archives@tulane.edu / Background: Chlamydia trachomatis (Ct) is the most common bacterial sexually transmitted infection in the United States. The rate among Black Americans is disproportionately higher than that of White Americans. Ct acquisition is influenced by factors at different levels of the social-ecological model. Methods: Data was collected through Check It, a community venue-based screening study for Black men aged 15-24 who have sex with women, in New Orleans, Louisiana between 05/17/2017-03/16/2020. Latent class analysis identified classes (patterns) of behaviors and relationship traits. Regression mixture modeling assessed associations between covariates and distal outcomes within classes. Path analysis examined mediation of neighborhood factors on Ct by sexual behaviors. Results: At the individual level, five classes of risk behaviors—including substance use, condomless sex, and multiple recent partners—were identified among 1872 men; Ct prevalence ranged from 7.3%-13.6%. Age and education beyond high school were significant risk factors for two classes and health insurance was significantly protective against Ct for two classes. Among 2906 partners reported, five relationship classes emerged with Ct prevalence ranging from 10.1%-18.3%. Community members provided diverse descriptions of the classes. Age, education, substance use, multiple partners, health insurance, and time in a detention facility were predictive of class membership. The effects of three neighborhood and institutional level factors (everyday discrimination, neighborhood safety, and time in a detention facility) on Ct prevalence were mediated by behaviors (substance use, multiple partners, and condomless sex). The three neighborhood and institutional factors were highly interrelated. Conclusion: This work describes the unique characteristics of this population and can be utilized in risk assessment and delivering sexual health services to young Black men who have sex with women. Understanding risk factors across levels of the social-ecological model supports the need for policy changes that address unequal environments and opportunities that increase the risk of Ct acquisition. / 1 / Megan Clare Craig-Kuhn

Chlamydia trachomatis is an important cause of immune-mediated damage to the reproductive tract of infected patients. Certain chlamydial antigens and host genetic factors have been identified as contributing to immunopathological events, but a comprehensive understanding of specific components involved in destructive vs. protective immune responses to chlamydial infections is far from clear. In this study, it is shown that C. trachomatis-infected patients generate antibodies against an iron-responsive chlamydial protein, YtgA. The identity of YtgA was confirmed by mass spectrometry following two-dimensional polyacrylamide gel electrophoresis and Western blot analysis. This finding underscores a necessity to examine patient sera samples to identify chlamydial antigens that are likely encountered and important to the immune response during human infections.

The obligate intracellular bacterium Chlamydia trachomatis requires iron in order to complete its developmental cycle. Addition of an iron-chelating drug, Desferal (deferoxamine mesylate), to infected cell culture causes Chlamydia to enter persistence. Here, we explore the ability of a stably-transfected cell line with inducible over-expression of the eukaryotic iron efflux protein ferroportin to starve C. trachomatis serovar E for iron. Ferroportin-induced iron removal is perhaps a more direct method of removing iron from the intracellular compartment versus exposure to an exogenous chemical chelator. Following induction, ferroportin-green fluorescent protein (Fpn-GFP) was detected in the plasma membrane, and cells expressing Fpn-GFP remained viable throughout the timescale required for Chlamydia to complete its developmental cycle. Following Fpn-GFP induction in infected cells, chlamydial infectivity remained unchanged, indicating chlamydiae were not in persistence. Ferritin levels indicate only a small decrease in cellular iron following Fpn-GFP expression relative to cultures exposed to Desferal. These data indicate that expression of Fpn-GFP in chlamydiae-infected cells is not capable of reducing iron below the threshold concentration needed to cause chlamydiae to enter persistence.