Chlamydia trachomatis is a human pathogen of the genital tract and ocular epithelium. It is an obligate intracellular parasite with a unique biphasic development cycle. C.trachomatis infection is the most common bacterial sexually transmitted disease in industrialized nations. Its ability to cause chronic disease makes it a serious economic burden and health threat to developed and developing countries. Although treatable, approximately 70% of C.trachomatis infections are asymptomatic, potentially leading to the development of chronic sequelae. Furthermore, chlamydial genital tract infection has been associated with an increased risk of cervical cancer and human immunodeficiency virus infection. Consequently, the development of an efficacious vaccine is the most convenient, potentially reliable and cost effective option to control chlamydial infection and disease complications. Anti-chlamydial protective immunity is essentially mediated by a T helper, type 1 (Th1), response that is dependent upon the presentation of antigen via major histocompatibility (MHC) class II molecules. While antibody secreting cells are not critical components of the primary effector response, they have been shown to be important for clearance of re-infection. Thus an ideal vaccine would be one capable of inducing both a strong Th1 T cell response and a strong mucosal antibody response. Currently there are very few efficacious vaccine candidates that have been identified and characterized. More specifically, there is only a limited number of known T cell antigens processed and presented by the human leukocyte antigen (HLA) class II molecules. This type of antigen is going to be essential to the development of an efficacious chlamydial vaccine. In this study we have identified a number of unique vaccine candidates using a novel in silico approach. In an attempt to overcome HLA polymorphism the whole chlamydial genome was screened for proteins containing epitopes predicted to bind multiple HLA class II molecules (i.e. predicted ‘promiscuous’ T cell epitopes). A wide range of HLA class II molecules were used in this screen to identify vaccine antigens that could potentially offer broad and ethnically balanced population coverage. This analysis identified a number of novel targets and was validated by the identification of a known chlamydial T cell epitope. A selection of these target proteins was cloned, expressed and purified. Recombinant protein was screened against serum samples from patients with both acute and chronic chlamydial infections. Two novel targets, hypothetical protein CT425 and ribonucleotide reductase small chain protein (NrdB) were identified as being immunoreactive. The in vivo protective efficacy of NrdB was analyzed using a mouse model. CD4+ T cells were harvested from NrdB immunized mice and adoptively transferred to naïve mice, which were subsequently infected at the genital site. NrdB immunization was found to confer a CD4+ T cell driven degree of protection similar to that seen with CD4+ T cells primed from a live challenge. The adjuvants and route of immunization used ensured immunological responses were initiated at both the systemic and local sites of infection. Immunization elicited a predominant Th1 response with primed T cells producing high levels of interferon gamma, an essential requirement for the development of an efficacious chlamydial vaccine. Furthermore, high titres of antigen specific IgG and IgA were produced following immunization, with sera derived antibodies demonstrating neutralization properties. NrdB is a highly conserved chlamydial protein with an essential role in the replication of chlamydiae and could play a central role in a multi-subunit vaccine against chlamydial genital tract infections.

The distinctive feature of the Human Immunodeficiency Virus (HIV) epidemic in the 21st century is the burden it places on women. Scientists believe that the best opportunities for successful interventions to prevent sexual HIV transmission lie in the initial stages of infection at the portal of entry, the genital tract (GT), which offers the greatest host advantages and viral vulnerabilities. However, understanding of the correlates of protection/vulnerability and innate immunity at the portal of entry is poor. First and foremost, there is no agreement about which GT sub-compartment is the primary site of HIV/SIV infection. Second, the epithelium, previously studied solely for its function as a barrier, has hardly been investigated for its role in innate immunity in the context of SIV/HIV infection. MIP-3α, a chemokine secreted by epithelial cells, was previously proposed to have a role in amplifying the early Simian Immunodeficiency Virus (SIV) infection events in the GT of female macaques. Specifically, MIP-3α was shown to be secreted by epithelial cells of the endocervix, accumulating subepithelially within the first 24 hours post exposure, following deposition of an intravaginal inoculum of SIV. Similar studies in humans have not been reported. We hence undertook to study MIP-3α for its role in early HIV infection events in the endocervix of humans. In order to achieve this, we first characterised MIP-3α constitutive secretion patterns in different sub-compartments of the GT before proceeding to determine its induced secretion patterns, stimulating with HIV-1 and various Toll-like receptor ligands. For completeness we determined constitutive and induced secretion patterns of multiple soluble proteins (SPs) and antimicrobial peptides (AMPs) in the endocervices of humans and macaques. The GT being an immunohormonal system, we further studied the influence of endogenous hormonal changes on the stability of MIP-3α and that of other innate immune markers. We quantified MIP-3α with a sandwich Elisa, and SPs and AMPs with the Luminex multiplex bead assay. Our results showed that the GT is a rich source of MIP-3α with its levels being among those of the highest SPs in the GT. Constitutive levels were highest in the endocervical sub-compartment of all the sub-compartments studied. Further, the GT is an inflammatory environment, which would explain the high levels of MIP-3α. The primary driver of MIP-3α levels appears to be inflammation rather than hormonal levels. MIP-3α levels are significantly higher in the GT of humans than in macaques. There was no evidence that MIP-3α levels are elevated on exposure to HIV and SIV in humans and macaques, respectively. We therefore concluded that since the endocervix is unlikely to respond to HIV/SIV by secreting MIP-3α in vivo, contrary to the previous reports, MIP-3α is hence not a key player in amplifying early events in infection. And as such, it should not be a prime target for preventive therapy. Further, the human GT having a pre-existing inflammatory profile may explain the high rates of HIV sexual transmission. Lastly, we concluded that the infection mechanisms described in the macaque model (i.e. the 'outside-in' signaling) are likely not required for human infection.

