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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.

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.

Thesis (Ph. D.)--Cornell University, May, 2007. / Vita. Includes bibliographical references (Leaves 270-320).

A Artrite Idiopática Juvenil (AIJ) abrange um grupo de doenças caracterizadas pela inflamação crônica sinovial com densa infiltração de linfócitos T. É uma das mais prevalentes entre as doenças reumáticas crônicas da infância, podendo acarretar incapacidade motora permanente. Não é conhecido fator causal até o momento. Há evidências da ação imunológica pela confirmação da elevação dos níveis séricos e no líquido sinovial de interleucinas e receptores de interleucinas tais como IL-6 e TNFα. Um receptor que está sendo associado ao estabelecimento do processo inflamatório nas doenças auto-imunes é o receptor de quimiocinas CCR5. As quimiocinas são moléculas pró-inflamatórias que funcionam através da ativação e atração química dos leucócitos. Elas exercem atividade quimiotática sobre neutrófilos, linfócitos e monócitos, tendo papel importante em todas as fases da inflamação. A variante polimórfica CCR5delta32 está descrita como protetora contra infecções por HIV-1 e doenças como Artrite Reumatóide (AR), mas há poucos estudos na AIJ. O objetivo deste estudo foi avaliar a associação da variante polimórfica delta32 do receptor de quimiocinas CCR5 com a Artrite Idiopática Juvenil e com seus subtipos oligoarticular, poliarticular e sistêmico. Trata-se de um estudo caso-controle com 101 pacientes que completaram critérios de AIJ oligoarticular, poliarticular e sistêmico, e 104 indivíduos saudáveis, caucasóides, acima de 16 anos, pareados por sexo. O DNA foi extraído e genotipado para a presença do alelo CCR5delta32 por reação em cadeia da polimerase. O teste de Qui-quadrado foi usado para comparar as diferenças entre as freqüências das variantes alélicas. Entre os 101 pacientes, 80 eram do sexo feminino (79,2%); a média de idade do início da doença foi 4,8 anos + 3,1; a média de tempo de doença foi 5 anos +2,6. A presença do alelo CCR5delta32 foi mais elevada nos pacientes com AIJ (9,4%; P=0,049) e no subtipo sistêmico (25%; P=0,004) em comparação com o grupo controle (4%), enquanto não houve diferença nos subtipos oligoarticular (4%; P=0,757) e poliarticular (10%; P=0,071). Este estudo permitiu concluir que a freqüência do alelo CCR5delta32 foi maior em pacientes com AIJ e em pacientes com o subtipo sistêmico, e semelhante aos controles nos pacientes com os subtipos oligoarticular e poliarticular. Os resultados também sugerem a existência de diferença na susceptibilidade genética e imunopatológica entre os subtipos de AIJ. / The Juvenile Idiopathic Arthritis (JIA) includes a group of diseases characterized by chronic synovial inflammation with dense T lymphocyte infiltration. It is one of the most prevalent among the rheumatic chronic diseases of the childhood, and could lead to permanent motive inability. There is not causal factor identified until the moment. There are evidences of the immunological action through the elevation of cytokine blood serum levels and interleukins receptor such as IL-6 and TNF. A receptor that is being associated to the establishment of the inflammatory process in auto-immune diseases is the chemokine receptor CCR5. Chemokines are pro-inflammatory molecules that act through the activation and chemoattraction of the leucocytes. They exercise chemotatic activity on neutrophyls, lymphocytes and monocytes and develop important functions in all phases of the inflammation. The polymorphic variant CCR5delta32 was described as protective against infections for HIV-1 and diseases as Rheumatoid Arthritis (RA), but there are few studies in JIA. The objective of this study was to evaluate the association of the polymorphic variant delta32 of the chemokine receptor CCR5 and Juvenile Idiopathic Arthritis subtypes: polyarticular, oligoarticular and systemic. It is a case-control study with 101 patients that fulfilled the criteria of oligoarticular, polyarticular and systemic JIA, and 104 healthy, caucasian individuals, above 16 years old, matched by gender. The DNA was extracted and genotyped for the presence of the CCR5delta32 allele by polimerase chain reaction. The Chi-square test was used to compare the differences among the frequencies of the allelic variants. Among the 101 patients, 80 were female (79,2%); the average of age on the beginning of the disease was 4,8 years + 3,1; the average of time of disease was 5 years + 2,6. The largest frequency of the CCR5delta32 allele was statistically significant in the patients with JIA (9,4%; P=0,049) and the subtype systemic arthritis (25%; P=0.004) as compared to the controls, while there was not difference between the oligoarticular (5%; P=0,757) and polyarticular subtypes (10%; P=0.071) and controls (4%). This study allowed concluding that the frequency of the CCR5delta32 allele was higher in patients with JIA and subtype systemic, and similar to the controls in the patients with oligoarticular and polyarticular subtypes. The result also suggests the existence of difference in the genetic immunopathologic susceptibility among the JIA subtypes.

