Orientadores: Sara Teresinha Olalla Saad, Carolina Louzão Bigarella / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-19T22:43:13Z (GMT). No. of bitstreams: 1 Melo_RitadeCassiaCarvalho_M.pdf: 3664788 bytes, checksum: 419eef7eff318d29e3e09f23e8b10461 (MD5) Previous issue date: 2012 / Resumo: A medula óssea é constituída por microambientes específicos denominados "nichos". O fator SDF-1 (Stromal derived factor-1) foi identificado como um importante fator quimioatrativo produzido por células estromais da medula óssea. Sua ação sobre seu receptor CXCR4 desempenha função primordial na migração, retenção e desenvolvimento dos progenitores hematopoiéticos na medula óssea. Células leucêmicas mielóides e linfóides expressam CXCR4 e aproveitam-se disso para acessar nichos medulares normalmente restritos ás células progenitoras, passando a residir em microambientes que propiciam sobrevivência e proliferação. Recentemente foi descoberto que o receptor CXCR7 é capaz de se ligar ao SDF-1. Ele é expresso em várias linhagens tumorais, mas em células hematopoiéticas seu papel é ainda pouco explorado. Em vista da escassez de dados na literatura o objetivo deste trabalho foi investigar a expressão e função de CXCR7 em síndromes mielodisplásicas e leucemias agudas. Neste estudo, foi mostrado que a expressão gênica de CXCR7 foi significativamente maior em leucemia linfoblástica aguda (LLA) em comparação com sindrome mielodisplásica (SMD), leucemia mielóide aguda (LMA) e indivíduos controles (p<0.0001, Mann-Whitney). A proteína CXCR7 também foi mais expressa em linhagens celulares linfoblástica T (Molt-4 e Jurkat) em comparação com linhagens mielóides. Em células linfoblásticas T, a localização subcelular de CXCR7 e CXCR4 por microscopia confocal e citometria de fluxo evidenciou CXCR7 mais próximo à membrana das células Molt-4 e mais frequentemente no citoplasma de células Jurkat enquanto CXCR4 está na membrana de ambas as linhas celulares. Curiosamente, notamos também que, depois da indução de SDF-1, células Molt-4 têm maior capacidade migratória comparada com Jurkat (mediana Molt 4 = 52,0 ± 5 vs Jurkat = 24,1 ± 3, p = 0,0079, teste de Mann-Whitney), que pode estar relacionado com a disponibilidade de membrana de CXCR7. Além disso, a inibição da CXCR7 ou CXCR4 resultou em mudanças significativas na resposta migratória de Molt4 e Jurkat (p<0,05 Mann-Whitney), no entanto, a inibição de ambos, CXCR7 e CXCR4 resultou em uma redução mais significativa na migração celular (p = 0.0079/Molt-4; p = 0.0043/Jurkat, Mann-Whitney). Uma vez que é bem estabelecido que células CD34+ de pacientes com SMD não são atraídas pelo gradiente de SDF-1, apesar de terem expressão normal de CXCR4, nos interessou investigar qual a localização de CXCR4 nas células SMD e se esta irresponsividade estava associada a CXCR7. Linhagens mielóides P39 e U937 foram usadas como modelo de SMD e LMA, respectivamente. Foram encontrados níveis similares de expressão de CXCR4 e CXCR7 em ambas as linhagens celulares, no entanto encontramos que CXCR4 está localizado no citoplasma de células P39 enquanto ele está na membrana das células U937. Uma vez que a proteína quinase C isotipo zeta (PKC'dzeta' está relacionada com a sinalização SDF-1/CXCR4 aumentando a expressão de CXCR4 e sua disponibilidade na membrana, resolvemos trabalhar também com células P39 hiperexpressando PKC'dzeta' (PKC'dzeta'wt). Este procedimento resultou na translocação de CXCR4 para a membrana de células P39, mas não alterou a localização subcelular de CXCR7. Ensaios de migração por tranwell mostraram que células P39 PKC'dzeta'wt apresentam maior capacidade de migração em relação a SDF-1 em comparação com células P39 controle (aumento de 35 vezes pcDNA3 PKC-'dzeta'-HA vs pcDNA3 transfectadas células P39, p = 0,0032, Mann-Whitney), sugerindo que a PKC'dzeta' restaura a capacidade quimiotática de células P39. Aumento da expressão de CXCR7, como aqui observado em células leucemicas linfoblásticas, é um fenômeno já descrito em uma variedade de linhagens de células tumorais sólidas, tais como cérebro, próstata e pulmão. Em tumores sólidos, CXCR7 principalmente aumenta a proliferação de células malignas. Estes resultados sugerem que a função biológica de CXCR7 depende tecido e órgãos que ele está localizado e que, na leucemia linfoblástica aguda pode ter um papel na migração