L’emtricitabine est une molécule centrale dans la stratégie de lutte contre l’infection à VIH. En effet, elle est recommandée en première intention pour le traitement de l’infection chez l’adulte, ainsi que pour la prévention de la transmission de l’infection. Bien que l’emtricitabine soit une molécule clé, elle reste jusqu’à présent peu étudiée. Il est donc primordial d’analyser et de caractériser la pharmacocinétique de l’emtricitabine dans les différentes populations susceptibles d’être exposées à cette molécule. Une approche de population a été utilisée pour réaliser les analyses pharmacocinétiques, nous permettant de décrire la pharmacocinétique avec peu de prélèvements par individu (contraintes éthiques, difficulté de recueil des prélèvements…) et nous permettant d’expliquer la variabilité observée. L’étude rapportant la pharmacocinétique de l’emtricitabine chez l’adulte a mis en évidence une modification du profil cinétique selon l’état de la fonction rénale. Le modèle développé nous a permis d’évaluer les recommandations posologiques actuelles. Afin d’optimiser l’efficacité, la simulation de schémas thérapeutiques alternatifs chez les patients avec une insuffisance rénale modérée a été réalisée. Dans le cadre de la prévention de la transmission mère-enfant, l’étude décrivant la pharmacocinétique de l’emtricitabine chez les femmes enceintes a mis en évidence une modification de la pharmacocinétique au cours de la période gestationnelle. Toutefois, il ne semblait pas nécessaire d’adapter la posologie au cours de la grossesse. La dernière étude a permis d’étudier la pharmacocinétique de l’emtricitabine dans le tractus génital masculin. L’étude de la pénétration dans le compartiment génital est d’un intérêt capital pour le traitement mais aussi pour la prévention de la transmission de l’infection à VIH dans le cadre de la prophylaxie pré-exposition. Notre étude a rapporté une distribution importante de l’emtricitabine dans le tractus génital, avec des concentrations séminales supérieures aux concentrations plasmatiques. / Emtricitabine is a key antiretroviral drug in the strategy to fight against HIV infection. Indeed, emtricitabine is recommended in first-line treatment for HIV infection in adults, as well as for the prevention of HIV transmission. Although emtricitabine is a key molecule, it remains poorly studied until now. It is therefore essential to analyze and characterize the pharmacokinetics of emtricitabine in the different populations that may be exposed to this drug. A population approach was used to perform pharmacokinetic analyses, allowing us to describe the pharmacokinetics with few samples per individual (ethical constraints, difficulty of samples collection...) and allowing us to explain the observed variability. The study reporting emtricitabine pharmacokinetics in adults highlighted a change in emtricitabine kinetic profile depending on the state of renal function. The developed model allowed us to evaluate the current dosing recommendations. To optimize efficiency, simulations of alternative regimens in patients with moderate renal impairment were performed. As part of the prevention of mother to child transmission, the study describing emtricitabine pharmacokinetics in pregnant women showed a change in pharmacokinetics during the gestational period. However, it did not seem necessary to adjust the dosage during pregnancy. The latest work focused on emtricitabine pharmacokinetics in the male genital tract. Studying the penetration in the genital tract is of major interest for treatment but also for prevention of HIV transmission in the context of pre-exposure prophylaxis. Our study reported a significant distribution of emtricitabine in the genital compartment, with concentrations in seminal plasma higher than concentrations in blood plasma.