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2016-08-09T13:56:09Z No. of bitstreams: 2 Dissertação - Kelly Deyse Segati - 2012.pdf: 1381150 bytes, checksum: 6c27c539f7ddade59c13124dfc688935 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-08-09T13:57:32Z (GMT) No. of bitstreams: 2 Dissertação - Kelly Deyse Segati - 2012.pdf: 1381150 bytes, checksum: 6c27c539f7ddade59c13124dfc688935 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-08-09T13:57:32Z (GMT). No. of bitstreams: 2 Dissertação - Kelly Deyse Segati - 2012.pdf: 1381150 bytes, checksum: 6c27c539f7ddade59c13124dfc688935 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2012-12-18 / Infection with Chlamydia trachomatis (CT) may be associated with persistent carcinogenic HPV types and the development of cervical neoplasia. There are indications that detection of CT serum antibodies rather than DNA is a better measure of cumulative exposure to CT or of exposure occurring several years prior to the development of cervical disease. The objective of this study was to compare the positivity for CT by ELISA and PCR and to correlate with the severity of cervical neoplasia in women with abnormal cervical smear. Between February 2007 and March 2009, 136 women were referred to the colposcopy clinic at the Santa Casa de Misericordia in Goiânia-GO. HPV DNA was detected by the polymerase chain reaction (PCR) and genotyping was performed by reverse line-blot hybridization assay. CT seropositivity was tested by ELISA for the detection of IgG antibodies and the detection of CT was done by PCR to amplify a sequence in the cryptic plasmid generating a fragment of 512 base pairs. The total prevalence of HPV infection was 85.2%. Seropositivity for CT was 26%. Thirty-one women 26.7 were tested positive for CT antibodies and HPV-DNA. Of these 10.3% had diagnosis of cervical intraepithelial neoplasia grade 1 (CIN1) or cervicitis, while 16.3% had histological diagnosis of CIN2 worse diagnosis. When employed PCR test positivity was found to be 8.8%. Eleven women 9.48% were tested positive for CT and HPV DNA. Of these 5.1% had diagnosis of cervicitis or CIN1 and 4.3% had a diagnosis of CIN2 or worse diagnosis. The agreement between serology and PCR tests for CT was considered poor (kappa=0.10 IC 95% 0.69-7.9). Taking as reference the cases negatives for HPV and CT, a positivity for HPV and CT seropositivity was significantly associated with a diagnosis of CIN2 or worse diagnosis, for all HPV types (OR=11.9 IC=2.00-91.5 p=0.0009) and types 16 and 18 (OR=7.50 IC=0.91-76.28 p=0.02). Significant association was observed after adjustment for HPV. A Borderline significance was observed considering other HPV types (OR=7.50 IC=0.91-76.28 p=0.02). CT seropositivity was associated with CIN2 worse diagnosis in women infected by HPV, mainly when the types 16 and 18 were involved. This study did not show any association between CT infection detected by PCR and CIN2 or worse diagnosis. These data support the hypothesis that seropositivity for CT compared to PCR positivity in HPV positive women, especially for types 16 and 18, is a better measure of previous exposure, which reflects a higher probability of association with the severity of cervical neoplasia. / A infecção por Chlamydia trachomatis (CT) pode estar associada com a persistência dos tipos de Papilomavírus humano (HPV) oncogênicos e desenvolvimento da neoplasia cervical. Há indicações de que a detecção de CT por sorologia seja uma melhor medida de exposição cumulativa ou da exposição passada quando comparada a detecção pela reação da polimerase em cadeia (PCR). O objetivo deste estudo foi comparar a positividade para CT pelos métodos de ELISA e PCR e relacionar com a gravidade da neoplasia cervical em mulheres com anormalidades citológicas. Entre fevereiro de 2007 e março de 2009, 136 mulheres, foram encaminhadas à Clínica de Colposcopia na