de células, potencializando a resposta de CXCR4 a SDF-1 e, portanto, poderia contribuir para o recrutamente de células leucêmicas para nichos uma vez já ocupados por células-tronco hematopoéticas normais. Além disso, nossos resultados levam a crer que um defeito na via PKC'dzeta'/CXCR4 está envolvido com a irresponsividade de células SMD a SDF-1, gerando uma hematopoese ineficaz. E confirma dados que sugerem que PKC'dzeta' é uma proteína central na via de sinalização SDF-1/CXCR4, muito importante para a migração de células hematopoéticas malignas / Abstract: Bone marrow is constituted of specific microenvironments called "niches". The factor SDF-1 (stromal derived factor-1) was identified as an important chemoattractant factor produced by bone marrow cells. SDF-1 acts on its receptor CXCR4 and plays primordial function in migration, retention and development of hematopoietic progenitors in bone marrow. CXCR4 is expressed in leukemic cells and enables them to access marrow niches that normally are restricted to quiescent stem cells, thereby ensuring its protection from cell death resulting in a worse prognosis. Recently, CXCR7 was identified as another SDF-1-binding receptor, but its contribution to SDF-1 - mediated effects in hematopoietic cells is still poorly explored, even though the CXCR7 relationship with tumor progression in non-hematopoietic malignancies is well established. Given that there is little information regarding CXCR7 we investigated its function and expression in MDS and acute leukemias. This work, was showed that CXCR7 is significantly higher expressed in ALL compared to MDS, AML and control subjects (p<0.0001, Mann-Whitney test). CXCR7 protein is also higher expressed in lymphoblastic cell lines (Molt-4 and Jurkat) compared with myeloid cells. In lymphoblastic cell lines, the subcellular location of CXCR7 and CXCR4 by confocal microscopy and flow cytometry evidenced CXCR7 closer in the membrane of Molt-4 cells and more frequently in the cytoplasm of Jurkat cells whereas CXCR4 was in the membrane of both cell lines. Interestingly, we also noticed that, after SDF-1 induction, Molt-4 cells have higher chemotactic ability compared with Jurkat (median Molt 4=52.0 ± 5 vs Jurkat=24.1 ± 3, p=0.0079, Mann-Whitney test) which may be related with the membrane availability of CXCR7. In addition, the inhibition of CXCR7 or CXCR4 resulted in significant changes in Molt4 and Jurkat chemotactic response (p<0,05, Mann-Whitney test), however, the inhibition of both CXCR7 and CXCR4 resulted in a more significant reduction in cell migration (p=0.0079/Molt-4; p=0.0043/Jurkat, Mann-Whitney test). Since it is well established that CD34 + progenitor cells from patients with myelodysplastic syndromes (MDS) are not attracted by gradient of SDF-1 despite of having CXCR4 normal expression, we addressed if MDS cells have an abnormal localization of CXCR4 or association with CXCR7. P39 and U937 cell line were used as a model of MDS and AML, respectively. Similar expression levels of CXCR4 and CXCR7 in both cell lines however we found, by confocal microscopy and flow cytometry, that CXCR4 was localized in the cytoplasm of P39 cells while it was in the membrane of U937 cells. Since the protein quinase C (PKC'dzeta') is related to the SDF-1/CXCR4 signaling by increasing CXCR4 expression and its membrane availability, we decided to work with cells P39 overexpressing PKC'dzeta' (PKC'dzeta'wt). This procedure resulted in translocation of CXCR4 to the membrane of P39 cells but did not change the CXCR7 subcellular localization. Transwell chemotaxis assay showed that P39 cells overexpressing PKC'dzeta' displayed higher chemotactic ability upon SDF-1 treatment compared with control P39 (35 fold increase pcDNA3-PKC'dzeta'-HA vs pcDNA3-HA transfected P39 cells, p=0.0032; Mann-Whitney), suggesting that PKC'dzeta' restored the chemotactic capacity of P39 cells. Increased expression of CXCR7, as here observed in lymphoblastic leukemia cells, is a phenomenon already described in a variety of solid tumor cell lines such as brain, prostate and lung. In solid tumors, CXCR7 mainly increases the proliferation of malignant cells. These results suggest that the biological function of CXCR7 depends on its tissue and organ localization and that, in acute lymphoblastic leukemia may have a role in cell chemotaxis, potentiating CXCR4 response to SDF-1 and thus, could contribute for leukemia initiating cell recruitment to niches once occupied by normal hematopoietic stem cells. Furthermore, our results lead us to believe that a defect in the PKC'dzeta'/CXCR4 pathway is involved with the unresponsiveness of MDS cells to SDF-1, generating an ineffective hematopoiesis. It confirms data that suggest that PKC'dzeta' is a central protein in the SDF-1/CXCR4 signaling pathway, important for the migration of malignant hematoietic cells / Mestrado / Fisiopatologia Médica / Mestre em Ciências