Made available in DSpace on 2015-03-26T13:47:25Z (GMT). No. of bitstreams: 1 texto completo.pdf: 459969 bytes, checksum: 684ba7361082f44d749b2627833c1178 (MD5) Previous issue date: 2014-02-21 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Morphofunctional studies on the testis allied to studies in endocrinology, embryology and cryopreservation of gametes, generate information that can be applied in assisted reproduction, favoring the development of methodologies used in conservation of species. The objectives of this study were to quantify morphological and functional structures of the testicular parenchyma, calculate gonadosomatic, tubulosomatic, leydigosomatic and Sertoli cell indices; describe the duration of the seminiferous epithelium cycle (SEC) by identifying the most advanced germ cells labeled by 5- bromodeoxyuridine (BrdU); characterize the various stages that constitute the seminiferous epithelium cycle based on tubular morphology and calculate daily sperm production per gram of testis of jaguarundi (Pumayagouaroundi). For this purpose we used five adult males, one from Centro de Triagem de AniamaisSilvestres at Universidade Federal de Viçosa and four from ParqueZoológico de São Paulo. After chemical restraint animals had their body weight, body size, and diameter of the chest measured, and underwent testicular biopsies in order to obtain biological material for histological and immunohistochemical evaluation. In the studied Jaguarundis the average weight was 6.6 kg, being that 0.032% of it was allocated in testis (gonadosomatic index), which has 57.36 m of seminiferous tubule, coming to a tubulosomatic index of 0.027% . The total number of Leydig cells found in this species was 72.96 million cells and leydigosomatic index was calculated at 0.001%. Eight stages of the SEC were described and had their relative frequencies determined based on the tubular morphology method. At SEC, pre- meiotic stage of the spermatogenic process was higher than the other ones (48.66 %). Through the use of a cell proliferation marker (BrdU) injected in the testicles, labeled cells were observed in round spermatids of stage V after 14 days of application. From this, we can conclude that a seminiferous epithelium cycle of the Jaguarundi lasts 8.4 days, and the total spermatogenic process duration is 37.8 days. The Jaguarundi presented in the seminiferous epithelium, in each cross section of the seminiferous tubule at stage I of the cycle, an average of 1.03 spermatogonia type A; 9.78 primary spermatocytes at pre-leptotene/leptotene; 10.68 primary spermatocytes in pachytene; 25.3 round spermatids. The spermatogenesis general yield in this species was approximately 18.9 cells, and each Sertoli cell was able to sustain and maintain 14.24 round spermatids. The daily sperm production found demonstrates that the Jaguarundi is an animal with a high level of production, since its production is about 51.52 million sperm per gram of testis. / As pesquisas morfofuncionais sobre o testículo aliadas aos estudos em endocrinologia, embriologia e criopreservação de gametas, produzem informações que podem ser aplicadas na reprodução assistida, favorecendo o desenvolvimento de metodologias utilizadas na conservação de espécies. Os objetivos deste estudo foram quantificar as estruturas morfofuncionais do parênquima testícular, calcular os índices gonadossomáticos, tubulossomáticos e leydigossomáticos e de célula de Sertoli; descrever a duração do ciclo do epitélio seminífero pela identificação das células germinativas mais avançadas marcadas pela 5-bromodeoxiuridina (BrdU); caracterizar os diversos estádios que constituem o ciclo do epitélio seminífero com base na morfologia tubular e calcular a produção espermática diária por grama de testículo de gato mourisco (Puma yagouaroundi). Para isso foram utilizados cinco animais machos adultos, sendo um proveniente do Centro de Triagem de Animais Silvestres da Universidade Federal de Viçosa e quatro do Parque zoológico de São Paulo. Após contenção químicaos animais tiveram suas medidas corporais de peso, tamanho corporal, e diâmetro de tórax mensurados, foram também submetidos a biópsias testiculares a fim de se obter material biológico para avaliação histológica e imunohistoquímica. Nos gatos mourisco estudados a média de peso foi de 6,6 kg sendo desses, 0,032% alocados em testículo (índice gonadossomático) que possui 57,36 m de túbulo seminífero perfazendo um índice tubulossomático de 0,027%. O número total de células de Leydig encontradas nessa espécie foi de 72,96 milhões de células e o índice leydigossomático foi calculado em 0,001%. Foram descritos e determinada a frequência relativa de oito estádios do ciclo do epitélio seminífero (CES) com base no método da morfologia tubular. No CES a fase pré-meiótica do processo espermatogênico foi maior que as outras (48,66%). Através do uso de um marcador de proliferação celular injetado intratesticularmente (BrdU) foram observadascélulas marcadas em espermátides arredondadas do estádio V, após 14 dias da aplicação. A partir daí, pode-se concluir que um ciclo do epitélio seminífero do gato mourisco dura 8,4 dias, e o processo espermatogênico duração total de 37,8 dias. O gato mourisco apresentou no epitélio seminífero, em cada secção transversal do túbulo seminífero no estádio I do ciclo, em média, 1,03 espermatogônias do tipo A; 9,78 espermatócitos primários em pré-leptóteno/leptóteno; 10,68 espermatócitos primários em paquíteno; 25,3 espermátides arredondadas. O rendimento geral da espermatogênese nesta espécie foi de aproximadamente 18,9 células, e cada célula de Sertoli foi capaz de sustentar e manter 14,24 espermátides arredondadas. A produção espermática diária encontrada demonstra que o gato mourisco é um animal com alto nível de produção, uma vez que sua produção é cerca de 51,52 milhões de espermatozoides por grama de testículo.