A Artrite Idiopática Juvenil (AIJ) abrange um grupo de doenças caracterizadas pela inflamação crônica sinovial com densa infiltração de linfócitos T. É uma das mais prevalentes entre as doenças reumáticas crônicas da infância, podendo acarretar incapacidade motora permanente. Não é conhecido fator causal até o momento. Há evidências da ação imunológica pela confirmação da elevação dos níveis séricos e no líquido sinovial de interleucinas e receptores de interleucinas tais como IL-6 e TNFα. Um receptor que está sendo associado ao estabelecimento do processo inflamatório nas doenças auto-imunes é o receptor de quimiocinas CCR5. As quimiocinas são moléculas pró-inflamatórias que funcionam através da ativação e atração química dos leucócitos. Elas exercem atividade quimiotática sobre neutrófilos, linfócitos e monócitos, tendo papel importante em todas as fases da inflamação. A variante polimórfica CCR5delta32 está descrita como protetora contra infecções por HIV-1 e doenças como Artrite Reumatóide (AR), mas há poucos estudos na AIJ. O objetivo deste estudo foi avaliar a associação da variante polimórfica delta32 do receptor de quimiocinas CCR5 com a Artrite Idiopática Juvenil e com seus subtipos oligoarticular, poliarticular e sistêmico. Trata-se de um estudo caso-controle com 101 pacientes que completaram critérios de AIJ oligoarticular, poliarticular e sistêmico, e 104 indivíduos saudáveis, caucasóides, acima de 16 anos, pareados por sexo. O DNA foi extraído e genotipado para a presença do alelo CCR5delta32 por reação em cadeia da polimerase. O teste de Qui-quadrado foi usado para comparar as diferenças entre as freqüências das variantes alélicas. Entre os 101 pacientes, 80 eram do sexo feminino (79,2%); a média de idade do início da doença foi 4,8 anos + 3,1; a média de tempo de doença foi 5 anos +2,6. A presença do alelo CCR5delta32 foi mais elevada nos pacientes com AIJ (9,4%; P=0,049) e no subtipo sistêmico (25%; P=0,004) em comparação com o grupo controle (4%), enquanto não houve diferença nos subtipos oligoarticular (4%; P=0,757) e poliarticular (10%; P=0,071). Este estudo permitiu concluir que a freqüência do alelo CCR5delta32 foi maior em pacientes com AIJ e em pacientes com o subtipo sistêmico, e semelhante aos controles nos pacientes com os subtipos oligoarticular e poliarticular. Os resultados também sugerem a existência de diferença na susceptibilidade genética e imunopatológica entre os subtipos de AIJ. / The Juvenile Idiopathic Arthritis (JIA) includes a group of diseases characterized by chronic synovial inflammation with dense T lymphocyte infiltration. It is one of the most prevalent among the rheumatic chronic diseases of the childhood, and could lead to permanent motive inability. There is not causal factor identified until the moment. There are evidences of the immunological action through the elevation of cytokine blood serum levels and interleukins receptor such as IL-6 and TNF. A receptor that is being associated to the establishment of the inflammatory process in auto-immune diseases is the chemokine receptor CCR5. Chemokines are pro-inflammatory molecules that act through the activation and chemoattraction of the leucocytes. They exercise chemotatic activity on neutrophyls, lymphocytes and monocytes and develop important functions in all phases of the inflammation. The polymorphic variant CCR5delta32 was described as protective against infections for HIV-1 and diseases as Rheumatoid Arthritis (RA), but there are few studies in JIA. The objective of this study was to evaluate the association of the polymorphic variant delta32 of the chemokine receptor CCR5 and Juvenile Idiopathic Arthritis subtypes: polyarticular, oligoarticular and systemic. It is a case-control study with 101 patients that fulfilled the criteria of oligoarticular, polyarticular and systemic JIA, and 104 healthy, caucasian individuals, above 16 years old, matched by gender. The DNA was extracted and genotyped for the presence of the CCR5delta32 allele by polimerase chain reaction. The Chi-square test was used to compare the differences among the frequencies of the allelic variants. Among the 101 patients, 80 were female (79,2%); the average of age on