Le Syndrome WHIM (SW) est un déficit immuno-hématologique rare qui se caractérise notamment par une profonde leucopénie circulante. Le SW résulte principalement de mutations hétérozygotes autosomiques dans CXCR4 qui tronquent partiellement le domaine C-terminal de la protéine et entraînent un défaut de désensibilisation de CXCR4 en réponse à CXCL12. L’impact in vivo d’un gain de fonction de CXCR4 sur le développement et la différenciation lymphocytaires restant à définir, nous avons généré un modèle murin hétérozygote pour la mutation ponctuelle identifiée chez certains patients. L’objectif de ma thèse a été de déterminer si un défaut de différenciation des cellules souches et des progéniteurs hématopoïétiques (CSPH) était à l’origine de ces anomalies de lymphopoïèse. Nous avons observé que le nombre et la clonogénicité des CSH sont préservés dans la moelle osseuse des souris mutantes. Toutefois, les CSH porteuses de la mutation gain de fonction de Cxcr4 ont une quiescence accrue alors que leur multipotence et leur auto-renouvellement sont réduits. Ces dérégulations du compartiment des CSH entraînent, in vivo et in vitro, une altération de la production et de la spécification lymphoïde des progéniteurs multipotents (MPP). En périphérie, nous avons mis en évidence une diminution des CSPH dans le sang des souris mutantes, anomalie également retrouvée à partir de prélèvements sanguins de quatre patients atteints du SW et porteurs d’une mutation hétérozygote dans CXCR4. Ces résultats indiquent une anomalie de circulation des CSPH, hypothèse confortée par le développement d’une hématopoïèse extra-médullaire intra-splénique chez les souris mutantes. Ces données suggèrent que la désensibilisation de Cxcr4 régule la quiescence, la multipotence et l’auto-renouvellement des CSH et serait requise pour la spécification lymphoïde des MPP. Ainsi, l’absence de ce processus pourrait être à l’origine de la lymphopénie observée dans notre modèle murin et, par extrapolation, chez les patients. / The Warts, Hypogammaglobulinemia, Infections and Myelokathexis Syndrome (WS) is a rare immuno-hematological disorder characterized notably by a pan-leukopenia. It is mostly caused by heterozygous autosomal dominant gain-of-function mutations in CXCR4, which engender a distal truncation in the carboxyl-terminal tail domain and lead to a desensitization-resistant receptor. The in vivo impact of a gain-of-CXCR4 function on lymphocyte development and differentiation remain to be established. Then we recently produced major breakthroughs in the pathophysiology of the WS by generating a mouse strain harbouring a WS-linked heterozygous Cxcr4 mutation. The aim of my PhD was to determine if a HSPC differentiation defect would account for the lymphopenia. We showed that the total number and the clonogenicity of HSC were not altered in the BM of mutant mice. However, HSC carrying the gain-of-function mutation in Cxcr4 have an increased quiescence but a decreased multipotency and self-renewal. These HSC compartment deregulations lead to a in vivo and in vitro decline of multipotent progenitors (MPP) production and lymphoid specification. Furthermore, we reported a decrease in the total number of HSPC in the blood of mutant mice, an anomaly also found in four WS patients carrying a heterozygous CXCR4 mutation. This suggested an abnormal HSPC circulation that was strengthened by an enhanced extramedullary haematopoiesis observed in the spleen of mutant mice. These results suggest that Cxcr4 desensitization regulates HSC quiescence, multipotency and self-renewal and is required for MPP lymphoid specification. In this respect, the absence of such process could account for the lymphopenia in this model and likely in patients.

No description available.