The phosphorylation and dephosphorylation of the tyrosine amino-acids in proteins play an important role in the regulation of many cellular processes in all eukariotic organisms, including the regulation of cell cycle control, growth control, cellular differentiation and gene and synaptic transmission. The involvement of the phosphatase genes in human carcinogenesis was long-suspected, but PTEN is the first important phosphatase gene proven to be a true tumour suppressor. The basic function of normal PTEN is the dephosphorylation of the kinases and inhibition of the integrin and growth factor mediated kinase signalling pathways. The central hypothesis of this study is that PTEN plays an important role in tumours of the upper female genital tract. The involvement of aberrations in the coding regions of this gene was studied in specific gynaecologic tumours and tissues using polymerase chain reaction based mutation analysis. The research model was to study both the malignant tumour and the closest available pre-malignant or benign counterpart to demonstrate different levels of involvement of PTEN in the evolving steps. The PTEN gene was found to be intimately involved in endometrial carcinogenesis. Involvement was demonstrated in hyperplasia and was common in endometroid carcinoma (54%). Pathogenic PTEN mutations were much more common in cancer than in hyperplasia (10%). Multiple mutations were found in some late stage tumours, suggesting that the already malignant tumour cells accumulate more genetic mutations over time. All tumours with more than one pathogenic mutation occurred in African patients. The latter twofindings are unique to the current study. Selective involvement of the PTEN gene was demonstrated in uterine soft tissue tumours. PTEN involvement was neither found in benign soft tissue tumours nor significantly in leiomyosarcoma or endometrial stromal sarcoma. However, PTEN plays a significant role in uterine carcinosarcoma (13%) and specifically in tumours with an endometroid epithelial component, where mutations were found in 17%. This finding is a highly significant and unique research result which supports the hypothesis of the endometrial origin of these tumours. It also supports the observation of a strong link between this gene and endometroid differentiation, with morphology strongly linked to cellular genetics. PTEN gene mutation was demonstrated in ovarian endometroid carcinoma in ~29% of cases investigated. This finding confirms PTEN involvement in carcinogenesis in this tumour type. The finding suggests that PTEN involvement is linked to endometroid epithelial morphology. We could not sufficiently test the involvement of the gene in benign or pre-malignant ovarian endometroid lesions and thus cannot comment on the chronology of mutations in this tissue type. When all tumour types were included, there was a tendency towards a lower frequency ofPTEN mutations in African women. PTEN mutations correlated with endometroid histology. In combination, these results confirm the racial disparity in tumour type distribution or morphology. In summary this study demonstrated significant though highly selective PTEN gene involvement and a strong and interesting association between genotype and histological phenotype was confirmed. The findings enhance our understanding of carcinogenesis and should lead to translational research into new anti-neoplastic drugs. AFRIKAANS: Fosforilering en defosforilering van die tirosien aminosure in proteine speel ‘n belangrike rol in die regulering van sellulêre prosesse in alle eukariotiese organismes. Dit sluit die regulering van selsikluskontrole, groeikontrole, sellulêre differensiasie sowel as genetiese en sinaptiese oordrag in. Dit word lank reeds gespekuleer dat die fosfatase-gene betrokke is in menslike karsinogenese, maar die PTEN geen is die eerste fosfatase geen wat bewys word om ‘n ware tumoronderdrukker geen te wees. As basiese funksie defosforileer normale PTEN die kinases en inhibeer dit die kinase sinjaal kontrolepaaie wat deur integrien en groeifaktor beheer word. Die sentrale hipotese van hierdie studie is dat PTEN ‘n belangrike rol speel in tumore van die boonste genitale traktus. Die frekwensie van abnormaliteite in die koderingsareas van hierdie geen is bestudeer in spesifieke ginekologiese tumore en weefsels met die gebruik van polimerase kettingreaksie gebaseerde mutasie-analise. Die maligne tumore sowel as die mees verwante pre-maligne of benigne weefsel- of tumortipes wat beskikbaar was, is gebruik as navorsingsmateriaal om sodoende die verskillende vlakke van PTEN betrokkenheid in die ontwikkeling van neoplasie te demonstreer.Intieme betrokkenheid van die PTEN geen is gevind in endometriële karsinogenese. PTEN mutasies is in hiperplasie gevind en dit was algemeen in endometroiede karsinoom (54%). Patogene mutasies was baie meer algemeen in kanker as in hiperplasie (10%). Veelvuldige mutasies is in sommige laat stadium tumore aangetoon, wat suggereer dat reeds maligne selle meer genetiese mutasies oor tyd verkry. Alle tumore waar meer as een patogeniese mutasie gevind is het voorgekom by swart pasiënte. Die laaste twee bevindinge is uniek tot hierdie studie.Selektiewe betrokkenheid van die PTEN geen is gevind in die ontwikkeling van sagte weefsel tumore van die uterus. PTEN mutasies is nie in benigne sagte weefsel tumore gevind nie en geen betekenisvolle betrokkenheid is in leiomiosarkome of