, que os indivíduos com periodontite agressiva generalizada apresentaram um padrão de hipometilação nos dinucleotídeos CpGs analisados no promotor IL8 / Abstract: Periodontitis is a complex and multifactorial disease that results from the interaction of the host defense mechanisms with the plaque microorganism. Studies of animals and humans indicate that genetic factors could impair inflammatory and immune responses in general, affecting periodontitis experience specifically. Constitutively produced by the epithelial cells, the Interleukin-8 (IL-8) chemokine is important in the regulation of the inflammatory response for its ability to mediate the activation and migration of neutrophils. Therefore, the aim was to investigate the epigenetic (methylation) and genetic (polymorphism) aspects and RNA expression of the IL8 gene promoter in chronic and aggressive periodontitis patients. The first chapter investigated the association of the single-nucleotide polymorphism (SNP) rs4073 and chronic periodontitis; in addition, the levels of IL-8 mRNA in gingival tissue were observed. The IL8 SNP rs4370 was detected and analyzed by PCR-RFLP assay in 289 genomic DNA samples of control (108) and generalized chronic periodontitis (181); analyses were adjusted by the multivariate logistic regression modeling. Total RNA from gingival cells was isolated (12 control subjects x 12 periodontitis patients) and real-time PCR performance was used to detect the levels of IL-8 mRNA. Analysis points to a statistical significant association of the generalized chronic periodontitis with TA genotype (p=4.78x10-3) and age (p=4.32x10-7); moreover, the results showed an increase in the frequency of the A allele in the diseased group. Farther, the higher levels of the IL-8 mRNA were found in the chronic PD group, mainly in individuals who presented the TA genotype; the difference amongst the groups was statistically significant (p=0.03). The IL8 SNP rs4073 is associated with generalized chronic periodontitis in non-smoker Brazilian subjects, since it was observed an increase in the production of IL-8 mRNA transcripts, in the presence of the periodontal inflammation. Importantly, this significance was preserved even after correcting for factors such as age, gender and ethnicity. The second chapter investigated the frequency and the transmission pattern of the A allele of the IL8 SNP rs4370 within Brazilian families affected with generalized aggressive. The polymorphism was detected and analyzed by PCR-RFLP assay in 13 nuclear families. The statistical analysis was performed using the transmission disequilibrium test (TDT). There was no statistically significant association of the IL8 SNP rs4370 with generalized aggressive periodontitis, with a balanced transmission between the alleles (allele T: Z=1.213, p=0.2252; allele A: Z=-1.213, p=0.2252). In addition, there is also similar allele frequency (allele T: 52% and allele A: 48%). The transmission pattern of the A allele and its genotype combination are balanced, in generalized aggressive individuals and in the control subjects. The third chapter aimed to observe the DNA methylation status in the IL8 gene promoter in cells of the oral epithelium of control subjects and to compare it with that of subjects affected by generalized aggressive periodontitis. Genomic DNA from epithelial oral cells of 37 generalized aggressive periodontitis patients and 37 controls were purified, modified by sodium bisulphite and submitted by PCR-MSP technique. Subjects who presented generalized aggressive periodontitis had a higher frequency of hypomethylation of the IL8 gene promoter than those controls (86.5% in the generalized aggressive periodontitis group versus 62% in the control group; p=0.016; ?2 test). The frequency of the individuals in the control group who presented only the unmethylated status was about 62%, while there was an increase in this frequency in the generalized aggressive periodontitis group to 86.5%. It was found a marked hypomethylated status in the subjects who presented generalized aggressive periodontitis, comparing to the controls, in the promoter region analyzed of the IL8 gene / Doutorado / Histologia e Embriologia / Doutor em Biologia Buco-Dental

Desonide, a new topical anti-inflammatory and antipruritic steroid, has been investigated for its clinical, vasoconstrictor and in vitro percutaneous penetration properties, and compared to betamethasone 17-valerate, triamcinolone acetonide and hydrocortisone. The clinical and vasoconstrictor bioassay tests place desonide quantitatively among the most effective topical anti-inflammatory agents, possibly because of its relatively rapid skin penetration rate. The significance of the study is (a) it provides definitive data on topical anti-inflammatory effectiveness of desonide and (b) it reveals that, contrary to current opinion, fluorination of the steroid molecule may be unnecessary for topical anti-inflammatory activity, and that 9 °C-fluorination in prednisolone acetonides impedes rather than favours their skin penetration rates. New data on octanol/water partition coefficients and an unsuccessful effort of adopting the Martin (1968) oxime derivative spectrophotofluorometric technique for desonide assays are also included. / Pharmaceutical Sciences, Faculty of / Graduate