Made available in DSpace on 2014-06-12T18:04:10Z (GMT). No. of bitstreams: 2 arquivo3738_1.pdf: 896817 bytes, checksum: 7cd3fad40aa580f879fae31cec4ad007 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2008 / O gene ccr5 codifica o receptor b-quimiocina 5 (CCR5), uma proteína transmembrânica que age como principal co-receptor para os vírus HIV-1, Variola major e para a bactéria Yersinia pestis, nos macrófagos e monócitos humanos. Uma deleção de 32 pares de bases neste gene dá origem ao alelo mutante ccr532 cuja presença, em homozigose, tem sido relatada em indivíduos resistentes à AIDS. A mutação ccr532 tem uma origem recente e se deu na Europa, e atinge suas maiores freqüências nas populações do norte (16% na Finlândia). Ocorrências isoladas foram descritas no resto do globo, entretanto resultariam de fluxo gênico recente para essas populações. Segundo informação verbal popular, Triunfo não se constitui numa cidade atrativa para imigrações e apresentaria certo grau de consangüinidade entre os seus 14 mil habitantes, por isso esta população tornou-se objeto de estudos das freqüências dos alelos ccr5 e ccr532 para que se pudesse determinar se essas freqüências divergem ou não das encontradas nos demais Estados Nordestinos. Foram analisados 345 indivíduos não aparentados desta população, e após extração por mini salting-out o DNA genômico foi amplificado por PCR e a partir da eletroforese por PAGE a 5% suas bandas foram visualizadas por impregnação com AgNO3. As freqüências genotípicas observadas foram 89,28% (ccr5/ccr5), 10,72% (ccr5/$32) e 0,0% ($32/$32). As freqüências alélicas foram 94,64% para o ccr5 e 5,36% para o ccr5Q32. A população encontrase em equilíbrio de Hardy-Weinberg (p= 0,61). A freqüência um pouco elevada do ccr532 encontrada na população de Triunfo pode ser resultado da ocorrência de efeito fundador nessa cidade, ou de um processo de deriva genética

Genetic variability within both the HIV-1 coreceptor, CCR5, and its ligand, CCL3L, has been shown to contribute towards differences between individuals in their susceptibility to HIV-1 infection and rate of disease progression. In this study we investigated the extent of genetic variation within the CCR5 gene as well as CCL3L, CCL3La and CCL3Lb gene copy number distribution in two healthy HIV-1 uninfected South African populations, South African Africans (SAA) and South African Caucasians (SAC). The impact of variations within these genes on the expression of CCR5 and CCL3 was subsequently assessed. Furthermore, CCR5 genetic variability, CCL3L gene copy number distribution and the expression of CCR5 and CCL3, was assessed in a similar way in a small cohort of HIV-1 infected long term nonprogressors (LTNPs). Genotyping of the CCR5 gene in SAA (n=41) and SAC (n=46) HIV-1 uninfected individuals revealed a high degree of genetic variation between the two population groups, both in terms of single nucleotide polymorphism (SNP) profiles and CCR5 haplotype distribution. Seven complex putative haplotypes spanning the length of the sequenced region were identified with only one of the identified haplotypes, SAA/C-HHC, common to both study populations. The effect of genetic variability on promoter activity of four different CCR5 promoter regions for three CCR5 haplotypes,SAA-HHA, SAA/C-HHC and SAC-HHE, were evaluated. Results showed variability in (i) promoter activity between different promoter regions tested, (ii) results obtained with different cells used for analysis, and (iii) the haplotype being analysed, thereby highlighting that both the cellular environment as well as genetic variability within the promoter region, have the capacity to influence the efficiency of a promoter and consequently CCR5 expression levels. Haplotype-specific promoter analysis demonstrated the SAA-HHA haplotype to have the strongest promoter activity in THP-1 and K562 cells for both P1A (downstream) and P2 (upstream) promoter regions, while in the other cell lines tested (Jurkat and U937), HHA demonstrated intermediate promoter strength.Differences seen between the haplotypes tested in this study and other published studies may be attributable to additional SNPs being tested in the promoter constructs used in this study. The two population groups differed significantly with regards to cell activation levels, as measured by HLA-DR expression, in CD4+ T cell (P=0.002) and CD56+ NK cell subsets (P<0.001). CCR5 expression, determined both as the number of CCR5 molecules per cell (density) and the percentage of CCR5-expressing cells, was found to differ between SAA and SAC individuals across all peripheral blood cell types. SAA individuals had larger proportions of CCR5-expressing natural killer (NK) cell subsets (P<0.01) but lower CCR5 molecules per cell density on CCR5+CD8+ T cell and CCR5+ NK cell subsets (CD56+, CD16+CD56+ and CD56dim) (all P<0.05) compared to SAC individuals. These differences were