La dimérisation des récepteurs couplés aux protéines G est un nouveau concept apparu dans la littérature au cours des quelques années qui ont précédé le début de notre travail. Bien qu’il soit clairement établi que les récepteurs sont capables de former des homo et des hétérodimères, les conséquences fonctionnelles de telles interactions demeurent souvent peu claires. Dans une étude précédente, le laboratoire d’accueil a montré que les récepteurs aux chimiokines CCR2 et CCR5 forment des homo et des hétérodimères de manière constitutive et identifié une coopérativité négative de liaison de nature allostérique entre les deux sites de liaison de CCR2 et CCR5 dans des cellules co-exprimant les deux récepteurs. Dans ce travail, nous avons étendu cette étude au récepteur CXCR4, structurellement plus éloigné que CCR2 et CCR5 entre eux. Nous montrons par une méthode biophysique se basant sur le transfert d’énergie de bioluminescence (le BRET) que CCR2, CCR5 et CXCR4 forment des homodimères et des hétérodimères de manière constitutive. De plus nous démontrons une coopérativité négative de liaison de nature allostérique des deux sites de liaisons pour les hétérodimères CCR2/CXCR4 et CCR5/CXCR4. lorsque CXCR4 est co-exprimé avec CCR2 ou CCR5, la chimiokine spécifique de CXCR4 (SDF-1α) inhibe la liaison du traceur spécifique de CCR2 (MCP-1) ou du traceur spécifique de CCR5 (MIP-1β), et vice-versa. La nature allostérique de ces interactions est démontrée par des expériences mesurant la dissociation de traceurs en présence ou non de compétiteurs. La coopérativité négative de liaison de nature allostérique des deux sites de liaisons est montrée également dans des cellules primaires, excluant tout effet indésirable dû à la surexpression de récepteurs. Nous montrons également que l’antagoniste spécifique de CXCR4 (AMD3100) inhibe la liaison du traceur spécifique de CCR2 (MCP-1) ou du traceur spécifique de CCR5 (MIP-1β), et vice-versa (TAK-779 vs SDF-1α), uniquement quand CXCR4 est co-exprimé respectivement avec CCR2 ou CCR5. Il s’agit là de la première preuve montrant que les interactions allostériques au sein d’hétérodimères de récepteurs aux chimiokines impliquent aussi des antagonistes, et qu’un antagoniste de récepteur aux chimiokines influence la réponse fonctionnelle d’un autre récepteur aux chimiokines auquel il ne se lie pas. De tels effets fonctionnels ont été montré dans des expériences de mobilisation de Ca++, de chimiotactisme sur lymphoblastes et dans des expériences d’air pouch in vivo.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 76-91).

CAVALCANTE, Galyléia Menezes. Estudo da imunoexpressão dos sistemas CXCR4-CXC112/SDF-1, CCR7-CCl21 e KI-67 no carcinoma de células escamosas oral e sua associação com indicadores clínicopatológicos, metástase linfonodal e sobrevida. 2013. 57 f. Dissertação (Mestrado em Odontologia) - Universidade Federal do Ceará. Faculdade de Farmácia, Odontologia e Enfermagem, Fortaleza, 2013. / Submitted by denise santos (denise.santos@ufc.br) on 2014-07-14T13:15:32Z No. of bitstreams: 1 2013_dis_gmcavalcante.pdf: 1293725 bytes, checksum: fa485fabd530db7d0d4df8d64c549656 (MD5) / Approved for entry into archive by denise santos(denise.santos@ufc.br) on 2014-07-14T13:16:38Z (GMT) No. of bitstreams: 1 2013_dis_gmcavalcante.pdf: 1293725 bytes, checksum: fa485fabd530db7d0d4df8d64c549656 (MD5) / Made available in DSpace on 2014-07-14T13:16:38Z (GMT). No. of bitstreams: 1 2013_dis_gmcavalcante.pdf: 1293725 bytes, checksum: fa485fabd530db7d0d4df8d64c549656 (MD5) Previous issue date: 2013 / Chemokines are responsible for the directed migration of leukocyte chemotactic cytokines, coordinating cell movement during inflammation and the transport of hematopoietic cells. In addition to leukocytes, chemokine receptors are also found in neoplastic cells and tumors associated with stromal cells. Among chemokines, and the CXCR4/CXCL12 CCR7/CCL21 systems have been shown the involvement of lymph node metastases or distant metastases in different cancers. Thus, aim of this study was to evaluate the expression of CXCR4, CXCL12, CCR7, CCL21 and Ki-67 in oral squamous cell carcinoma (SCC) and to correlate these markers with clinicopathological indicators, lymph node metastasis and survival. We