Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

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No. of bitstreams: 1 Tese - Norma Suely de Lima Freitas.pdf: 8476174 bytes, checksum: b8e0fdb20a7b419aea33bc83321484fa (MD5) Previous issue date: 2012-08-28 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Despite the high prevalence and the risks associated with Chlamydia trachomatis in Brazil and other countries, little is known about the distribution of genotypes in Brazil and the biological variability of this important transmitting agent of sexually transmitted diseases (STDs). The absence of an inquiry routine for C. trachomatis and effective treatments can cause serious complications and consequences for individuals as pelvic inflammatory disease, infertility, ectopic pregnancy and neonatal infections. Currently, C. trachomatis presents 19 serotypes A-C associated with trachoma, D-K responsible for urogenital infections and L1, L2 and L3, agents responsible for lymphogranuloma venereum syndrome. The MOMP, which is recognized as the immunodominant antigen encoded by the ompA gene displays a large area of nucleotide variation of four variable domains (VDI to VDIV). Studies suggest that mutations occur frequently among genotypes and that these mutations may indicate differences between the immunogenic MOMP genotypes of C. trachomatis. The present work aims to identify the genotypes from samples positive by PCR for C. trachomatis in women diagnosed with infertility and with pregnant women in the city of Manaus, Amazonas - Brazil, in addition to sequence and analyze the ompA gene. The study population consisted of 96 infertile women and 53 pregnant women. Genotyping was performed by the technique of Polymerase Chain Reaction (PCR) from the ompA gene sequence and the following genotypes were identified in pregnant women: D [50.0%], E [25.0%] and F [12.5%] and I [12.5%]. In infertile women genotypes were identified: E [16.7%] F [16,7%] and K [66,7%]. The frequency of genotype K and D found in this study are considered high (66,7%) and (50,0%) and for phylogenetic analysis, we found that the genotypes analyzed shares the same ancestor. It is suggested that these variations in the sequences of genotypes identified arise from point mutations, or possibly by VD recombination in MOMP. The VDII region showed to be the most variable in the sequences analyzed. / Apesar da alta prevalência e dos riscos associados à Chlamydia trachomatis no Brasil e outros países do mundo, pouco se conhece sobre a distribuição dos genótipos no Brasil e a variabilidade biológica deste importante agente transmissor de doenças sexualmente transmissíveis (DST). A ausência de uma investigação rotineira para C. trachomatis e tratamentos efetivos pode originar sérias complicações e conseqüências para os indivíduos como doença inflamatória pélvica, infertilidade, gravidez ectópica e infecções neonatais. Atualmente, a C. trachomatis apresenta 19 sorotipos: A-C associados ao tracoma, D-K responsáveis por infecções urogenitais e L1, L2 e L3, agentes responsáveis pela síndrome do linfogranuloma venéreo. A MOMP, reconhecida como o antígeno imunodominante codificado pelo o gene ompA, exibe uma extensa área de variação nucleotídica sendo por sua vez, conferido por quatro domínios variáveis (VDI a VDIV). Estudos sugerem que as mutações ocorrem frequentemente entre os genótipos e que essas mutações podem indicar diferenças imunogênicas entre as MOMP de genótipos de C. trachomatis. O presente trabalho tem por objetivo identificar os genótipos a partir de amostras positivas por PCR para C. trachomatis de mulheres com diagnóstico de infertilidade e em gestantes na cidade de Manaus, Amazonas – Brasil, além de sequenciar e analisar o gene ompA. A população de estudo consistiu de 96 mulheres inférteis e 53 mulheres gestantes. A genotipagem foi feita pela a técnica de Reação em Cadeia de Polimerase (PCR) a partir da seqüência do gene ompA e os seguintes genótipos foram identificados em gestantes: D [50,0%]; E [25,0%]; F [12,5%] e I [12,5%]. Em mulheres inférteis os genótipos identificados foram: E [16,7%], F [16,7,%] e K [66,7%]. A freqüência de genótipo K e D encontrada neste estudo são consideradas elevadas (66,7%) e (50,0%) e quanto à análise filogenética, verificamos que os genótipos analisados compartilham do mesmo ancestral. Sugere-se que as variações encontradas nas sequências dos genótipos identificados surgem dos pontos de mutação ou possivelmente pela recombinação dos VD na MOMP. A região VDII foi que mais apresentou variações nas seqüências analisadas.