maintained even after CCR3D32 heterozygous SAC individuals were included in the analyses. Furthermore, the previously described haplotypes, HHA and HHC, associated with differences in CCR5 expression on different cell subsets between individuals within the same population group. SAA individuals with the HHA haplotype had significantly lower percentages of CCR5-expressing CD8+ T cells compared to SAA individuals that lacked the haplotype (P=0.001). SAC individuals with the HHC haplotype had significantly higher density on NK (CD56+) and CD16+CD56+ NK cell subsets (P=0.030 and P=0.024, respectively) compared to SAC individuals without this haplotype. The latter observation suggests that the protective effect of the HHC haplotype in Caucasians might be explained by higher density of CCR5 expression on NK cells that is not evident in HHC+ SAA individuals, thus highlighting the potential role of CCR5-expressing cells other than CD4+ T cells in protection from HIV-1 acquisition and disease progression. Despite significant differences in CCL3La (CCL3L chemokine coding) and CCL3Lb (nonchemokine coding) copy number between SAA and SAC populations, no difference in CCL3 production by peripheral blood mononuclear cells (PBMCs) was noted between the two study populations. Assuming equal contribution of CCL3 and each copy of CCL3La to CCL3 production,we found that SAC individuals produced higher levels of CCL3 per functional copy of CCL3La compared to SAA individuals (P<0.001). Although, when SAA and SAC individuals with comparable CCL3La and CCL3Lb gene copy numbers were compared, there was no difference in production per functional copy between the two groups (P=0.974). We also determined CCL3La and CCL3Lb gene copy number for a previously established cohort of HIV-1 intrapartum-infected (IP) and exposed uninfected (EU) infants and found that differences previously seen in cord blood mononuclear cell (CBMC) CCL3 production between IP and EU infants with comparable CCL3L copy numbers could not be attributed to differences in CCL3Lb copy number. The potential role of differences in CCR5 genotype, CCR5 expression, CCL3 genotypes and CCL3 production levels in the control of HIV-1 infection was then examined by comparing a small group (10 SAA and 4 SAC) of LTNPs to the respective background population. No polymorphisms in the CCR5 open reading frame were detected in these LTNP individuals. However, the HHA haplotype frequency was significantly higher in SAC LTNP individuals compared to SAC control individuals (P=0.010). Interestingly, CCR5 density on CD4+ T cells and monocytes was significantly lower in SAA LTNP individuals (P=0.025 and P=0.022, respectively) with a trend towards a similar relationship in CD8+ T cells (P=0.058), while the proportions of CCR5-expressing CD8+ T cells were elevated compared to SAA controls (P=0.043). This latter finding reflects the increased immune activation in these individuals compared to uninfected individuals, as evidenced by increased proportions of HLA-DR-expressing T cells (CD8+ and CD4+, P<0.0001). In addition,PHA-induced CCL3 production by PBMCs was significantly lower in LTNP (SAA and SAC combined) compared to control individuals (P=0.004). SAA LTNP individuals had higher proportions of CD8+ T cells (P<0.0001) and lower proportions of natural killer cells (CD56+,P=0.002) compared to control SAA individuals. Thus, CCL3 production differences may be partially explained by differences in the distribution of immune cell subsets between the two study groups.Furthermore, PBMCs of LTNP individuals with low viral loads (<400 copies/ml) produced CCL3 at lower levels than those from individuals with higher viral loads, irrespective of whether or not the cells were stimulated (P=0.005 and P=0.035, respectively). In summary, this study demonstrates that: (i) two ethnically divergent populations show marked differences in CCR5 genetic variability, cell activation and CCR5 expression which are likely to impact on both susceptibility to HIV-1 infection and the rate of HIV-1 disease progression, (ii) both CCR5 genotypic differences and differences in baseline cellular activation levels appear to be contributing towards the observed differences in CCR5 protein expression, and (iii) the two study populations do not differ with respect to CCL3 production by PBMC cultures which suggests that either the two copy per diploid genome gene, CCL3, may play a significant role in CCL3 production and/or that as yet undefined mechanisms regulate production of CCL3 from variable CCL3L copy number. In addition, a pilot study conducted in a small group of LTNP individuals demonstrates that two major determinants of HIV-1 disease progression, CCR5 and CCL3, are both expressed at lower levels in LTNPs individuals compared to healthy uninfected controls and has identified CCR5 haplotypes which are potentially associated with disease progression.