conducted a survey of reports and paraffin blocks of excisional biopsies of patients with SCC treated at the Hospital Haroldo Juaçaba (2001-2009). Data on anatomic location of the lesion, sex, age, patient survival, degree of histological differentiation of the tumor, tumor stage and presence or absence of lymph node metastasis, lymphovascular and perineural invasion, nuclear grade and depth of invasion were collected. For immunohistochemical analysis, followed by the technique of streptavidin-biotin-peroxidase using the anti-CXCR4, anti-CXCL12, anti-CCR7, anti-CCL21 and Ki-67 antibody. Histological sections were photomicrographed in 10 fields chosen randomly and measured for the number of labeled tumor cells and determined the percentage of each labeling antibody. The marking of CXCR4 was detected in the cytoplasm and nucleus, CXCL12, CCR7 and CCL21 were only cytoplasmic, their expression was observed in 18 (60%) 8 (22.66%) 16 (53.3%) and 3 (12%) cases, respectively. We found a significant positive association between lymphovascular invasion and immunostaining of CXCR4 (p = 0.007) and CCR7 (P = 0.01) and among these cases metastasis was present in 62.5% and 37.5%, respectively. When in combination with Ki67, we found a significant positive correlation between CXCR4 (p = 0.0086), CXCL12 (p = 0.036) and CCR7 (p = 0:04). Among patients CXCR4 + over 111 months, only 38.4% were alive (p = 0.845), whereas both patients CCR7 + (p = 0.398) as well as CXCR4 +, and CCR7 + (p = 0.441) after 62 months, everyone had already died. We conclude that these chemokines are associated with lymphovascular invasion and cell proliferation, perhaps favoring the development of metastasis and poor prognosis. / As quimiocinas são citocinas quimiotáticas responsáveis pela migração direcionada de leucócitos, coordenando o movimento celular durante a inflamação e o transporte de células hematopoiéticas. Além dos leucócitos, os receptores de quimiocinas também são encontrados em células neoplásicas e em tumores associados com células estromais. Dentre as quimiocinas, os sistemas CXCR4/CXCL12 e CCR7/CCL21 têm sido demonstrado no envolvimento de metástases linfonodais ou à distância em diferentes tipos de câncer. Dessa forma, foi objetivo desse trabalho avaliar a expressão de CXCR4, CXCL12, CCR7, CCL21 e Ki-67 em carcinoma de células escamosas orais (CEC) e correlacionar estes marcadores com indicadores clínicopatológicos, metástase linfonodal e sobrevida. Realizou-se um levantamento de laudos e blocos parafinados de biopsias excisionais de pacientes portadores de CEC tratados no Hospital Haroldo Juaçaba (2001 a 2009). Foram coletados dados sobre localização anatômica da lesão, sexo, idade, sobrevida do paciente, grau de diferenciação histopatológica do tumor, estadiamento tumoral e presença ou ausência de metástase linfonodal, invasão linfovascular e perineural, grau nuclear e profundidade de invasão. Para reação de imunohistoquímica, seguiu-se a técnica da estreptavidina-biotina-peroxidase, utilizando os anticorpos anti-CXCR4, anti-CXCL12, anti-CCR7, anti-CCL21 e Ki-67. As secções histológicas foram fotomicrografadas em 10 campos escolhidos aleatoriamente e quantificadas quanto ao número de células tumorais marcadas e determinado o percentual de marcação de cada anticorpo. A marcação de CXCR4 foi detectada em citoplasma e núcleo, CXCL12, CCR7 e CCL21 tiveram marcação apenas citoplasmática, sendo observada suas expressões em 18 (60%), 8 (22,66%), 16 (53,3%) e 3 (12%) casos, respectivamente. Encontrou-se uma associação significativa positiva entre a invasão linfovascular e a imunomarcação do CXCR4 (p=0.007) e CCR7 (p=0.01) e dentre esses casos a metástase esteve presente em 62,5% e 37,5%, respectivamente. Quando em associação com o Ki67, encontrou-se uma correlação positiva significante entre o CXCR4 (p=0.0086), CXCL12 (p=0.036) e CCR7 (p=0.04). Dentre os pacientes CXCR4+, ao longo de 111 meses, apenas 38,4% estavam vivos (p=0.845), ao passo que tanto para pacientes CCR7+ (p = 0.398), quanto CXCR4+ e CCR7+ (p = 0.441), após 62 meses, todos haviam ido a óbito. Conclui-se que essas quimiocinas estão associadas com a invasão linfovascular e proliferação celular, talvez favorecendo o desenvolvimento de metástases e um pior prognóstico.

