Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-07-30T18:02:27Z No. of bitstreams: 1 Marcelo Magalhaes Silva Estudo de alterações genéticas....pdf: 2168493 bytes, checksum: 48c50f165408a4314aa5ad8de3c72ca0 (MD5) / Made available in DSpace on 2012-07-30T18:02:27Z (GMT). No. of bitstreams: 1 Marcelo Magalhaes Silva Estudo de alterações genéticas....pdf: 2168493 bytes, checksum: 48c50f165408a4314aa5ad8de3c72ca0 (MD5) Previous issue date: 2012 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / A leucemia/linfoma de células T do adulto (ATL) é uma severa doença linfoproliferativa de células T CD4+ associada ao HTLV-1. Por apresentar diferentes manifestações clínicas, essa neoplasia pode ser classificada em cinco formas: aguda, crônica, smoldering, linfomatosa e tumoral primária de pele. Embora alguns trabalhos venham estudando o processo oncogênico mediado pelo HTLV-1, diversos fatores responsáveis pelo desenvolvimento da ATL ainda permanecem desconhecidos. Este estudo teve como objetivo a investigação de alterações genéticas em células ATL (mutações pontuais em genes supressores de tumor e alterações microssatélites) e sua associação com a evolução clínica da doença e sobrevida dos pacientes. A presença de mutações pontuais nos supressores de tumor TP53, p15INK4B e p16INK4A foram avaliadas em 31 pacientes com diferentes formas da ATL (16 agudos, dez crônicos e cinco smoldering) por análise de seqüenciamento de DNA. Cinco pacientes (16%) apresentaram mutações pontuais no gene TP53, sendo que quatro dentre os mesmos foram classificados com a forma aguda. A presença de mutações nos genes avaliados foi associada com pior prognóstico em pacientes com a forma aguda. Em um dos pacientes incluídos neste trabalho (forma aguda) foi verificada a presença de alteração no éxon 2 do gene p16INK4A. Mutações pontuais não foram detectadas no gene p15INK4B em nenhum dos pacientes incluídos. Os marcadores D10S190, D10S191, D11S1391 e D18S21 foram utilizados para a análise de alterações microssatélites por metodologia semi-automatizada. Dentre os 25 pacientes ATL avaliados (seis agudos, oito crônicos, dez smoldering e um linfomatoso), sete apresentaram alterações microssatélites. Três desses pacientes apresentaram instabilidade (MSI), três pacientes apresentaram perda de heterozigosidade (LOH) e em um paciente foi verificado ambas as alterações. Na Bahia, mutações pontuais em TP53 foram detectadas principalmente na forma aguda da ATL e parece estar associada com pior prognóstico. Além disso, de acordo com nossos conhecimentos, este é o primeiro estudo a descrever tanto MSI com LOH em pacientes portando formas crônica e smoldering da ATL. / Adult T-cell leukemia/lymphoma (ATL) is a severe CD4+ lymphoproliferative disease associated to human T-cell lymphotropic virus type 1 (HTLV-1). ATL has different clinical manifestations and is classified in five clinical forms: acute, chronic, smoldering, lymphoma and primary cutaneous tumoral. Although the mechanisms of oncogenesis of the HTLV-1 have been investigated, the factors related to ATL development are still unknown. The goal of this study was to investigate genetic alterations in ATL cells (point mutations in tumor suppressor genes and microsatellite alterations) and their association with the clinical evolution of the disease and survival. The presence of point mutations in TP53, p15INK4B e p16INK4A were evaluated in 31 ATL patients (16 acute, ten chronic and five smoldering) by direct sequencing. Five of them (16%) had TP53 point mutations, four of them with the acute form of ATL. The presence of point mutations in this gene was associated to poor prognosis in acute patients. Only one case (acute form) has an alteration in the exon 2 of the p16INK4A gene. No point mutations of the p15INK4B were found in the patients included. The markers D10S190, D10S191, D11S1391 and D18S21 were considered for the microsatellite analysis using a semiautomated technique. From the twenty-five ATL cases included (six acute, eight chronic, ten smoldering and one lymphoma), seven showed microsatellite alteration. Among them, three patients had microsatellite instability (MSI), three had loss of heterozygosity and one patient presented both alterations. In Bahia, point mutations in TP53 were detected mainly in acute form of ATL and were associated to poor prognosis. The presence of MSI and LOH in the smoldering and chronic forms of ATL were demonstrated for the first time in this study.