A sepse é uma resposta inflamatória sistêmica resultante da inabilidade do sistema imune em controlar uma infecção, onde a taxa de sobrevida está associada ao recrutamento de neutrófilos para o local da infecção. Tem sido demonstrado que a expressão de receptores quimiotáticos pode ser alterada durante a sepse. Neutrófilos de animais naives respondem às quimiocinas CXC, mas são irresponsivos às quimiocinas CC. Entretanto, dados do nosso laboratório mostram que a expressão de CXCR2 é reduzida na sepse, prejudicando a migração de neutrófilos para o foco da infecção. Além disso, ocorre o aparecimento do receptor CCR2 nos neutrófilos, levando à infiltração dessas células no pulmão e outros órgãos. Nesse contexto, o nosso objetivo foi investigar a possível expressão do receptor CCR5 em neutrófilos e seu papel na evolução da sepse. Demostramos que animais sham-operados expressam baixos níveis de CCR5 e altos níveis de CXCR2. Entretanto, sob a condição de sepse experimental induzida por ligação e perfuração do ceco (CLP), neutrófilos circulantes e que migraram para a cavidade peritoneal expressam altos níveis de CCR5 em paralelo com a internalização de CXCR2. Além disso, animais deficientes para CCR5 (CCR5-/-), submetidos à CLP, apresentam diminuição na taxa de sobrevida, redução na migração de neutrófilos para o foco da infecção, aumento da disseminação bacteriana, aumento no infiltrado de neutrófilos no pulmão e aumento nos níveis de marcadores de lesão do coração e rim, quando comparados com animais selvagens (WT). Adicionalmente, a incubação de neutrófilos isolados da medula óssea com LPS aumentou a expressão de CCR5 e os tornou responsivos à MIP-1? (ligante de CCR5), induzindo quimiotaxia. Também demonstramos que o receptor CCR5 possui importante papel durante a adesão de neutrófilos ao endotélio vascular para posterior migração. Em conjunto, esses resultados indicam que durante a CLP, o aumento da expressão de CCR5 em neutrófilos tem um papel protetor, visto que animais CCR5-/- sépticos apresentam reduzida migração de neutrófilos para o foco infeccioso, inflamação sistêmica acentuada e baixa taxa de sobrevivência. / Sepsis is a systemic inflammatory response resulted from the inability of the innate immune system to control infections, being the survival rate associated to the recruitment of neutrophils to the infection site. It has been demonstrated that chemokine receptors expression profile can be altered under sepsis conditions. Neutrophils from naïve mice respond to CXC chemokines, but are usually unresponsive to CC chemokines. However, data from our laboratory show that CXCR2 expression is down regulated, impairing the neutrophil migration to infection focus. In addition, CCR2 appears on the surface of neutrophils, mediating the accumulation of these cells in the lung and other organs. In this context, we aimed to investigate the possible expression of CCR5 receptor on neutrophils and its role on sepsis evolution. We showed that neutrophils from sham mice express high levels of CXCR2 and low levels of CCR5. However, during experimental sepsis, induced by cecal ligation and puncture (CLP), in parallel with CXCR2 internalization, neutrophils from the circulation or from the peritoneal cavity express higher levels of CCR5. Interestingly, deficient mice for the CCR5 receptor (CCR5-/-), undergone to CLP show decreased survival rate, reduction in the neutrophil migration to the site of infection, increase in the numbers of bacteria, increase in the neutrophil infiltration in lung and heart and increase in the levels of markers of injuries in heart and kidney, when compared to wild type mice (WT).In addition, the incubation of bone marrow derivedneutrophils with LPS enhances the expression of CCR5 and renders them responsive to CCL4 (a ligant of CCR5)-induced chemotaxis. Moreover, we demonstrated that CCR5 receptor has an important role during neutrophil adhesion to the vascular endothelium before transmigration. Together, these results indicate that during CLP-induced sepsis, the increase of the expression of CCR5 on neutrophils plays a host protective role, since CCR5-/- mice under sepsis present reduced neutrophil migration to infection focus, high systemic inflammation and low survival rate.