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Objetivos: determinar a prevalÃncia de infecÃÃo genital por Chlamydia trachomatis em gestantes, comparando o subgrupo com diagnÃstico positivo com o de diagnÃstico negativo quanto aos fatores bio-sÃcio-demogrÃficos, histÃria ginecolÃgica e exame fÃsico ginecolÃgico, avaliando-se os fatores associados à presenÃa de infecÃÃo genital por Chlamydia trachomatis. Sujeitos e mÃtodos: submeteram-se ao teste de captura hÃbrida para Chlamydia trachomatis 446 gestantes do ambulatÃrio de prÃ-natal do Hospital Geral Dr CÃsar Cals, da Secretaria Estadual de SaÃde-CearÃ, no perÃodo de Agosto de 2003 a Maio de 2004. A idade mÃdia do grupo foi de 25,98 anos, idade gestacional mÃdia de 19 semanas. Aplicou-se questionÃrio diretamente Ãs gestantes, independente da idade gestacional e de estarem sintomÃticas ou nÃo, excluindo-se aquelas que tivessem feito uso de antibiÃticos ou de qualquer substÃncia quÃmica intravaginal nos quinze dias anteriores à coleta, ou que tivessem mantido relaÃÃes sexuais nos dois dias anteriores à consulta de prÃ-natal, com coleta de swab endocervical para realizaÃÃo de teste de captura hÃbrida II, com material colhido em tubo com soluÃÃo conservadora utilizando o sistema de micro placa, conforme procedimento descrito pelo fabricante. Os dados foram analisados utilizando o software STATA 13.0, procedendo-se anÃlise descritiva e analÃtica atravÃs do teste de qui-quadrado e regressÃo logÃstica, subtraindo-se variÃveis. Resultados: A prevalÃncia de Chlamydia trachomatis entre as gestantes foi de 2.91%. Identificou-se como fatores de risco independentemente associados à infecÃÃo genital por Chlamydia trachomatis a histÃria de dor pÃlvica ou doenÃa inflamatÃria pÃlvica, presenÃa de corrimento vulvar ao exame fÃsico e nÃo uso de preservativo com parceiro eventual. Calculou-se o Odds-ratio (OR), para cada um destes fatores, com respectivos intervalos de confianÃa. ConclusÃes: O subgrupo com rastreamento positivo para Chlamydia trachomatis caracterizou-se por apresentar uma faixa etÃria e renda familiar menor que o subgrupo com sorologia negativa, alÃm de apresentar maior frequencia de pacientes separadas, que usam menos preservativos com parceiros eventuais e com mais antecedentes de corrimento genital e dor pÃlvica. A OR para presenÃa de infecÃÃo genital por Chlamydia trachomatis foi de 1,7 para aquelas que nÃo usam preservativos e foi de 0,10 e 0,17, respectivamente, para ausÃncia de dor pÃlvica/DIP e corrimento vulvar. / Objectives: To estimate the prevalence of Chlamydia trachomatis in pregnant women, comparing the positive group to the negative one in respect to socio-demographic factors, gynecologic history and exam, evaluating the risk factors associated to Chlamydia trachomatis genital infection. Subjects and methods: Hybrid capture test for Chlamydia trachomatis was performed in 446 pregnant women at Hospital Geral Dr CÃsar Cals, from the Health Secretary of the State of CearÃ, from August, 2003 to May, 2004. Medium age in the group was 25.98 years and 19 weeks was the medium age of pregnancy. A structured questionnaire was applied, no matter the age of pregnancy, whether they were or not symptomatic, excluding those who had used antibiotics or any other substance into the vagina, during the previous fifteen days or who had kept sexual relationship until two days before the consultation, with a endocervical swab being performed, in order to have a hybrid capture test for the presence of Chlamydia trachomatis, as indicated by the manufacturer. Data were analysed by STATA 13.0, performed by means of the qui-square and logistic regression tests with descriptive and analytic presentation. Results: The prevalence of Chlamydia trachomatis among the pregnant women was 2.91%. Risk factors independently associated to Chlamydia trachomatis genital infection were history of pelvic pain or pelvic inflammatory disease, vulvar discharge and not using condom with an eventual sex partner. Respective odds ratio and confidence intervals were calculated to these variable. Conclusions: The positive group was younger, had smaller salaries and presented a greater frequency of divorced women, with less preservative use and more positive history of genital discharge and pelvic pain in the past. The OR to the presence of Chlamydia trachomatis genital infection was 1, 7 for those women not using condom and 0, 10 and 0, 17, respectively for a negative history of pelvic pain / PID, and the absence of vulvar discharge