endometriële stromale sarkome aangetoon nie. PTEN was egter betekenisvol betrokke in karsinosarkome van die uterus (13%) en veral in tumore met ‘n endometrioiede epiteelkomponent waar mutasies in 17% gevind is. Hierdie bevinding is ‘n hoogs betekenisvolle en unieke navorsingsbevinding wat die hipotese ondersteun dat hierdie tumore uit die endometrium ontstaan. Dit onderskryf ook die indruk dat ‘n sterk band bestaan tussen hierdie geen en endometroiede differensiasie, met morfologie sterk gekoppel aan sellulêre genetika.Mutasie in die PTEN geen is aangetoon in ovariële endometroiede karsinoom in ~29% van gevalle wat ondersoek is. Die bevinding bevestig PTEN betrokkenheid in karsinogenese in hierdie tumortipe. Weereens toon die resultaat dat PTEN betrokkenheid gekoppel is aan endometroiede morfologie. Die ondersoek van benign of pre-maligne letsels in hierdie orgaan was nie voldoende om kommentaar oor die tydsberekening van mutasie te kan lewer nie.Met alle tumortipes in ag genome, is daar ‘n tendens aangetoon van minder PTEN mutasies in swart vroue. PTEN mutasies korreleer met endometroiede histologie. In kombinasie bevestig hierdie resultaat ‘n rasse-diskrepansie in die distribusie van tumourtipe of morfologie. In opsomming is die bevinding van hierdie studie dat daar betekenisvolle dog hoogs selektiewe PTEN geen betrokkenheid in boonste genitale traktus tumore is. ‘n Sterk en interessante verband is bevestig tussen genotipe en histologiese fenotipe. Hierdie resultate verbeter die begrip van karsinogenese en behoort ‘n bydrae te lewer in die soeke na nuwe anti-neoplastiese middels. / Thesis (PhD)--University of Pretoria, 2011. / Obstetrics and Gynaecology / unrestricted

A disfunção erétil (DE) tem alta prevalência entre hipertensos e tem sido considerada marcador precoce de risco cardiovascular. A presença e gravidade da DE bem como a resposta clínica aos inibidores da fosfodiesterase tipo 5 (PDE5) parecem depender da biodisponibilidade do óxido nítrico (NO) endotelial e da extensão da doença aterosclerótica. O objetivo deste estudo foi avaliar a resposta clínica da vardenafila usada em dois regimes terapêuticos em hipertensos com DE vasculogênica e sem doença cardiovascular maior, correlacionando a gravidade da DE e a eficácia da vardenafila com dados antropométricos, laboratoriais, escore de risco cardiovascular e parâmetros vasculares funcionais e estruturais. A resposta clínica à vardenafila nos dois regimes foi avaliada conforme o percentual de respostas positivas à questão 3 do Perfil do Encontro Sexual (PES3). Os parâmetros vasculares considerados foram a espessura médio-intimal (EMI) da carótida comum, a dilatação mediada pelo fluxo (DMF) da artéria braquial e a dilatação nitrato-mediada (DNM). Foram incluídos 100 homens hipertensos com idade entre 50 e 70 anos, sendo 74 portadores de DE vasculogênica e 26 com função erétil normal que serviram de grupo controle. Nos pacientes com DE, o índice de massa corporal, relação cintura-quadril, EMI da carótida, níveis séricos de triglicerídeos, colesterol total e LDL foram significativamente maiores que no grupo controle. Após o uso de vardenafila on demand (fase 1), os pacientes com mais de 50% de respostas positivas ao PES3 ou 50% de respostas afirmativas e um incremento de 6 pontos ou mais em relação ao Índice Internacional de Função Erétil (IIEF-FE) basal e/ou resposta positiva a Questão de Avaliação Global (QAG), foram considerados respondedores. O escore do IIEF-FE basal se correlacionou negativamente com a EMI da carótida (r=-0,48, P<0,001) e com o escore de Framingham (r= -0,41, P<0,001) no grupo com DE. Houve forte correlação positiva entre a resposta clínica à vardenafila com a DMF (r= 0,70, P<0,001), que não se observou entre o sub-grupo de diabéticos. Os 35 pacientes considerados não-respondedores na fase 1 foram randomizados e, em desenho duplo-cego, receberam vardenafila ou placebo diariamente durante cinco semanas, podendo usar 10 mg de vardenafila uma hora antes da atividade sexual (fase2). Houve resposta clínica positiva em 38,8% dos que receberam a vardenafila na fase 2 e esta resposta se correlacionou com a frequência sexual (r= 0,68, P<0,01) e com o escore de Framingham (r= -0,65, P<0,01), com a EMI da carótida (r= -0,61, P=0,01) e com o LDL-colesterol (r= -0,64, P<0,01). A vardenafila foi bem tolerada em ambos os regimes terapêuticos. Concluímos que nessa amostra de hipertensos, a gravidade da DE foi relacionada a parâmetros vasculares estruturais (EMI), enquanto a resposta clínica à vardenafila on demand foi mais diretamente dependente da função vascular momentânea (DMF). Houve benefício na utilização de vardenafila diariamente com o objetivo de resgatar a eficácia do inibidor quanto à melhora do desempenho sexual. A falta de eficácia clínica ao inibidor da PDE5 em ambos os regimes terapêuticos pode servir como marcador clínico que identifica homens hipertensos com um risco cardiovascular aumentado. / Erectile dysfunction (ED) is a high prevalent disease in hypertensive subjects and has been considered an early cardiovascular risk marker. EDs presence and severity, as well as clinical response to phosfodiesterase type 5 (PDE5) inhibitors, vary according to nitric oxide (NO) availability and atherosclerosis extension. We investigated whether vasculogenic ED severity and clinical response to vardenafil used on demand or continuously were associated with structural and functional vascular changes