A sepse é uma resposta inflamatória sistêmica resultante da inabilidade do sistema imune em controlar uma infecção, onde a taxa de sobrevida está associada ao recrutamento de neutrófilos para o local da infecção. Tem sido demonstrado que a expressão de receptores quimiotáticos pode ser alterada durante a sepse. Neutrófilos de animais naives respondem às quimiocinas CXC, mas são irresponsivos às quimiocinas CC. Entretanto, dados do nosso laboratório mostram que a expressão de CXCR2 é reduzida na sepse, prejudicando a migração de neutrófilos para o foco da infecção. Além disso, ocorre o aparecimento do receptor CCR2 nos neutrófilos, levando à infiltração dessas células no pulmão e outros órgãos. Nesse contexto, o nosso objetivo foi investigar a possível expressão do receptor CCR5 em neutrófilos e seu papel na evolução da sepse. Demostramos que animais sham-operados expressam baixos níveis de CCR5 e altos níveis de CXCR2. Entretanto, sob a condição de sepse experimental induzida por ligação e perfuração do ceco (CLP), neutrófilos circulantes e que migraram para a cavidade peritoneal expressam altos níveis de CCR5 em paralelo com a internalização de CXCR2. Além disso, animais deficientes para CCR5 (CCR5-/-), submetidos à CLP, apresentam diminuição na taxa de sobrevida, redução na migração de neutrófilos para o foco da infecção, aumento da disseminação bacteriana, aumento no infiltrado de neutrófilos no pulmão e aumento nos níveis de marcadores de lesão do coração e rim, quando comparados com animais selvagens (WT). Adicionalmente, a incubação de neutrófilos isolados da medula óssea com LPS aumentou a expressão de CCR5 e os tornou responsivos à MIP-1? (ligante de CCR5), induzindo quimiotaxia. Também demonstramos que o receptor CCR5 possui importante papel durante a adesão de neutrófilos ao endotélio vascular para posterior migração. Em conjunto, esses resultados indicam que durante a CLP, o aumento da expressão de CCR5 em neutrófilos tem um papel protetor, visto que animais CCR5-/- sépticos apresentam reduzida migração de neutrófilos para o foco infeccioso, inflamação sistêmica acentuada e baixa taxa de sobrevivência. / Sepsis is a systemic inflammatory response resulted from the inability of the innate immune system to control infections, being the survival rate associated to the recruitment of neutrophils to the infection site. It has been demonstrated that chemokine receptors expression profile can be altered under sepsis conditions. Neutrophils from naïve mice respond to CXC chemokines, but are usually unresponsive to CC chemokines. However, data from our laboratory show that CXCR2 expression is down regulated, impairing the neutrophil migration to infection focus. In addition, CCR2 appears on the surface of neutrophils, mediating the accumulation of these cells in the lung and other organs. In this context, we aimed to investigate the possible expression of CCR5 receptor on neutrophils and its role on sepsis evolution. We showed that neutrophils from sham mice express high levels of CXCR2 and low levels of CCR5. However, during experimental sepsis, induced by cecal ligation and puncture (CLP), in parallel with CXCR2 internalization, neutrophils from the circulation or from the peritoneal cavity express higher levels of CCR5. Interestingly, deficient mice for the CCR5 receptor (CCR5-/-), undergone to CLP show decreased survival rate, reduction in the neutrophil migration to the site of infection, increase in the numbers of bacteria, increase in the neutrophil infiltration in lung and heart and increase in the levels of markers of injuries in heart and kidney, when compared to wild type mice (WT).In addition, the incubation of bone marrow derivedneutrophils with LPS enhances the expression of CCR5 and renders them responsive to CCL4 (a ligant of CCR5)-induced chemotaxis. Moreover, we demonstrated that CCR5 receptor has an important role during neutrophil adhesion to the vascular endothelium before transmigration. Together, these results indicate that during CLP-induced sepsis, the increase of the expression of CCR5 on neutrophils plays a host protective role, since CCR5-/- mice under sepsis present reduced neutrophil migration to infection focus, high systemic inflammation and low survival rate.