the beginning of the disease was 4,8 years + 3,1; the average of time of disease was 5 years + 2,6. The largest frequency of the CCR5delta32 allele was statistically significant in the patients with JIA (9,4%; P=0,049) and the subtype systemic arthritis (25%; P=0.004) as compared to the controls, while there was not difference between the oligoarticular (5%; P=0,757) and polyarticular subtypes (10%; P=0.071) and controls (4%). This study allowed concluding that the frequency of the CCR5delta32 allele was higher in patients with JIA and subtype systemic, and similar to the controls in the patients with oligoarticular and polyarticular subtypes. The result also suggests the existence of difference in the genetic immunopathologic susceptibility among the JIA subtypes.

A Artrite Idiopática Juvenil (AIJ) abrange um grupo de doenças caracterizadas pela inflamação crônica sinovial com densa infiltração de linfócitos T. É uma das mais prevalentes entre as doenças reumáticas crônicas da infância, podendo acarretar incapacidade motora permanente. Não é conhecido fator causal até o momento. Há evidências da ação imunológica pela confirmação da elevação dos níveis séricos e no líquido sinovial de interleucinas e receptores de interleucinas tais como IL-6 e TNFα. Um receptor que está sendo associado ao estabelecimento do processo inflamatório nas doenças auto-imunes é o receptor de quimiocinas CCR5. As quimiocinas são moléculas pró-inflamatórias que funcionam através da ativação e atração química dos leucócitos. Elas exercem atividade quimiotática sobre neutrófilos, linfócitos e monócitos, tendo papel importante em todas as fases da inflamação. A variante polimórfica CCR5delta32 está descrita como protetora contra infecções por HIV-1 e doenças como Artrite Reumatóide (AR), mas há poucos estudos na AIJ. O objetivo deste estudo foi avaliar a associação da variante polimórfica delta32 do receptor de quimiocinas CCR5 com a Artrite Idiopática Juvenil e com seus subtipos oligoarticular, poliarticular e sistêmico. Trata-se de um estudo caso-controle com 101 pacientes que completaram critérios de AIJ oligoarticular, poliarticular e sistêmico, e 104 indivíduos saudáveis, caucasóides, acima de 16 anos, pareados por sexo. O DNA foi extraído e genotipado para a presença do alelo CCR5delta32 por reação em cadeia da polimerase. O teste de Qui-quadrado foi usado para comparar as diferenças entre as freqüências das variantes alélicas. Entre os 101 pacientes, 80 eram do sexo feminino (79,2%); a média de idade do início da doença foi 4,8 anos + 3,1; a média de tempo de doença foi 5 anos +2,6. A presença do alelo CCR5delta32 foi mais elevada nos pacientes com AIJ (9,4%; P=0,049) e no subtipo sistêmico (25%; P=0,004) em comparação com o grupo controle (4%), enquanto não houve diferença nos subtipos oligoarticular (4%; P=0,757) e poliarticular (10%; P=0,071). Este estudo permitiu concluir que a freqüência do alelo CCR5delta32 foi maior em pacientes com AIJ e em pacientes com o subtipo sistêmico, e semelhante aos controles nos pacientes com os subtipos oligoarticular e poliarticular. Os resultados também sugerem a existência de diferença na susceptibilidade genética e imunopatológica entre os subtipos de AIJ. / The Juvenile Idiopathic Arthritis (JIA) includes a group of diseases characterized by chronic synovial inflammation with dense T lymphocyte infiltration. It is one of the most prevalent among the rheumatic chronic diseases of the childhood, and could lead to permanent motive inability. There is not causal factor identified until the moment. There are evidences of the immunological action through the elevation of cytokine blood serum levels and interleukins receptor such as IL-6 and TNF. A receptor that is being associated to the establishment of the inflammatory process in auto-immune diseases is the chemokine receptor CCR5. Chemokines are pro-inflammatory molecules that act through the activation and chemoattraction of the leucocytes. They exercise chemotatic activity on neutrophyls, lymphocytes and monocytes and develop important functions in all phases of the inflammation. The polymorphic variant CCR5delta32 was described as protective against infections for HIV-1 