Santa Casa de Misericórdia em Goiânia-GO por exame citológico alterado. A detecção de DNA do HPV foi realizada por PCR utilizando os iniciadores PGMY09/PGMY11, e a genotipagem foi realizada por hibridização reversa em pontos. A positividade para CT foi avaliada por ELISA para detecção de anticorpos IgG e por PCR empregando iniciadores cujo alvo é uma região de plasmídeo críptico, gerando um fragmento de aproximadamente 512 pares de bases. A prevalência total da infecção por HPV foi 85,2%. A positividade para CT por sorologia foi de 25%. Trinta e uma amostras 26,7% foram positivas para HPV e CT. Destas 10,3% tinham diagnóstico de neoplasia intraepitelial cervical grau 1 (NIC1) ou cervicite, enquanto 16,3% tinham diagnóstico histológico de NIC 2 ou pior diagnóstico. Quando empregado o teste de PCR a positividade encontrada foi de 8,8%. Onze amostras 9,48% foram positivas para HPV e CT por PCR, sendo que 5,1% das pacientes apresentavam diagnóstico de NIC1 ou cervicite e 4,3% tinham diagnóstico de NIC 2 ou pior diagnóstico. A concordância entre os testes de sorologia e PCR para CT foi considerado ruim (kappa=0,10 IC 95% 069-7.9). Tomando como referência casos negativos para HPV/CT, a positividade para HPV/CT por sorologia foi significantemente associada com diagnóstico de NIC2 ou pior diagnóstico, para todos os tipos de HPV (OR=11.9 IC=2.00-91.5 p=0.0009) e para os tipos 16 e 18 (OR=16.25 IC=2.28-148.57 p=0.0005). Uma associação limítrofe foi observada considerando outros tipos de HPV (OR=7.50 IC=0.91-76.28 p=0.02). Houve associação estatisticamente significante após o ajustamento para infeção por HPV entre as infecções pelos tipos 16 e 18 e soropositividade para CT com a gravidade da neoplasia cervical. Quando empregado o teste de PCR, não houve associação entre a coinfecção HPV/CT e a gravidade da neoplasia cervical. Estes dados reforçam a hipótese de que a soropositividade para CT quando comparada a positividade por PCR em mulheres HPV positivas, especialmente para os tipos 16 e 18, é uma melhor medida de exposição anterior, o que reflete maior probabilidade de associação com a gravidade da neoplasia cervical.

Submitted by Alisson Mota (alisson.davidbeckam@gmail.com) on 2015-07-08T20:15:35Z No. of bitstreams: 1 Tese - Norma Suely de Lima Freitas.pdf: 8476174 bytes, checksum: b8e0fdb20a7b419aea33bc83321484fa (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2015-07-09T13:56:33Z (GMT) No. of bitstreams: 1 Tese - Norma Suely de Lima Freitas.pdf: 8476174 bytes, checksum: b8e0fdb20a7b419aea33bc83321484fa (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2015-07-09T13:54:31Z (GMT) No. of bitstreams: 1 Tese - Norma Suely de Lima Freitas.pdf: 8476174 bytes, checksum: b8e0fdb20a7b419aea33bc83321484fa (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2015-07-09T14:05:09Z (GMT) No. of bitstreams: 1 Tese - Norma Suely de Lima Freitas.pdf: 8476174 bytes, checksum: b8e0fdb20a7b419aea33bc83321484fa (MD5) / Made available in DSpace on 2015-07-09T14:05:09Z (GMT). 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

Chlamydien sind Gram-negative, obligat-intrazelluläre Bakterien, die für ein weites Spektrum an relevanten Krankheiten verantwortlich sind. Auf Grund ihres zweiphasigen Entwicklungszyklusses sind Chlamydien von einer intakten Wirtszelle abhängig, um sich erfolgreich vermehren und im Organismus ausbreiten zu können. Daher haben Chlamydien anspruchsvolle Strategien entwickelt, um das Immunsystem des Wirtes auszuschalten oder den programmierten Zelltod ihrer Wirtszelle zu verhindern. In der vorliegenden Arbeit wurde untersucht, ob eine Infektion mit C. trachomatis einen Einfluss auf die zelluläre Antwort auf dsRNA nehmen kann. Die Synthese von dsRNA ist ein charakteristisches Merkmal der Replikation von Viren, welche sowohl die Apoptose induzieren als auch das Immunsystem aktivieren kann. Um eine chlamydiale und virale Co-Infektion zu simulieren, wurden Chlamydien-infizierte Epithelzellen mit der synthetischen dsRNA Polyinosin-Polycytidinsäure (polyI:C) transfiziert. Im ersten Teil der Arbeit wurde untersucht, ob Chlamydien die durch dsRNA eingeleitete Apoptose verhindern