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-07-25T20:46:06Z No. of bitstreams: 1 Giselle Calasans Investigação de mutacçoes nos genes....pdf: 17299730 bytes, checksum: b9919577a31cf080e622e852dc432578 (MD5) / Made available in DSpace on 2012-07-25T20:46:06Z (GMT). No. of bitstreams: 1 Giselle Calasans Investigação de mutacçoes nos genes....pdf: 17299730 bytes, checksum: b9919577a31cf080e622e852dc432578 (MD5) Previous issue date: 2012 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / O HIV-1 é o agente etiológico da AIDS. Sabe-se que fatores virais e do hospedeiro podem influenciar na susceptibilidade à infecção pelo vírus e na progressão para a AIDS. Em relação aos fatores intrínsecos ao hospedeiro, tem sido demonstrado que algumas alterações na CCR5 podem afetar sua ligação com a gp120 do HIV-1, influenciando na infecção pelo vírus. Além disso, a proteína Langerina, encontrada na superfície das Células de Langerhans (LCs), apresenta um papel importante em relação à infecção pelo HIV-1, por ter a capacidade de se ligar à gp120 viral através de seu Domínio de Reconhecimento de Carboidrato (CRD). Esta interação permite que as LCs internalizem o vírus em Grânulos de Birbeck, os quais degradam a partícula viral, inibindo a apresentação do HIV-1 para os linfócitos T. Desta forma, diferenças na função da langerina, devido a mutações no promotor do gene Langerina e na região codificante do CRD, por exemplo, podem influenciar a susceptibilidade à infecção pelo HIV-1. Sendo assim, o objetivo principal deste trabalho foi verificar a existência de mutações nas regiões do gene CCR5 que codificam para os domínios N e Cterminal da proteína, no promotor do gene da Langerina e na região codificante do CRD, bem como verificar a existência de possíveis associações entre as mutações encontradas e a infecção pelo HIV-1. Para tal, através de sequenciamento, foi analisado um total de 128 amostras de DNA de indivíduos infectados pelo HIV-1 de Feira de Santana, Bahia e 197 amostras de DNA de indivíduos não-infectados de Salvador, Bahia. Os possíveis sítios de ligação para fatores de transcrição da região promotora do gene da Langerina foram analisados pela ferramenta MatInspector implementada no software Genomatix. Análises físico-químicas, de domínios protéicos potenciais, de predição da estrutura proteica secundária e de modelagem tridimensional proteica foram também realizadas, utilizando diferentes ferramentas de bioinformática. Os estudos na região N-terminal da CCR5 revelaram a existência de uma mutação de sentido trocado no aminoácido 55 (p.L55Q) apenas em indivíduos não-infectados, com uma frequência do alelo mutante de 1,8%. Análises físico-químicas demonstraram que esta mutação aumentou a flexibilidade e a acessibilidade da CCR5 e a modelagem protéica demonstrou que a mutação levou a um pequeno desvio para a direita, bem como alterou levemente a carga eletrostática dessa região da proteína. Foi observada diferença estatisticamente significante entre as frequências alélicas (p=0,039) e genotípicas (p=0,038) da mutação p.L55Q quando os indivíduos infectados e não-infectados foram comparados. Os estudos na região C-terminal da CCR5 demonstraram a existência de três mutações silenciosas em indivíduos infectados pelo HIV- 1: c.3,765C>T, c.3,777A>T e c.3,831A>G. Em relação à análise da região promotora do gene da Langerina, foram observadas três mutações (-577T>C, -517T>C e -160T>C) que, segundo o MatInspector, criaram novos sítios de ligação para fatores de transcrição, tais como: NFAT5, HOXB9.01 e STAT6.01. Entretanto, comparando as frequências alélicas e genotípicas dessas mutações entre os indivíduos infectados e não-infectados, não foi observada diferença estatisticamente significante. Já as análises realizadas na região gênica que codifica o CRD da Langerina demonstraram a existência de três mutações: p.K313I (c.937T>A), c.941C>T (g.4728C>T) e c.983C>T (g.4770C>T) As análises físico-químicas revelaram que a mutação p.K313I aumentou a hidrofobicidade e as hélices transmembranares e diminuiu a hidrofilicidade, a acessibilidade e a antigenicidade da proteína. Entretanto, a presença do alelo mutante não alterou a predição da estrutura secundária da Langerina e não foi observada diferença estatisticamente significante nas frequências alélicas e genotípicas quando os dois grupos estudados foram comparados. Estes resultados sugerem que a mutação p.L55Q, encontrada no domínio N-terminal da CCR5, pode afetar a entrada do HIV-1 na célula. Foi possível, também, observar que as mutações encontradas no gene da Langerina não apresentam associação com a infecção pelo HIV-1. No entanto, é importante que novos estudos sejam realizados com o intuito de compreender melhor o verdadeiro papel da mutação p.L55Q na