and diseases as Rheumatoid Arthritis (RA), but there are few studies in JIA. The objective of this study was to evaluate the association of the polymorphic variant delta32 of the chemokine receptor CCR5 and Juvenile Idiopathic Arthritis subtypes: polyarticular, oligoarticular and systemic. It is a case-control study with 101 patients that fulfilled the criteria of oligoarticular, polyarticular and systemic JIA, and 104 healthy, caucasian individuals, above 16 years old, matched by gender. The DNA was extracted and genotyped for the presence of the CCR5delta32 allele by polimerase chain reaction. The Chi-square test was used to compare the differences among the frequencies of the allelic variants. Among the 101 patients, 80 were female (79,2%); the average of age on the beginning of the disease was 4,8 years + 3,1; the average of time of disease was 5 years + 2,6. The largest frequency of the CCR5delta32 allele was statistically significant in the patients with JIA (9,4%; P=0,049) and the subtype systemic arthritis (25%; P=0.004) as compared to the controls, while there was not difference between the oligoarticular (5%; P=0,757) and polyarticular subtypes (10%; P=0.071) and controls (4%). This study allowed concluding that the frequency of the CCR5delta32 allele was higher in patients with JIA and subtype systemic, and similar to the controls in the patients with oligoarticular and polyarticular subtypes. The result also suggests the existence of difference in the genetic immunopathologic susceptibility among the JIA subtypes.

No description available.

CCR5 et CXCR4 sont les corécepteurs d'entrée du VIH utilisés par le virus in vivo en plus du récepteur principal CD4 pour infecter les cellules. Au début et tout le long de l'infection, on retrouve chez les patients infectés par le VIH, des virions R5 utilisant le corécepteur CCR5 pour infecter les cellules. Dans les stades tardifs de l'infection et chez environ la moitié des personnes infectées par le VIH, on observe en plus de ces souches R5, l'apparition de souches X4, utilisant le corécepteur CXCR4 pour infecter les cellules. Cette apparition de souches X4 est un facteur d'aggravation de la maladie. Les causes de cette commutation de R5 vers X4 sont mal définies. Le but de mon travail de thèse a été de trouver de nouvelles stratégies thérapeutiques visant l'un ou l'autre de ces corécepteurs. La première partie de mon travail compare les deux isoformes de CXCR4 en tant que corécepteurs du VIH. Ces deux isoformes, CXCR4-A et CXCR4-B, diffèrent de 9 acides aminés en NH2 terminal suite à un épissage alternatif. Nous avons montré que l'isoforme CXCR4-B est la plus performante en tant que corécepteur du VIH mais que ces deux variants sont équivalents pour la migration vers leur ligand commun SDF-1. Ainsi, nous proposons qu'en ciblant exclusivement l'isoforme B qui est la plus favorable à l'infection, via par exemple des siRNA, il serait possible de limiter les infections par des souches X4 tout en gardant une partie des fonctions essentielles de ce récepteur dans l'organisme, assurées par l'isoforme A. Nos résultats suggèrent également que l'infection par des souches R5 augmente le ratio en ARNm CXCR4-B / CXCR4-A dans des PBMC, et que ce ratio est en partie responsable de la commutation de R5 vers X4 associée à une aggravation de la maladie. Cibler cette isoforme CXCR4-B pourrait donc se révéler bénéfique. La deuxième partie de cette thèse étudie la modulation de la fonction de corécepteur du VIH de CCR5 par S1P1, un autre membre de la famille des récepteurs couplés aux protéines G qui permet la remise en circulation des lymphocytes après leur séjour dans les organes lymphoïdes secondaires par chimiotactisme vers son ligand S1P, abondant dans le sang. Nous montrons que S1P1 interagit physiquement avec CCR5 et gêne l'entrée des virus R5 dans la cellule-hôte. A l'inverse, S1P1 active les étapes post-entrée du cycle viral, notamment l'expression génique virale. La résultante de ces effets opposés est une augmentation de la production virale par des cellules infectées in vitro. Ce projet a également montré que l'utilisation de FTY720, un antagoniste fonctionnel de S1P1, diminue l'infection de cellules