in patients with uncomplicated hypertension. Our main efficacy criterion was per patient percentage of positive answers on Sexual Encounter Profile question 3(SEP3). Vascular parameters considered were intima-media thickness (IMT), flow-mediated dilation (FMD) on brachial artery and nitrate-mediated dilation. A total of 100 hypertensive men aging between 50 and 70 years were included. Among these patients, 74 had vasculogenic ED and 26 presented normal erectile function according to erectile domain of International Index of Erectile Function (IIEF-EF). Among those with ED, body mass index, waist-rip ratio, carotid IMT, triglycerides, total cholesterol and LDL-cholesterol were significantly higher than controls. After vardenafil on demand usage during phase 1, patients with more than 50% of positive answers on SEP3 or 50% and more than 6 points on IIEF basal score or positive answer to global evaliation question were considered responders. IIEF basal score correlated inversely with carotid IMT (r=-0.48, P<0.001) and with Framingham risk score (r= -0.41, P<0.001) in ED group. Clinical response to vardenafil strongly correlated with FMD (r= 0.70, P<0.001), except among diabetics. Non responders (n=35) on phase 1 were included on phase 2 when, after randomization, they received vardenafil 10 mg nightly or placebo during five weeks. Open vardenafil on demand were allowed one hour before sexual intercourse, and 38.8% of active group improved and became responders to vardenafil. Clinical response on phase 2 correlated with sexual frequency (r= 0.68, P<0.01), Framingham score (r= -0.65, P<0.01), carotid IMT (r= -0.61, P=0.01) and LDL-cholesterol (r= -0.64, P<0.01). We concluded that in hypertensive males with vasculogenic ED and no other clinical evidence of atherosclerosis, ED severity correlated with structural parameters (carotid IMT), while phosphodiesterase-5 effectiveness correlated with functional vascular aspects (brachial FMD). There were positive impact with continuous vardenafil on non responders to on demand regime and that could be an option to salvage strategy. Lack of PDE5 inhibitor efficacy in both therapeutic strategies could point out to higher cardiovascular risk and could be considered a useful clinical marker.

A disfunção erétil (DE) tem alta prevalência entre hipertensos e tem sido considerada marcador precoce de risco cardiovascular. A presença e gravidade da DE bem como a resposta clínica aos inibidores da fosfodiesterase tipo 5 (PDE5) parecem depender da biodisponibilidade do óxido nítrico (NO) endotelial e da extensão da doença aterosclerótica. O objetivo deste estudo foi avaliar a resposta clínica da vardenafila usada em dois regimes terapêuticos em hipertensos com DE vasculogênica e sem doença cardiovascular maior, correlacionando a gravidade da DE e a eficácia da vardenafila com dados antropométricos, laboratoriais, escore de risco cardiovascular e parâmetros vasculares funcionais e estruturais. A resposta clínica à vardenafila nos dois regimes foi avaliada conforme o percentual de respostas positivas à questão 3 do Perfil do Encontro Sexual (PES3). Os parâmetros vasculares considerados foram a espessura médio-intimal (EMI) da carótida comum, a dilatação mediada pelo fluxo (DMF) da artéria braquial e a dilatação nitrato-mediada (DNM). Foram incluídos 100 homens hipertensos com idade entre 50 e 70 anos, sendo 74 portadores de DE vasculogênica e 26 com função erétil normal que serviram de grupo controle. Nos pacientes com DE, o índice de massa corporal, relação cintura-quadril, EMI da carótida, níveis séricos de triglicerídeos, colesterol total e LDL foram significativamente maiores que no grupo controle. Após o uso de vardenafila on demand (fase 1), os pacientes com mais de 50% de respostas positivas ao PES3 ou 50% de respostas afirmativas e um incremento de 6 pontos ou mais em relação ao Índice Internacional de Função Erétil (IIEF-FE) basal e/ou resposta positiva a Questão de Avaliação Global (QAG), foram considerados respondedores. O escore do IIEF-FE basal se correlacionou negativamente com a EMI da carótida (r=-0,48, P<0,001) e com o escore de Framingham (r= -0,41, P<0,001) no grupo com DE. Houve forte correlação positiva entre a resposta clínica à vardenafila com a DMF (r= 0,70, P<0,001), que não se observou entre o sub-grupo de diabéticos. Os 35 pacientes considerados não-respondedores na fase 1 foram randomizados e, em desenho duplo-cego, receberam vardenafila ou placebo diariamente durante cinco semanas, podendo usar 10 mg de vardenafila uma hora antes da atividade sexual (fase2). Houve resposta clínica positiva em 38,8% dos que receberam a vardenafila na fase 2 e esta resposta se correlacionou com a frequência sexual (r= 0,68, P<0,01) e com o escore de Framingham (r= -0,65, P<0,01), com a EMI da carótida (r= -0,61, P=0,01) e com o LDL-colesterol (r= -0,64, P<0,01). A vardenafila foi bem tolerada em ambos os regimes terapêuticos. Concluímos que nessa amostra de hipertensos, a gravidade da DE foi relacionada a parâmetros vasculares estruturais (EMI), enquanto a resposta clínica à vardenafila on demand foi mais diretamente dependente da função vascular momentânea (DMF). Houve benefício na utilização de vardenafila diariamente com o objetivo de resgatar a eficácia do inibidor quanto à melhora do desempenho sexual. A falta de eficácia clínica ao inibidor da PDE5 em ambos os regimes terapêuticos pode servir como marcador clínico que identifica