infecção pelo HIV-1, assim como, novas análises voltadas para a busca de variações no gene da Langerina também devam ser conduzidas, uma vez que este é o primeiro estudo que investiga mutações na Langerina em indivíduos infectados pelo HIV-1. / HIV-1 is the etiologic agent of AIDS. It has been demonstrated that the mechanisms underlying HIV-1 infection and pathogenesis require a combination of viral and host factors. Concerned to the host intrinsic factors, some mutations at CCR5 human gene can affect the interaction between CCR5 protein and HIV-1 gp120, influencing the virus infection. Besides, the Langerin protein, located exclusively on Langerhans cells surface, has an important role in HIV-1 infection due to its ability of binding to gp120 through its Carbohydrate Recognition Domain (CRD). This interaction ables HIV-1 internalization into Birbeck granules, which degrade the virus and prevent LC infection and viral dissemination. So, differences in Langerin function due to mutations at promoter and CRD encoding regions of the human Langerin gene, for example, might influence the susceptibility to HIV-1 infection. Thus, the main purpose of this study is to investigate the existence of mutations at the regions of CCR5 gene that encodes the N- and C-terminal protein domains and at promoter and CRD encoding regions of the human Langerin gene, and finally to stablish possible associations among the observed mutations and HIV-1 infection. Using DNA sequencing, it was studied a total of 128 DNA samples of HIV-1 infected individuals from Feira de Santana, Bahia and 197 DNA samples of HIV-1 non-infected individuals from Salvador, Bahia. The transcription factors binding sites were analyzed using the MatInspector tool implemented in the Genomatix software. Physico-chemical, potential protein domain, prediction of protein secondary structure and protein modeling analyses were also performed, using Bioinformatic tools. The studies into the N-terminal protein domain revealed a new missense mutation at aminoacid 55 (p.L55Q), only in HIV-1 non-infected individuals, with allelic frequency of 1.8%. Physico-chemical analysis revealed that this mutation magnified the flexibility and accessibility profiles and the modeling of CCR5 structures showed that this mutation resulted in a small deviation to the right, as well as, a hydrophobic to hydrophilic property alteration. When HIV-1 infected and non-infected groups were compared, the allelic and genotypic frequencies of the p.L55Q mutation were statistically significant (p=0.039 and 0.038, respectively). Three novel silent mutations were found at encoding region of C-terminal protein domain in the HIV-1 infected individuals: c.3,765C>T, c.3,777A>T and c.3,831A>G. Concerned to the analyses at promoter Langerin region, the studies revealed three mutations: -577T>C, -517T>C and -160T>C. The search for possible transcription factors binding sites using MatInspector demonstrated that these promoter mutations created new binding sites to some transcription factors, such as NFAT5, HOXB9.01 and STAT6.01. However, when HIV- 1 infected and non-infected groups were compared, the allelic and genotypic frequencies of the analyzed promoter sites were not statistically significant. It was observed three mutations at Langerin gene region that encodes to the protein CRD: p.K313I (c.937T>A), c.941C>T (g.4728C>T) and c.983C>T (g.4770C>T). The physico-chemical analysis revealed that the p.K313I polymorphism magnified the hydropathy and transmembrane profiles and reduced the hydrophilicity, accessibility and anitigenicity profiles. However, this mutation did not modify the protein secondary structure prediction and when HIV-1 infected and non-infected individuals were compared, it was not observed a statistically significant difference in the allelic and genotypic frequencies from the p.K313I polymorphism. These results suggest that the p.L55Q mutation can affect HIV-1 infection through CCR5 entry. It was also observed that the mutations detected at the promoter and CRD encoding regions of the human Langerin gene are not associated with HIV-1 infection susceptibility. However, it is important to accomplish future studies in order to better understand the role of the p.L55Q mutation at HIV-1 infection, as well as, conduct new search for variations at Langerin gene that could influence HIV-1 infection, since this is the first study that analyzes mutations at promoter and encoding regions of Langerin gene in a HIV-1 infected population.