können. Eine signifikante Reduktion der dsRNA-induzierten Apoptose konnte in infizierten Zellen beobachtet werden. Es zeigte sich, dass die Prozessierung der Initiator-Caspase-8 in infizierten Zellen unterblieb. Dies war von der frühen bakteriellen Proteinsynthese abhängig und für die dsRNA-vermittelte Apoptose spezifisch, da der durch TNFalpha bewirkte Zelltod nicht auf der Ebene der Caspase-8 verhindert werden konnte. Die Aktivierung von zellulären Faktoren, die bei der Apoptoseinduzierung eine wichtige Rolle spielen, beispielsweise PKR und RNase L, war in infizierten Zellen jedoch unverändert. Stattdessen konnte durch RNA Interferenz-vermittelte Depletion gezeigt werden, dass der zelluläre Caspase-8-Inhibitor cFlip eine entscheidende Rolle bei der chlamydialen Blockierung der dsRNA-vermittelten Apoptose spielt. Mittels Co-Immunopräzipitation konnte ein erster Hinweis darauf gefunden werden, dass C. trachomatis eine Anreicherung von cFlip im dsRNA-induzierten Komplex von Caspase-8 und FADD bewirkt. Im zweiten Teil der Arbeit wurde untersucht, ob Chlamydien die Immunantwort auf virale Infektionen beeinflussen, welche vor allem die Expression von Interferonen und Interleukinen beinhaltet. Es stellte sich heraus, dass die Aktivierung des Interferon regulatory factor 3 (IRF-3) und des zur Familie von NF-kappaB Trankriptionsfaktoren gehörenden p65, zwei zentralen Regulatoren der Immunantwort auf dsRNA, in infizierten Epithelzellen verändert war. Die Degradation von IkappaB-alpha, des Inhibitors von NF-kappaB, war in infizierten Zellen beschleunigt, begleitet von einer Veränderung der Translokation des Transkriptionsfaktors in den Zellkern. Im Gegensatz dazu wurde die nukleäre Translokation von IRF-3 durch die Infektion signifikant verhindert. Die hier vorgestellten Daten zeigen erstmals, dass eine Infektion mit C. trachomatis die zelluläre Antwort auf dsRNA signifikant verändern kann und implizieren einen Einfluss von chlamydialen Infektionen auf den Ausgang von viralen Superinfektionen. / Chlamydia are Gram-negative obligate intracellular bacteria responsible for a wide spectrum of relevant diseases. Due to their biphasic developmental cycle Chlamydia depend on an intact host cell for replication and establishment of an acute infection. Chlamydia have therefore evolved sophisticated strategies to inhibit programmed cell death (PCD) induced by a variety of stimuli and to subvert the host immune system. This work aimed at elucidating whether an infection with C. trachomatis can influence the cellular response to double-stranded RNA (dsRNA). The synthesis of dsRNA is a prominent feature of viral replication inside infected cells that can induce both PCD and the activation of a cellular innate immune response. In order to mimic chlamydial and viral co-infections, Chlamydia-infected cells were transfected with polyinosinic:polycytidylic acid (polyI:C), a synthetic dsRNA. In the first part of this work it was investigated whether C. trachomatis-infected host cells could resist apoptosis induced by polyI:C. A significant reduction in apoptosis, determined by PARP cleavage and DNA fragmentation, could be observed in infected cells. It could be shown that processing of the initiator caspase-8 was inhibited in infected host cells. This process was dependent on early bacterial protein synthesis and was specific for dsRNA because apoptosis induced by TNFalpha was not blocked at the level of caspase-8. Interestingly, the activation of cellular factors involved in apoptosis induction by dsRNA, most importantly PKR and RNase L, was not abrogated in infected cells. Instead, RNA interference experiments revealed the crucial role of cFlip, a cellular caspase-8 inhibitor, for chlamydial inhibition of dsRNA-induced apoptosis. First data acquired by co-immunoprecipitation experiments pointed to an infection-induced concentration of cFlip in the dsRNA-induced death complex of caspase-8 and FADD. In the second part of this work, the chlamydial influence on the first line of defense against viral infections, involving expression of interferons and interleukins, was examined. Activation of the interferon