dendritiques par des virus HIV-R5 in vitro, ainsi que la virémie dans un modèle de souris SCID infectées après reconstitution immunologique. La mise en évidence des interactions entre CCR5 et S1P1 ouvre donc des perspectives thérapeutiques. / CCR5 and CXCR4 are the two HIV entry coreceptors used by the virus in addition to the main receptor CD4 in vivo to infect cells. R5 virions, that use CCR5 as a coreceptor to infect cells, are detected in most HIV patients. At late stages of infection and in about half of HIV infected persons, there is an emergence of X4 virions that use CXCR4 as a coreceptor, in addition to R5 virions. This emergence is associated with an increase in disease progression. The reasons for this R5 to X4 switch are poorly understood. The goal of my PhD work was to find new therapeutic strategies that target these coreceptors.The first part of this work compares the two CXCR4 isoforms as HIV coreceptors. Those two isoforms, CXCR4-A and CXCR4-B, differ by 9 amino acids at their NH2 terminal extremity as a consequence of an alternative splicing. We have shown that CXCR4-B isoform is more efficient as an HIV coreceptor but that those two variants are equivalent in terms of chemotaxis toward their common ligand SDF-1. Thus, we propose that by targeting specifically the B isoform that supports infection, via siRNA by example, it is possible to limit X4 development while keeping essential functions of this receptor. Our results also suggest that R5 infection increases CXCR4-B / CXCR4-A mRNA ratio in PBMC and that this ratio is in part responsible for R5 to X4 switch. Thus, targeting CXCR4-B isoform could be beneficial.The second part of this PhD thesis studies the effect on CCR5 coreceptor function of S1P1, another G protein-coupled receptor that enables lymphocytes egress from lymph nodes by chemotaxis toward its ligand S1P that is abundant in blood. We have shown that S1P1 physically interacts with CCR5 and blocks R5 virus entry. On the other hand, S1P1 activates post-entry steps of the viral cycle, in particular gene expression. The resulting effect is an increase in viral production by infected cells in vitro. We also showed that the use of FTY720, a S1P1 functional antagonist, decreases dendritic cell infection by R5 viruses in vitro, and in vivo infection in a SCID mouse model. The emphasis of CCR5 and S1P1 interactions opens new therapeutic strategies.

De nombreuses études des RCPGs révèlent que leur activation n’implique pas que deux états conformationnels, l’un activé et l’autre inactivé, mais une diversité plus importante de ces états, impliquant également des états intermédiaires. Nous avons utilisé la simulation par dynamique moléculaire et l’analyse des interactions intramoléculaires non-covalentes pour étudier la plasticité structurale du récepteur CCR5 sous sa forme monomérique et dimérique. En couplant notre analyse avec diverses données expérimentales, nous avons pu proposer trois architectures dimériques du récepteur et associer des mouvements et des interactions clefs aux états conformationnels de CCR5 libre, lié à un agoniste, lié à un agoniste inverse et constitutivement activé ou inactivé par mutation d’acides aminés. Nous avons également développé une méthode d’identification des motifs d’interactions intramoléculaires transmembranaires, permettant de discriminer les états d’activations des RCPGs. / Many studies reveal that GPCR activation does not simply involve two conformational states, one activated and the other inactivated, but a variety of these states coupled to intermediate states. By using molecular dynamics simulations and analyzing non-covalent intramolecular interactions, we studied the structural plasticity of monomeric and dimeric CCR5. By coupling our analysis with various experimental data, we identified three dimeric organizations states of the receptor and associated key motions and intramolecular interactions to free CCR5, bound to an agonist, bound to an inverse agonist and constitutively activated or inactivated by mutated residues. We have also developed a method to identify intramolecular transmembrane interactions patterns, which allow the discrimination of GPCRs activation states.