Submitted by Caroline Falcao (caroline.rfalcao@ufpe.br) on 2017-04-06T17:05:06Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2012-dissertação-EvônioBarrosCampeloJunior.pdf: 1895687 bytes, checksum: 45a651f79dfdcc2ce3328e3201f91911 (MD5) / Made available in DSpace on 2017-04-06T17:05:06Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2012-dissertação-EvônioBarrosCampeloJunior.pdf: 1895687 bytes, checksum: 45a651f79dfdcc2ce3328e3201f91911 (MD5) Previous issue date: 2012-12-27 / Desde 1967, momento do seu surgimento no mundo científico, o GB vírus C (GBV-C) permanece conhecido como vírus indolente, todavia não obstante,pesquisas têm reveladoadespeito da ausência de vinculação do GBV-C com alguma doença, que a associação entre o GBV-C e HIV promove uma progressão mais lenta na doença pelo HIV em indivíduos coinfectados. Essa interaçãoteriacomo mecanismo a competição com o vírus da imunodeficiência humana (HIV) na ligação aos correceptores CCR5 e CXCR4 edessa formatem sidoobjeto de análise com intuito de promover a compreensão da dinâmica imunológica do HIV. Portanto, admitindoaimportância do GBV-C na pandemia de HIV/aids, esse trabalho determinou a prevalência da infecção pelo GBV-Cem pessoas vivendo com HIV/aids acompanhadas noServiço de Doenças Infecciosas e Parasitárias do HC-UFPE, verificou o perfil sócio-demográficoda população, identificou fatores de riscoe fez a associação entre a contagem de linfócitos TCD4 e a infecção peloGBV-C.Representou um estudode corte transversal, analisado como caso-controle, no qual a presença do GBV-C RNA foi investigada em249 pessoas vivendo com HIV/aids, de acordo com a demanda espontânea do ambulatório de Doenças Infecciosas e Parasitárias HC-UFPE,no período de junho adezembro de 2011 e que preenchiam os critérios de inclusão. Foram coletadas amostras de sangue de cada participante, os quais também assinaram um termo de consentimento livre e esclarecido e responderam questionário elaborado especificamente para a pesquisa. Adetecção do RNA do GBV-C foi realizado por PCR (Reação em CadeiadaPolimerase).O resultado da pesquisa indicou uma prevalência de 24,3%, compatível com outros estudos nacionais e internacionais,indicou o perfil sócio-demográfico, apontoua via parenteral como a mais prevalente na infecção pelo GBV-Ceassociou a contagem de linfócitos T CD4 e a infecção peloGBV-C,em pessoas vivendo com HIV/aids na fase inicial da infecção. Também a discussão apontou a necessidade de realização de trabalhos de coorte para melhor compreensão de fatores implicados na transmissão do GBV-C. / Since 1967, the time of its emergence in the scientific world, the GB virus C (GBV-C) virus remains known as indolent, but nevertheless, research has revealed despite the absence of binding of GBV-C with some disease, the association between GBV-C and HIV promotes a slower progression in HIV disease in coinfected individuals. This interaction mechanism would compete with the human immunodeficiency virus (HIV) in binding to CCR5 and CXCR4coreceptorsand thus has been the subject of analysis in order to promote understanding of the dynamics of HIV immune.Therefore, admitting the importance of GBV-C in the HIV/AIDS pandemic, this study estimated the prevalence of GBV-C infection in people living with HIV/AIDS followed in the Service of Infectious and Parasitic HC-UFPE, there the socio-demographic profile of the population, identified risk factors and made the association between CD4 count and GBV-C infection.Represented a cross-sectional study, analyzed as case-control study in which the presence of GBV-C RNA was investigated in 249 people living with HIV/AIDSat the outpatient of Infectious and Parasitic Diseases HC-UFPE, between June and December 2011 and who met the inclusion criteria. Blood samples were collected from each participant, who also signed a consent form and a questionnaire developed specifically for research. Detection of GBV-C RNA was performed by PCR (Polymerase Chain Reaction).The result of the survey indicated a prevalence of 24.3%, consistent with other national and international studies, said the socio-demographic profile, pointed the parenteral route as the most prevalent in the GBV-C infection and associated with CD4 count and GBV-C infection in people living with HIV/AIDS in the early phase of infection. Also the discussion pointed to the need to perform cohort studiesto better understand the factorsinvolvedinthe transmission of GBV-C.