regulatory factor 3 (IRF-3) and the NF-kappaB transcription factor family member p65, both central regulators of the innate immune response to dsRNA, was altered in Chlamydia-infected epithelial cells. polyI:C-induced degradation of IkappaB-alpha, the inhibitor of NF-kappaB, was accelerated in infected cells which was accompanied by a change in nuclear translocation of the transcription factor. Translocation of IRF-3, in contrast, was significantly blocked upon infection. Together the data presented here demonstrate that infection with C. trachomatis can drastically alter the cellular response to dsRNA and imply an impact of chlamydial infections on the outcome of viral super-infections.

Expedited partner therapy (EPT) is a strategy for treating the partners of chlamydia index cases by which a health care provider gives an index patient drugs or a prescription for treatment of chlamydia to deliver to their sex partner without an intervening medical evaluation of the partner. Despite routine offer of EPT in New York City Department of Health and Mental Hygiene (DOHMH) sexual health clinics, the majority of patients who are eligible for EPT do not receive it, largely because EPT eligibility requires lab confirmation of chlamydial infection, which is lacking in situations where patients are treated for chlamydia on the same day they are tested for chlamydia (i.e., presumptive treatment). These patients become eligible for EPT after they leave the clinic and often do not return for EPT. This dissertation includes three papers: one systematic review and two original analyses. The objective of the systematic review was to synthesize existing estimates of EPT effectiveness to better understand the impact of biases on these estimates; a meta-analysis provided an aggregate estimate of the effectiveness of EPT for preventing index patient reinfection with chlamydia and/or gonorrhea. We found 6 studies that included some measure of EPT effectiveness. Meta-analysis revealed that EPT significantly reduced the risk of reinfection from chlamydia and/or gonorrhea, but it also revealed a substantial amount of heterogeneity. Systematic review revealed that inclusion of patients whose sex partners were at the clinic or already treated for infection was a common source of bias among existing estimates of EPT effectiveness. The two original analyses used data from NYC DOHMH sexual health clinics where EPT is routinely offered as patient delivered partner therapy (PDPT), a form of EPT where medication is given directly to the index patient. The objective of the first analysis was to identify predictors of presumptive treatment and predictors of being offered PDPT among patients eligible for PDPT in the NYC clinics. This analysis demonstrated that patient diagnosis as a contact to a sexually transmitted infection (STI) that would warrant treatment with azithromycin or doxycycline (termed STI contacts) was the best predictor of presumptive treatment in NYC DOHMH sexual health clinics. Patients who were not contacts to such STIs or who were STI contacts with more than one sex partner were more likely to be offered PDPT compared to patients who were STI contacts and reported ≤ 1 sex partner. Males not diagnosed as STI contacts were identified as a target population for increasing rates of PDPT offer. The objective of the last analysis was to provide an estimate of PDPT effectiveness for preventing index patient reinfection with chlamydia. This analysis was novel compared to existing estimates of EPT effectiveness in that we excluded patients whose sex partners were at the clinics at the time of chlamydia testing, treatment, or PDPT offer. We found that PDPT significantly reduced the risk of repeat chlamydial infection at both 6 months and 1 year after initial infection. This result was unchanged by multiple sensitivity analyses that assessed the validity of our estimate. In this dissertation, we were able to fill gaps in the literature regarding EPT implementation. The results may help to decrease missed opportunities for offering patients EPT and to support the continued scale-up and optimization of EPT.