homens hipertensos com um risco cardiovascular aumentado. / Erectile dysfunction (ED) is a high prevalent disease in hypertensive subjects and has been considered an early cardiovascular risk marker. EDs presence and severity, as well as clinical response to phosfodiesterase type 5 (PDE5) inhibitors, vary according to nitric oxide (NO) availability and atherosclerosis extension. We investigated whether vasculogenic ED severity and clinical response to vardenafil used on demand or continuously were associated with structural and functional vascular changes in patients with uncomplicated hypertension. Our main efficacy criterion was per patient percentage of positive answers on Sexual Encounter Profile question 3(SEP3). Vascular parameters considered were intima-media thickness (IMT), flow-mediated dilation (FMD) on brachial artery and nitrate-mediated dilation. A total of 100 hypertensive men aging between 50 and 70 years were included. Among these patients, 74 had vasculogenic ED and 26 presented normal erectile function according to erectile domain of International Index of Erectile Function (IIEF-EF). Among those with ED, body mass index, waist-rip ratio, carotid IMT, triglycerides, total cholesterol and LDL-cholesterol were significantly higher than controls. After vardenafil on demand usage during phase 1, patients with more than 50% of positive answers on SEP3 or 50% and more than 6 points on IIEF basal score or positive answer to global evaliation question were considered responders. IIEF basal score correlated inversely with carotid IMT (r=-0.48, P<0.001) and with Framingham risk score (r= -0.41, P<0.001) in ED group. Clinical response to vardenafil strongly correlated with FMD (r= 0.70, P<0.001), except among diabetics. Non responders (n=35) on phase 1 were included on phase 2 when, after randomization, they received vardenafil 10 mg nightly or placebo during five weeks. Open vardenafil on demand were allowed one hour before sexual intercourse, and 38.8% of active group improved and became responders to vardenafil. Clinical response on phase 2 correlated with sexual frequency (r= 0.68, P<0.01), Framingham score (r= -0.65, P<0.01), carotid IMT (r= -0.61, P=0.01) and LDL-cholesterol (r= -0.64, P<0.01). We concluded that in hypertensive males with vasculogenic ED and no other clinical evidence of atherosclerosis, ED severity correlated with structural parameters (carotid IMT), while phosphodiesterase-5 effectiveness correlated with functional vascular aspects (brachial FMD). There were positive impact with continuous vardenafil on non responders to on demand regime and that could be an option to salvage strategy. Lack of PDE5 inhibitor efficacy in both therapeutic strategies could point out to higher cardiovascular risk and could be considered a useful clinical marker.

La plupart des infections par le VIH-1 sont acquises lors de rapports hétérosexuels. En Afrique subsaharienne on observe 71 % des infections mondiales et 60 % des nouvelles infections par le VIH-1 touchent les femmes. Le tractus génital féminin (TGF) constitue la principale porte d’entrée pour le VIH-1 et joue un rôle important dans la défense de l’organisme contre les microorganismes pathogènes tout en maintenant une tolérance de la flore commensale. On y trouve les cellules épithéliales qui participent à l’élaboration des réponses immunes en collaboration avec les cellules dendritiques (DCs), mais également d’autres types de cellules immunitaires qui confèrent une protection à la muqueuse vaginale, notamment à travers la production de cytokines et de chimiokines. Nous avons établi une cohorte de travailleuses du sexe (CSWs) au Bénin et nous avons identifié des femmes hautement exposées et séronégatives au VIH-1 (HESN), qui demeurent séronégatives après plus de sept années actives dans le travail du sexe. Les personnes HESN étant un excellent modèle d’immunité naturelle contre le VIH-1, le but de notre projet consiste donc à étudier les cellules immunitaires impliquées dans la protection de l’hôte face au VIH-1, au niveau du tractus génital féminin. Nous émettons l’hypothèse que le maintien de faibles conditions inflammatoires dans le TGF des femmes HESN préviendrait une activation immunitaire excessive en préservant l’intégrité de la barrière de la muqueuse vaginale et contribuerait ainsi à maintenir une protection contre l’infection par le VIH-1. Des études antérieures sur les HESN béninoises et kenyanes ont démontré que ces femmes présentent de faibles niveaux d’inflammation dans leur TGF inférieur. En accord avec cela, nous avons observé de faibles niveaux de BLyS/BAFF dans la muqueuse vaginale des HESN comparativement aux travailleuses du sexe séropositives (CSWs+ HIV+). BLyS/BAFF est une molécule importante pour la différenciation des cellules B et pour la sélection de cellules B de première ligne de la zone marginale (MZ). De ce fait, nous rapportons pour la première fois la présence de cellules B CD1c+ de type MZ qui sont capables de se lier naturellement à la gp120 glycosylée, au niveau de la muqueuse vaginale. Or, des cellules B CD1c+ exprimant IgG sont augmentées chez les CSWs+ HIV+ comparativement aux HESN, ce qui pourrait contribuer à l’hyperglobulinémie observée dans le TGF inférieur des CSWs+ HIV+. Les faibles niveaux de BLyS/BAFF retrouvés dans la muqueuse vaginale des HESN semblent donc préserver une homéostasie au sein du compartiment B et des cellules B CD1c+ du TGF. De