Introduction Yearly, 90 million people are infected with C. trachomatis. Even though it is easily treated with antibiotics the often-asymptomatic infection often spreads prior to detection. A vaccine is therefore of great interest. A chimeric major outer membrane protein (MOMP) of C. trachomatis has in earlier studies proved to contain the epitopes necessary for immunization. In this thesis the chimeric MOMP gene was cloned and expressed in E. coli. Furthermore, the expression of the protein was analyzed in previously transformed A. thaliana. Materials and Methods The chimeric MOMP gene was cloned into E.coli. Following vector amplification, the gene was expressed and the protein purified by affinity chromatography.  Seeds from different lines of previously transformed A. thaliana were screened by PCR. Hits were then analyzed by western blot.  Results The results show successful cloning and expression of the chimeric MOMP gene in E. coli. The following protein purification did result in purified protein, however in low concentration. For the A.thaliana lines, the presence and correct orientation of the gene was verified in some of the lines screened. The B7 line was verified to express the protein. Discussion The low concentration of purified protein in E.coli was probably due to un-optimized imunnoprecipitation conditions. In expression analysis of A. thaliana, purification of plant samples by immunoprecipitation prior to running western blot gave results, whereas running un-purified samples in urea buffer did not, probably due to interfering proteins in wild type plants.

Chlamydial attachment and infectivity in vitro and ascending disease and sequelae in vivo have been reported to be enhanced/modulated by estrogen. Endometrial carcinoma cell lines Ishikawa and HEC-1B and the breast cancer lines MCF-7 and HCC-1806 were examined for Chlamydia trachomatis E infectivity. Estrogen receptor (ER) presence was confirmed by Western blot and qRT-PCR analyses. FACS analysis was used to determine the percent of plasma membrane-localized ERs (mERs), and their activity was tested by estrogen binding and competitive estrogen antagonists assays. Chlamydiae grew in all cell lines with HEC (90%) ≫ MCF-7 (57%) > Ishikawa (51%) ≫ HCC-1806 (20%). The cell line ER isoform composition was re-defined as: ERα + ERβ + for MCF-7, HCC-1806 and Ishikawa; and ERβ only for HEC-1B. HeLa cells were also tested and found to express ERβ, but not ERα. A small percentage of both ERs were surface-exposed and functionally active. The endometrium- predominant ERβ isoform was found in all cell lines, including those most representative of the common sites of C. trachomatis infection. Thus, the role of chlamydial attachment/infectivity will now be analyzed in ERβ + and - isogenic HEC-1B cells.

Diss. (sammanfattning) Linköping : Univ., 2004. / Härtill 5 uppsatser.

MSc (Microbiology) / Department of Microbiology / See the attached abstract below

Indiana University-Purdue University Indianapolis (IUPUI) / Progression of Chlamydia into upper reproductive tract epithelium and the induction of subsequent immune responses to infection are major contributors to Chlamydia-induced pathogenesis of the genital tract. We reported that C. muridarum infection of the oviduct epithelial cells (OEs) secrete IFN-β in a TLR3 dependent manner. However, we showed that the C. muridarum infected TLR3-deficient OEs were still able to secrete minimal amounts of IFN-β into the supernatants, which is suggestive that there are other signaling pathways that contribute to Chlamydia-induced IFN-β synthesis in these cells. Previous studies describing the activation of the JAK/STAT signaling pathway during Chlamydia infection of cervical epithelial cells proposes a putative role for STAT1 in the synthesis of type I IFNs during Chlamydia infection. The present study investigated the role of STAT1 in Chlamydia-induced IFN-β production in OEs. OEs were infected with Chlamydia muridarum and analyzed at 24 hours by RT-PCR and western blot to determine STAT1 expression. STAT (-/-) OEs were infected and IFN-β production measured by ELISA. Quantitative real-time PCR analyses were performed at 6 and 16 hour post-infection to elucidate the mechanisms involved in IFN-β production during infection. Fluorescent microscopy was used to observe changes in Chlamydia replication. STAT1 activation and expression were significantly increased in wild-type (WT) OEs upon infection. TLR3 (-/-) OEs showed diminished STAT1 protein activation and expression. Augmented STAT1 protein expression corresponded to STAT1 mRNA levels. ELISA analyses revealed significantly less IFN-β production in infected STAT1 (-/-) OEs compared to WT OEs. Quantitative real-time PCR data showed that gene expression of IFN-β and of type I IFN signaling components were significantly increased during late stage Chlamydia infection, dependent on STAT1. Temporal regulation and increases in expression of IFN-α subtypes during infection were STAT1-dependent. Our results implicate STAT1-mediated signaling as a contributor to the C. muridarum-induced synthesis of IFN-β and other type I IFNs in OEs. We previously described a major role for TLR3 in the early-stage Chlamydia-induced synthesis of IFN-β in OEs; the results from this study suggest a role for STAT1 in the synthesis of type I IFNs that occurs during early and late stages of infection.