The obligate human pathogen Neisseria gonorrhoeae is responsible for the widespread sexually transmitted disease gonorrhoea, which in rare cases also leads to the development of disseminated gonococcal infection (DGI). DGI is mediated by PorBIA-expressing bacteria that invade host cells under low phosphate condition by interaction with the scavenger receptor-1 (SREC-I) expressed on the surface of endothelial cells. The interaction of PorBIA and SREC-I was analysed using different in vitro approaches, including surface plasmon resonance experiments that revealed a direct phosphate-independent high affinity interaction of SREC-I to PorBIA. However, the same binding affinity was also found for the other allele PorBIB, which indicates unspecific binding and suggests that the applied methods were unsuitable for this interaction analysis. Since N. gonorrhoeae was recently classified as a “super-bug” due to a rising number of antibiotic-resistant strains, this study aimed to discover inhibitors against the PorBIA-mediated invasion of N. gonorrhoeae. Additionally, inhibitors were searched against the human pathogen Chlamydia trachomatis, which causes sexually transmitted infections as well as infections of the upper inner eyelid. 68 compounds, including plant-derived small molecules, extracts or pure compounds of marine sponges or sponge-associated bacteria and pipecolic acid derivatives, were screened using an automated microscopy based approach. No active substances against N. gonorrhoeae could be identified, while seven highly antichlamydial compounds were detected. The pipecolic acid derivatives were synthesized as potential inhibitors of the virulence-associated “macrophage infectivity potentiator” (MIP), which exhibits a peptidyl prolyl cis-trans isomerase (PPIase) enzyme activity. This study investigated the role of C. trachomatis and N. gonorrhoeae MIP during infection. The two inhibitors PipN3 and PipN4 decreased the PPIase activity of recombinant chlamydial and neisserial MIP in a dose-dependent manner. Both compounds affected the chlamydial growth and development in epithelial cells. Furthermore, this work demonstrated the contribution of MIP to a prolonged survival of N. gonorrhoeae in the presence of neutrophils, which was significantly reduced in the presence of PipN3 and PipN4. SF2446A2 was one of the compounds that had a severe effect on the growth and development of C. trachomatis. The analysis of the mode of action of SF2446A2 revealed an inhibitory effect of the compound on the mitochondrial respiration and mitochondrial ATP production of the host cell. However, the chlamydial development was independent of proper functional mitochondria, which excluded the connection of the antichlamydial properties of SF2446A2 with its inhibition of the respiratory chain. Only the depletion of cellular ATP by blocking glycolysis and mitochondrial respiratory chain inhibited the chlamydial growth. A direct effect of SF2446A2 on C. trachomatis was assumed, since the growth of the bacteria N. gonorrhoeae and Staphylococcus aureus was also affected by the compound. In summary, this study identified the severe antichlamydial activity of plant-derived naphthoquinones and the compounds derived from marine sponges or sponge-associated bacteria SF2446A2, ageloline A and gelliusterol E. Furthermore, the work points out the importance of the MIP proteins during infection and presents pipecolic acid derivatives as novel antimicrobials against N. gonorrhoeae and C. trachomatis. / Neisseria gonorrhoeae ist ein obligat humanpathogenes Bakterium, das für die weltweit verbreitete sexuell übertragbare Krankheit Gonorrhoe verantwortlich ist. In seltenen Fällen kann es auch zur Ausbildung der Disseminierten