Infection with the human immunodeficiency virus (HIV) constitutes a global pandemic, and South Africa forms part of the region known to house over two-thirds of HIV infected individuals worldwide. In the early stages of infection, the C-C chemokine receptor type five (CCR5) is the major HIV-1 co-receptor. The importance of this receptor in HIV infection and disease progression was recognised with the discovery of the CCR5 delta 32 (Δ32) allele. Individuals homozygous for this mutation lack functional CCR5 receptors. Consequently, they are almost completely resistant to HIV infection, while the absence of CCR5 has minimal effects on health. Heterozygous individuals display decreased cell surface CCR5 which slows disease progression. Phenotypic expression of CCR5 is heterogeneous and its relation to genetic mutations in the CCR5 gene is not currently known for the South African population. This together with the effect of CCR5 expression on HIV infection provided the rationale for investigating both the phenotypic and genotypic distribution of CCR5. The aim of this study was therefore 1) to investigate CCR5 phenotypic expression on cluster differentiation four (CD4) T-lymphocytes in a group of South African individuals and 2) to analyse the genetic variation in a South African cohort. Flow cytometric methods were used to measure the phenotypic distribution of CCR5 in 245 individuals by assessing both the percentage of CD4+CCR5+ T-cells and CCR5 density. Sixty five individuals, mostly found within the lower CCR5 receptor density range were selected for DNA sequencing. The study found considerable variability in CCR5 expression with South African individuals expressing relatively high CD4+CCR5+ T-cell percentages. Ethnicity was established as a significant variable affecting CCR5 expression with Black African individuals displaying higher (p <0.05) CD4+CCR5+ T-cell percentages and densities than Caucasians. Genotypic data revealed 70 single nucleotide polymorphisms (SNPs), four insertions and the ∆32 deletion. Results showed that Black African individuals have greater genetic diversity with 39 mutations exclusive to this group. The ∆32 mutation was not detected in the Black African group but was identified in the Caucasian group at a frequency of 18.6 %. Twelve novel mutations were identified in this study with two in the open reading frame (ORF). It is evident from the data that the variability in CCR5 phenotypic expression is difficult to correlate with specific mutations in the gene. This thesis provides information on CCR5 distribution and diversity in the South African population which will be of value to patients, clinicians and health policy officials. / Dissertation (MSc)--University of Pretoria, 2011. / Immunology / Unrestricted

Malgré de nombreux progrès thérapeutiques, la seule réelle option thérapeutique des malades atteints d’une cirrhose terminale ou d’hépatite aiguë fulminante est la transplantation hépatique, cependant limitée par la pénurie d’organes. De même, la prise en charge des pathologies pancréatiques aigues et chroniques consiste essentiellement en traitements supportifs et des complications. La réaction inflammatoire au cours des maladies hépatiques et pancréatiques joue un rôle majeur dans l’évolution de ces maladies car elle influence la sévérité de l’affection aiguë et se complique fréquemment de fibrose et de cirrhose. Les chimiokines constituent une famille de peptides possédant des propriétés chimiotactiques et activatrices sur les leucocytes, et de ce fait jouent un rôle primordial dans la réaction inflammatoire en recrutant des cellules inflammatoires vers un site lésé. Les chimiokines exercent leurs activités en se liant à une famille de récepteurs à 7 hélices transmembranaires situés sur les leucocytes. Le CCR5 est un récepteur pour les chimiokines CCL3 (MIP-1α), CCL4 (MIP-1β), CCL5 (RANTES) et CCL8 (MCP-2). Le CCR5 joue un rôle important de corécepteur dans l’infection par le virus de l’immunodéficience humaine. Chez l’humain, il existe une mutation relativement fréquente du CCR5, appelée CCR5Δ32, qui confère chez les patients homozygotes pour la mutation une protection presque complète contre l’infection par le virus de l’immunodéficience humaine. Plus récemment, la mutation CCR5Δ32 a été rapportée comme étant associée à certaines maladies hépatiques et des traitements expérimentaux chez l’homme par inhibiteurs du CCR5 ont entraînés des cas d’hépatotoxicité sévère. De même, il a été rapporté que l’expression pancréatique de CCR5 était augmentée chez les patients atteints de pancréatite chronique. Cependant, le rôle du récepteur CCR5 et de ses ligands dans la pathogénie des maladies hépatiques et pancréatiques n’est pas connu. Dans un premier temps, nous avons démontré dans un modèle expérimental d’hépatite médiée par les lymphocytes T qu’il existe une production hépatique de CCL3, CCL4 et CCL5 au cours de la maladie et que le foie des souris malades est caractérisé par une infiltration accrue de cellules CCR5+. En utilisant des souris CCR5-déficientes, nous avons ensuite montré que l’absence de CCR5 est associée à une maladie plus sévère, à une production accrue de cytokines pro-inflammatoires et des chimiokines liant le CCR5 (CCL3, CCL4 et CCL5), ainsi que par un recrutement plus important de cellules inflammatoires, particulièrement des cellules CCR1+. Nous avons ensuite montré que la production accrue des ligands du CCR5 joue un rôle important dans l’exacerbation de la maladie observée chez les souris CCR5-déficientes, puisque leur neutralisation réduit fortement la sévérité de la maladie ainsi que le recrutement hépatique de cellules inflammatoires. Dans un second temps, nous avons étudié l’expression et le rôle du CCR5 et de ses ligands dans un modèle murin de pancréatite aiguë sécrétagogue, induite par des injections répétées d’un analogue de la cholécystokinine. Précocément après l’induction de la maladie, nous avons observé une augmentation de l’expression de CCL2 (MCP-1), CCL3 et CCL4 alors que l’augmentation d’expression de CCL5 est observée plus tardivement au cours de la maladie. Nous avons ensuite montré que les souris CCR5-déficientes développent une pancréatite plus sévère, ainsi qu’une production accrue de CCL2, CCL3 et CCL4, et un infiltrat inflammatoire plus marqué que les souris ‘’wild-type’’. Nous avons également montré que la production accrue de ces chimiokines joue un rôle dans l’exacerbation de la pancréatite aiguë chez les souris CCR5-déficientes. En effet, la neutralisation simultanée de ces chimiokines par des anticorps monoclonaux réduit significativement la sévérité de la maladie pancréatique chez ces souris. De même, la neutralisation simultanée des ligands du CCR5 chez des souris wild-type réduit également la sévérité de la pancréatite aiguë, suggérant un rôle de ces molécules dans la pathogénie de la pancréatite aiguë. En conclusion, nous avons montré que l’absence du récepteur CCR5 augmente la susceptibilité aux maladies inflammatoires hépatiques et pancréatiques expérimentales. Le développement d’inhibiteurs du CCR5 dans l’arsenal thérapeutique contre le virus de l’immunodéficience humaine devra tenir compte de ces données, d’autant plus que des cas d’hépatotoxicité sévère ont été récemment rapportés avec certains inhibiteurs en développement et que l’association du virus de l’immunodéficience humaine avec la présence de maladies hépatiques est fréquente. Enfin, ces travaux ouvrent de nouveaux champs d’investigation au niveau de l’étude d’association du CCR5Δ32 avec les maladies inflammatoires pancréatiques et hépatiques, et des perspectives thérapeutiques ciblant CCL3, CCL4 et CCL5.

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.

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