CXCR4 is a chemokine receptor that has been linked to several disease related pathways including: HIV-1 proliferation, autoimmune disorders, inflammatory disease and cancer metastasis. The interaction of the C-X-C chemokine receptor type 4 (CXCR4) with C-X-C chemokine ligand 12 (CXCL12) plays a key role in triggering these disease related pathways. Various antagonists for these receptors have been synthesized and tested, but many are not useful clinically either because of toxicity or poor pharmacokinetics. Some of the most extensive CXCR4 antagonist libraries stem from a class of compounds, p-xylyl-enediamines, which all feature a benzene ring as the core of the compound. This work focuses on the design and synthesis of a new class of compounds that show potential as CXCR4 antagonists by using heterocyclic aromatic rings (2,6-pyridine, 2,5-furan, 2,5-pyrazine and 3,4-thiophene) as the core of the scaffold. After synthesis, these analogues were probed through a variety of assays and techniques by our collaborators in the Shim lab at Emory University including: preliminary binding assays, Matrigel invasion assays, carrageenan mouse paw edema tests, and in silico analysis. In silico analysis also probed 2,5-thiophene-based analogues previously synthesized. This work has produced the beginnings of a new library of CXCR4 antagonists and has identified fifteen hit compounds that are promising leads for further testing and modification.

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PhD - Pathology / The entry stage of the HIV-1 viral life cycle has become a prime target for preventing HIV-1 infection. This has led to the development of a new class of anti-retroviral agents termed entry inhibitors, which have proven effective in vitro and in the clinic. These new agents target three different stages of entry, namely CD4 binding, coreceptor interaction with either CCR5 or CXCR4 and the fusion process. Here we studied isolates from patients with HIV-1 subtype C infection and the effectiveness of different coreceptor and fusion inhibitors in vitro. Further we examined resistance profiles to the first licensed entry inhibitor, T-20. In Chapter 2 we examined coreceptor usage of HIV-1 subtype C isolates and their sensitivity to CCR5 and CXCR4 inhibitors. Twenty-nine viral isolates with different coreceptor usage profiles were isolated from patients with advanced AIDS. The CCR5-specific agents, PRO140 an anti-CCR5 monoclonal antibody and RANTES, the natural ligand for CCR5 inhibited all 24 R5 isolates, while the two X4 and the three R5X4 viruses were sensitive to the CXCR4-specific inhibitor, AMD3100. The five X4 or R5X4 viruses were all able to replicate in peripheral blood mononuclear cells (PBMC) that did not express CCR5 confirming their ability to use CXCR4 on primary cells. When tested using coreceptor-transfected cell lines, one R5 virus was also able to use CXCR6, and another R5X4 virus could use CCR3, Bob/GPR15 and CXCR6. The R5X4 and X4 viruses contained more diverse V3 loop sequences with a higher overall positive charge, compared to the R5 viruses. Hence, HIV-1 subtype C viruses are able to use CCR5, CXCR4 or both for entry, and they are sensitive to specific inhibitors of entry via these coreceptors. In Chapter 3 we analyzed isolates from 10 acutely infected individuals, who were followed longitudinally for up to three years. Two of these individuals (Du151 and Du179) underwent a coreceptor switch and were studied more intensively. The other eight individuals retained CCR5 usage throughout the duration of the study. The initial 4 isolates from Du151 were able to utilize CCR5 but the later isolates were able to use both CCR5 and CXCR4 (R5X4). Du179 used both CCR5 and CXCR4 (R5X4) initially, but the later isolate was found to be monotropic and used CXCR4 (X4) exclusively. Viral isolates were tested for their sensitivity to small molecule inhibitors of CCR5, CXCR4 and the fusion process in a PBMC assay. All of the R5 isolates were sensitive to RANTES and PRO140 and insensitive to the two CXCR4 coreceptor inhibitors AMD3100 and T-140. There was a tendency for later isolates to become slightly less sensitive to the CCR5 inhibitors and more sensitive to the CXCR4 entry inhibitors. None of the R5X4 Du179 isolates were effectively inhibited by PRO140 and RANTES, but the X4 isolate of Du179 became sensitive to CXCR4 entry inhibitors. Both Du151 and Du179 underwent amino acid changes in their V3 sequences that included an increased charge associated with CXCR4 usage. This indicates that coreceptor switching can occur in subtype C infections and is associated with changes in the V3 loop. However, both Du151 and Du179 were subsequently found to be dually infected with another subtype C strain, which may account for some of the phenotypic and genotypic changes seen in these individuals including the appearance of CXCR4-virus variants. In Chapter 4 we explored two HIV-1 isolates (CM4 and CM9) able to use alternate HIV-1 coreceptors for entry (i.e. coreceptors other than CCR5 or CXCR4) on transfected cell lines. These isolates were tested for their sensitivity to inhibitors of HIV-1 entry on primary cells. Both isolates were from