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2015-07-23T16:56:03Z No. of bitstreams: 1 Igor Ives Santos Fraga Associação... 2014.pdf: 1141300 bytes, checksum: 79bef8840226ef6a639a9c8c9e118161 (MD5) / Approved for entry into archive by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2015-07-23T16:56:29Z (GMT) No. of bitstreams: 1 Igor Ives Santos Fraga Associação... 2014.pdf: 1141300 bytes, checksum: 79bef8840226ef6a639a9c8c9e118161 (MD5) / Made available in DSpace on 2015-07-23T16:56:29Z (GMT). No. of bitstreams: 1 Igor Ives Santos Fraga Associação... 2014.pdf: 1141300 bytes, checksum: 79bef8840226ef6a639a9c8c9e118161 (MD5) Previous issue date: 2014 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / O controle da carga proviral do HTLV-1 depende em parte da lise de células infectadas por células citotóxicas mediada pelos linfócitos T CD8⁺ e pelas células NK (Natural killer). A família de receptores KIR (killer-cell immunoglobulin-like receptor) interage com as moléculas de HLA de classe I, principalmente os alelos do HLA C do grupo 1 (C*01, C*03, C*07, C*08, C*12, C*13, C*14 e C*16), ativando ou inibindo a função destas células.O objetivo do presente estudo foi avaliar se os genes KIR2DL2/KIR2DL3 e os alelos de HLA-C do grupo 1 estão associados ao controle da carga proviral do HTLV-1 e ao diagnóstico de HAM/TSP. O estudo foi realizado no Centro de HTLV da Escola Bahiana de Medicina e Saúde Púbica, em Salvador-Bahia. A presença dos genes KIR2DL2 e KIR2DL3 foi determinada por PCR em tempo real (Syber Green). Foram incluídos 248 indivíduos infectados pelo HTLV-1 (161 assintomáticos e 87 com HAM/TSP) cujos alelos de HLA de classe I haviam sido previamente determinados. A carga proviral (quantificada por PCR em tempo real) e as frequências de indivíduos assintomáticos e com diagnóstico de HAM/TSP (Possível, Provável e Definido) foram comparadas de acordo com a presença ou ausência dos genes KIR avaliados. As frequências dos genes KIR2DL2 e KIR2DL3 foi 84,3% e 96,8%, respectivamente. Não foram observadas diferenças estatisticamente significantes na frequência de indivíduos que possuíam os genes (KIR2DL2 ou KIR2DL3) nos grupos clínicos, assim como na frequência de indivíduos que tinham simultaneamente os genes KIR e os alelos de HLA-C do grupo 1. Os indivíduos do grupo HAM/TSP possível que apresentavam o gene KIR2DL2 tinham menor carga proviral (2,9% de células infectadas) que os indivíduos sem este gene (19,2% de células infectadas) (p<0,001). Quando avaliamos a combinação da presença do gene KIR2DL2 com os alelos de HLA-C do grupo 1, menor carga proviral (2,1%) foi observada nos indivíduos que apresentavam algum dos alelos de HLA-C do grupo 1,comparados aqueles que portavam apenas KIR2DL2 (5,0%) (p=0,013). Menor carga proviral também foi observada nos indivíduos assintomáticos que portavam simultaneamente o gene KIR2DL2 e o alelo HLA-C*07, comparados aos indivíduos com apenas o gene KIR2DL2 (p=0,03), enquanto que os indivíduos com HAM/TSP-PB que tinham essa combinação (KIR2DL2/HLA-C*07) apresentaram tendência de menor carga proviral (p=0,051). Em conclusão, a presença da combinação do gene KIR2DL2 e de algum alelo de HLA-C do grupo 1 está associada ao controle da carga proviral. Este estudo quantificou pela primeira vez as frequências de genes KIR em uma coorte de indivíduos infectados pelo HTLV-1 do estado da Bahia. Estudos futuros são necessários para confirmar estes achados em outras populações e avaliar o valor prognóstico da associação de KIR2DL2 e HLA-C do grupo 1. / The control of proviral load of HTLV-1 depends in part of the lysis of infected cells mediated by cytotoxic CD8⁺T lymphocytes and NK (Natural killer) cells. The family of KIR (killer-cell immunoglobulin-like receptor) interacts with HLA class I molecules, especially those HLA-C alleles in-group 1 (C*01, C*03, C*07, C*08, C*12, C*13, C*14 and C*16) by activating or inhibiting the function of these cells. The aim of this study was to evaluate if the KIR2DL2, KIR2DL3 genes and group 1 HLA-C alleles are associated with the control of proviral load of HTLV-1 and the diagnosis of HAM/TSP. The study was performed at Bahiana School HTLV Center of Medicine and Health Public, in Salvador, Bahia. The presence of KIR2DL2 and KIR2DL3 genes was