Gonokokken-Infektion (DGI) kommen, die mit der Expression des Gonokokken Oberflächenproteins PorBIA assoziiert ist. PorBIA-exprimierende Bakterien invadieren in die Wirtszelle unter phosphatfreien Bedingungen, was durch eine Interaktion mit dem zellulären Oberflächenrezeptor scavenger receptor-1 (SREC-I) vermittelt wird. Die direkte Interaktion zwischen PorBIA und SREC-I wurde mittels verschiedenster Methoden analysiert, einschließlich einer Oberflächenplasmonresonanz-analyse, die eine direkte Bindung von PorBIA zu SREC-I in einem phosphatunabhängigen Schritt aufzeigte. Allerdings wurde dieselbe Affinität auch zu PorBIB gefunden, was auf eine unspezifische Bindung hindeutet und dafür spricht, dass die verwendeten Methoden für diese Interaktionsanalyse ungeeignet sind. N. gonorrheae wurde vor kurzem wegen der stetig steigenden Anzahl antibiotikaresistenter Stämme als „Superkeim“ bezeichnet. Aufgrund dessen wurden Inhibitoren gegen die PorBIA-vermittelte Invasion von N. gonorrhoeae, aber auch gegen Chlamydia trachomatis, den humanpathogenen Erreger von sexuell übertragbaren Infektionen und chronisch-follikulärer Bindehautentzündung, gesucht. 68 niedermolekulare Substanzen wurden mittels eines automatisierten Fluoreszenzmikroskopieverfahrens auf ihre inhibitorische Wirkung hin analysiert. Zu den getesteten Substanzen zählten pflanzenabstammende Stoffe, Isolate aus marinen Schwämmen oder Schwamm-assoziierten Bakterien, sowie Pipecolinsäure-Derivate. Gegen N. gonorrheae konnten keine Substanzen identifiziert werden, während sieben antichlamydiale Inhibitoren detektiert wurden. Pipecolinsäurederivate wurden synthetisiert als potentielle Inhibitoren des virulenz-assoziierten Proteins “macrophage infectivity potentiator” (MIP), das eine Peptidyl-Prolyl-cis-trans-Isomerase Aktivität besitzt. Diese Arbeit untersuchte die Rolle des MIP Proteins von N. gonorrhoeae und C. trachomatis während einer Infektion. Die zwei Inhibitoren PipN3 und PipN4 senkten die PPIase Aktivität des rekombinanten Chlamydien und Neisserien MIPs. Beide Substanzen beeinträchtigten das chlamydiale Wachstum und die Entwicklung in Epithelzellen. Ebenso konnte eine tragende Rolle des N. gonorrhoeae MIPs für das Überleben der Bakterien in Gegenwart von Neutrophilen aufgezeigt werden, das durch PipN3 und PipN4 inhibiert wurde. SF2446A2 war einer der Inhibitoren, der einen erheblichen Effekt auf das Wachstum und die Entwicklung von C. trachomatis aufgewiesen hat. Während der Analyse des Wirkmechanismus von SF2446A2 konnte eine Hemmung der mitochondrialen Atmungskette und eine Abnahme der mitochondrialen ATP Produktion in der Wirtszelle festgestellt werden. Allerdings war die Entwicklung von C. trachomatis unabhängig von der Funktionsfähigkeit der Mitochondrien. Eine Verbindung zwischen der antichlamydialen Wirkung von SF2446A2 und der Inhibierung der Mitochondrienatmungskette konnte damit ausgeschlossen werden. Nur das Reduzieren von zellulärem ATP durch Blockieren der Glykolyse und mitochondrialen Atmungskette verursachte eine Beeinträchtigung des Chlamydienwachstums. Eine direkte Auswirkung von SF2446A2 auf C. trachomatis wurde angenommen, da die Substanz auch das Wachstum von anderen Bakterien wie N. gonorrhoeae und Staphylococcus aureus inhibierte. Zusammengefasst identifizierte diese Studie die antichlamydiale Aktivität pflanzenabstammender Naphthochinone und der Isolate aus marinen Schwämmen oder Schwamm-assoziierten Bakterien SF2446A2, ageloline A und gelliusterol E. Ebenso verdeutlicht die Arbeit die Bedeutung der MIP Proteine während der Infektion und legt Pipecolinsäurederivate als mögliche neue Antibiotika gegen N. gonorrhoeae und C. trachomatis nahe.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2009.