<p>Chlamydia trachomatis is the most common sexually transmitted bacterial pathogen and is the leading cause of preventable blindness worldwide. Chlamydia is particularly intriguing from the perspective of cell biology because it is an obligate intracellular pathogen that manipulates host cellular pathways to ensure its proliferation and survival. This is achieved through a significant remodeling of the host cell’s internal architecture from within a membrane-bound vacuole, termed the inclusion. However, given a previous lack of tools to perform genetic analysis, the mechanisms by which Chlamydia induces host cellular changes remained unclear. Here I present genetic and molecular mechanisms of chlamydial manipulation of the host cytoskeleton and organelles. Using a forward genetics screen, InaC was identified as a necessary factor for the assembly of an F-actin structure surrounding the inclusion. InaC associated with the vacuolar membrane where it recruited Golgi-specific ARF-family GTPases. Actin dynamics and ARF GTPases regulate Golgi morphology and positioning within cells, and InaC acted to redistribute the Golgi to surround the Chlamydia inclusion. These findings suggest that Chlamydia places InaC at the inclusion-cytosolic interface to recruit host ARF GTPases and F-actin to form a platform for rearranging intracellular organelles around the inclusion. The inclusion is also surrounded by the intermediate filament vimentin and the chlamydial protease CPAF cleaves vimentin in vitro. CPAF-dependent remodeling of vimentin occurred selectively in late stages of the infection. In living cells, this cleavage occurred only after a loss of inclusion membrane integrity, suggesting that CPAF cleaves intermediate filaments specifically during chlamydial exit of host cells. In summary, I have implemented recent forward and reverse genetic approaches in Chlamydia to reveal how it employs effector proteins to manipulate the internal organization of cells in novel ways.</p> / Dissertation

La infección por Chlamydia trachomatis es la infección de transmisión sexual bacteriana más frecuente en todo el mundo. La Organización Mundial de la Salud estima que 90 millones de casos ocurren a nivel mundial. El objetivo de este estudio fue evaluar el desempeño de la prueba rápida HEXAGON CHLAMYDIA para el diagnóstico de infección por Chlamydia trachomatis en muestras de secreción endocervical de mujeres usuarias que asisten al consultorio de planificación familiar del “Hospital Nacional Dos de Mayo” y trabajadoras sexuales del servicio PROCETTS del Centro de Salud “Alberto Barton”. Se estudiaron un total de 101 muestras de secreción endocervical, 50 muestra de mujeres que asistieron al consultorio de planificación familiar del “Hospital Nacional Dos de Mayo” y 51 muestras de trabajadoras sexuales del servicio PROCETTS del Centro de Salud “Alberto Barton”. A todas las mujeres se les tomó dos muestras de secreción endocervical, una de las muestras fue procesada por la prueba rápida inmunocromatografica HEXAGON CHLAMYDIA en evaluación y la otra por una prueba validada de amplificación de los ácidos nucleicos (nucleic acid amplification, NAATs, por sus siglas en inglés): APTIMA COMBO 2 basada en la metodología de amplificación mediada por transcripción (transcription mediated amplification, TMA, por sus siglas en inglés) Se detectaron 4 casos de infección por C. trachomatis, todos estos casos se encontraron en el grupo de usuarias del consultorio de planificación familiar mientras en el grupo de trabajadoras sexuales no se encontró ninguno. La sensibilidad y la especificidad de la prueba rápida HEXAGON CHLAMYDIA fue 75% y 84,5%.El valor predictivo positivo fue 16,7% y el valor predictivo negativo fue 98,7%. El valor Kappa fue 0,21. La prueba rápida HEXAGON CHLAMYDIA en este estudio tuvo un pobre desempeño al ser comparada con la prueba APTIMA COMBO 2