patients with cryptococcal meningitis, a severe AIDS defining condition. CM4 was able to use CCR5 and Bob/GPR15 efficiently in transfected cells. This isolate grew in D32/D32 CCR5 PBMC in the presence of AMD3100, indicating that it used a receptor other than CCR5 or CXCR4 on primary cells. It was insensitive to the CCR5 entry inhibitors RANTES and PRO140, but was partially inhibited by vMIP-1, a chemokine that binds CCR3, CCR8, Bob/GPR15 and CXCR6. The coreceptor used by this isolate on primary cells is thus currently unknown. CM9 used CCR5, CXCR4, Bob/GPR15, CXCR6 and CCR3 on transfected cells and was able to replicate in the presence of AMD3100 in D32/D32 CCR5 PBMC. It was insensitive to vMIP-1, eotaxin and I309 used individually, but was inhibited completely when vMIP-1 or I309, the ligand for CCR8, were combined with AMD3100. These results strongly suggest that this isolate can use CCR8 on primary cells. Collectively these data suggest that some HIV-1 isolates can use alternate coreceptors on primary cells, which may have implications for strategies that aim to block viral entry using coreceptor inhibitors. In Chapter 5 we examined the effectiveness of T-20 to inhibit HIV-1 subtype C isolates. T-20 blocks the fusion stage of the viral entry cycle and it is the first entry inhibitor to be licensed for clinical use. T-20 consists of 36 amino acids and was designed based on the HR-2 region of HIV-1 subtype B. A total of 23 HIV-1 subtype C isolates were tested for their ability to replicate in the presence and absence of T-20. This included five isolates with multiple genotypic drug resistance mutations to reverse transcriptase and protease inhibitors. Among the 23 subtype C isolates there were 10-16 amino acid changes in the 36 amino acid region corresponding to T-20. However, all isolates were effectively inhibited by T-20 at 1 mg/ml, including the 5 isolates resistant to other anti-retroviral drugs. The gp41 region was sequenced and the HR-1 and HR-2 amino acids analyzed. All isolates had the amino acids GIV at positions 36-38 in gp41, which are associated with sensitivity to T-20. One X4 had a GVV motif but this did not affect its sensitivity. Thus, T-20 inhibited subtype C viruses despite significant genetic differences in the HR-2 regions of subtypes B and C. These data suggest that T-20 would be highly effective in patients with HIV-1 subtype C infection including those failing existing anti-retroviral drug regimens. In Chapter 6 we examined the in vitro resistance patterns of HIV-1 subtype C to T-20. Resistance to T-20 is a consequence of persistent exposure to the antiretroviral peptide. To establish if patterns of resitance to T-20 were similar to resistance mutations occurring in subtype B viruses, 11 subtype C and 4 subtype B viruses were passaged in the presence of increasing concentrations of T-20. The subtype C isolates showed varying levels of replication at 1 mg/ml T-20 by day 18, but by day 29 all replicated efficiciently at 10 mg/ml T-20. All isolates showed evidence of genotypic changes in gp41 HR-1 following exposure to T-20 that included G36S/E/D, I37T, V38M/A/L/E, N42D, N43K/S, L45R/M and A50T/V. Five viruses had compensatory changes in the HR-2 region, which corresponds to the T-20 sequence, and two isolates had changes in the V3 region. Mutational patterns among the 4 subtype B viruses were similar to those for subtype C and those previously published in the literature. These data indicate that in vitro resistance to T-20 develops rapidly among HIV-1 subtype C isolates. In general, mutational patterns for subtype C were similar to those described for subtype B, suggesting that the mechanism of action for T-20 is similar for HIV-1 subtype B and C isolates. Observations from these studies indicate that HIV-1 subtype C predominantly use the CCR5 coreceptor to enter cells. CXCR4 usage is rare compared to other subtypes, although such isolates are found in patients with advanced AIDS. The two cases of coreceptor switching reported here were dually infected. Subtype C isolates were sensitive to coreceptor and fusion inhibitors except for two isolates able to utilize alternate coreceptors. However, alternate coreceptor usage is very rare and unlikely to impact on the utility of coreceptor inhibitors. Given the propensity for CCR5 usage this may imply that CCR5 coreceptor inhibitors may be more effective in countries where HIV-1 subtype C predominate. Entry inhibitors may be useful for prevention and treatment strategies and have the potential to provide sterilizing immunity. These agents could be used as microbicides and as an adjunct to existing antiretroviral therapies for use in HIV-1 subtype C infected individuals. However resistance to entry inhibitors can emerge and should be used in combination with other antiretrovirals to minimize this outcome. While entry inhibitors provide a new line of defence against HIV-1, their cost may prevent their use in developing countries in the immediate future. Nevertheless, this is the first comprehensive study of the sensitivity of HIV-1 subtype C isolates to entry inhibitors providing a data-driven rationale for their use in individuals infected with HIV-1 subtype C.