plus, nous y détectons une réactivité des IgG1 avec la gp-41 de l’enveloppe virale, qui pourrait contribuer à leur immunité naturelle. Avec les cellules épithéliales, les DCs sont l’une des premières à être en contact avec le virus dans le TGF. Elles jouent un rôle essentiel dans l’orchestration des réponses immunitaires. Nous pensons que les DCs contribuent au maintien de faibles conditions inflammatoires dans le TGF des HESN, prévenant ainsi l’activation immunitaire excessive et préservant l’intégrité de la barrière muqueuse de façon à maintenir une protection/contrôle contre le virus. Nous avons caractérisé une population myéloïde endocervicale « tolérogénique » HLA-DR+CD14+CD11c+ exprimant HLA-G, ILT4, CD103 et de forts taux d’IFN-α et d’IL-10 dont la fréquence relative était augmentée au niveau du col de l’utérus des HESN comparativement aux CSWs+ HIV+. De plus, des populations Tregs/Tr1 étaient aussi augmentées chez les HESN. Ces données reflètent à la fois une réponse antivirale et une contribution au contrôle des conditions inflammatoires dans le TGF des HESN. Afin de mieux comprendre la nature des cellules myéloïdes tolérogéniques, nous avons voulu dériver des monocytes en cellules dendritiques (MoDCs). Toutefois, nous avons remarqué que la différenciation des MoDCs des HESN était altérée. Suite à cela, nous avons caractérisé le profil transcriptomique des monocytes. Les résultats préliminaires mettent en lumière l’éventuel rôle des récepteurs nucléaires NR4A dans la modulation des MoDCs et, possiblement, sur le plan des cellules myéloïdes tolérogéniques chez les HESN. Dans l’ensemble, ces résultats nous ont permis d’acquérir de nouvelles connaissances sur les mécanismes mis en place chez les HESN dans l’immunité naturelle contre le VIH-1. / Most HIV-1 infections are acquired through heterosexual intercourse. In sub-Saharan Africa, 71% of global infections are observed and 60% of new HIV-1 infections affect women. The female genital tract (FGT) constitutes a main portal of entry for HIV-1 and plays an important role in protecting the host against pathogens while maintaining a tolerance to a commensal flora. FGT immunity involves genital epithelial cells as well as dendritic cells (DCs) and many other types of immune cells which confer protection, through the production of chemokines and cytokines. We established a cohort of commercial sex workers (CSWs) in Benin and identified HIV-1 highly exposed seronegative (HESN) individuals, who remain uninfected after more than seven years of active prostitution. These HESN individuals being an exceptional model of natural immunity against HIV-1, the aim of our project is to characterize immune cells involved in protection from HIV-1 infection, in the female genital tract. We hypothesize that maintenance of low inflammatory conditions in the FGT of HESN women helps to prevent excessive immune activation likely preserving the mucosal barrier integrity and would help to maintain a protection against HIV infection. Previous studies of Beninese and Kenyan HESN have shown that these women have a low inflammatory profile in their lower FGT. Accordingly, we found that vaginal mucosa of HESN had lower soluble BLyS/BAFF levels when compared to HIV-infected CSWs (CSWs+ HIV+). BLyS/BAFF is highly recognized for its role in B-cell ontogenesis, as well as cell fate decision towards the innate marginal zone (MZ) B-cell pool. For the first time, we report the presence of genital MZ-like CD1c+ B-cells that naturally bind to fully glycosylated gp120 in the vaginal mucosa. However, CD1c+ B-cells expressing IgG are increased in the lower FGT of CSWs+ HIV+ when compared to HESN, suggesting that these cells could contribute to the hyperglobulinemia observed in the lower FGT of CSWs+ HIV+. The low levels of BLyS/BAFF found in the vaginal mucosa of HESN thus appear to preserve homeostasis of the FGT B cell compartment and CD1c+ B-cells. In addition, we detect a reactivity of IgG1 to HIV-gp41 in cervico-vaginal lavages (CVL) supernatants of HESN, which could contribute to their natural immunity. Epithelial cells and DCs are one of the earliest cell types to sense the virus in the FGT. They play a key role in the orchestration of immune responses. We characterized a "tolerogenic" endocervical myeloid HLA-DR+CD14+CD11c+ population expressing HLA-G, ILT4, CD103 and high levels of IFN-α and IL-10, that was increased in the cervix of HESN when compared to CSWs+ HIV+. In addition, frequencies of Tregs/Tr1 cells were also increased in HESN. We believe that DCs contribute to maintaining low inflammatory conditions in the FGT of HESN, preventing excessive immune activation and preserving the integrity of the mucosal barrier to maintain a protection/control against the virus. These data reflect both an antiviral response and a contribution to the control of inflammatory conditions in the FGT of HESN. To better understand the nature of tolerogenic myeloid cells, we wanted to derive monocyte-derived dendritic cells (MoDCs). However, derivation of blood MoDCs was impaired in HESN. As a result, we decided to characterize the transcriptomic profile of total blood monocytes. Preliminary results appear to demonstrate the possible role of NR4A nuclear receptors in MoDCs modulation, and possibly in tolerogenic myeloid cells in HESN. Overall, our results contribute to a better understanding of the mechanisms established by HESN in natural immunity to HIV-1.