determined by real-time PCR (Syber Green). The study included 248 subjects infected with HTLV-1(161 and 87 asymptomatic with HAM/TSP) whose HLA class I alleles were previously determined. The proviral load (quantified by real-time PCR) and the frequency asymptomatic individuals diagnosed with HAM/TSP (possibly, probably and definitive) were compared according to the presence or absence of KIR genes evaluated. The frequencies of KIR2DL2 and KIR2DL3 genes were 84.3% and 96.8%, respectively. No statistically significant differences were observed in the frequency of individuals who possessed the genes (KIR2DL2 or KIR2DL3) in clinical groups, as well as the frequency of individuals who had both the KIR genes and HLA-C alleles group 1. Individuals in the group HAM/TSP possible to KIR2DL2 showed that the gene had lower proviral load (2.9%of cells infected) individuals without this gene (19.2% infected cells) (p<0.001). When we evaluated the combination of the presence of KIR2DL2 and 2DL3 genes with HLA-C genes in group 1, lower proviral load (2.1%) was observed in individuals with any of the alleles of HLA-C group 1, compared who those which harbored only KIR2DL2 (5.0%) (p=0.013) .Minor proviral load was also observed in asymptomatic individuals which carried both the KIR2DL2 gene and HLA-C*07 allele when compared to individuals with only KIR2DL2 gene (p=0.03), whereas patients with HAM/TSP-PB that had this combination (KIR2DL2/HLA-C*07) tended to lower proviral load(p=0.051). In conclusion, the presence of the combination of KIR2DL2 gene and a HLA-C group1allele is associated with proviral load control. This study quantified for the first time the frequencies of KIR genes in a cohort of individuals infected with HTLV-1 in Bahia. Future studies are needed to confirm these findings in other populations and to evaluate the prognostic value of KIR2DL2 association and HLA-C group 1.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2015-12-30T14:45:37Z No. of bitstreams: 1 Regina Santos Nascimento Avaliação de celulas...2015.pdf: 3386534 bytes, checksum: 28a4bcbab904a339ca7717124724aa89 (MD5) / Approved for entry into archive by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2015-12-30T14:45:53Z (GMT) No. of bitstreams: 1 Regina Santos Nascimento Avaliação de celulas...2015.pdf: 3386534 bytes, checksum: 28a4bcbab904a339ca7717124724aa89 (MD5) / Made available in DSpace on 2015-12-30T14:45:53Z (GMT). No. of bitstreams: 1 Regina Santos Nascimento Avaliação de celulas...2015.pdf: 3386534 bytes, checksum: 28a4bcbab904a339ca7717124724aa89 (MD5) Previous issue date: 2015 / Fundação Oswaldo Cruz, Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / O HTLV-1 é o agente etiológico da leucemia /linfoma de células T do adulto (ATLL), da paraparesia espástica tropical/ mielopatia associada ao HTLV-1 (HAM/TSP) e da uveíte. Além destas, a ceratoconjutivite seca (CCS), doença multifatorial da lágrima e da superfície ocular, tem sido descrita com maior frequência em indivíduos infectados pelo HTLV-1. Assim como em outras doenças associadas, a CCS tem sido relacionada a uma elevada carga proviral. As células T regulatórias (Treg) são importantes na manutenção da homeostase do sistema imunológico e um comprometimento da imunorregulação exercido por elas pode contribuir para o ambiente inflamatório observado na CCS. Este estudo objetivou avaliar os linfócitos Treg de pacientes com CCS associada à infecção pelo HTLV-1. Foram realizados ensaios de imunofenotipagem por citometria de fluxo para avaliar a frequência de linfócitos T ativados (HLA-DR+) e de células T CD4+ e CD8+ regulatórios (FOXP3+), bem como a produção de IL-10 e TGF-β por estas células. Foram avaliados 37 pacientes infectados pelo HTLV-1 e assintomáticos para HAM/TSP, sendo 27 com diagnóstico positivo para a manifestação ocular (CCS), 10 com diagnóstico negativo (ASS), além de 17 voluntários não infectados pelo vírus (NI). As frequências de linfócitos T CD4+FOXP3+, CD8+FOXP3+, CD4+HLA-DR+ e CD8+HLA-DR+ foram significativamente maiores nos grupos CCS e ASS, quando comparados aos indivíduos não infectados. Quanto à produção das citocinas imunossupressoras, foi observada uma maior frequência de linfócitos T CD4+FOXP3+ duplo produtores de IL-10 e TGF-β no grupo CCS quando comparado ao grupo ASS. Com relação aos linfócitos CD8+FOXP3+, o grupo CCS apresentou uma maior frequência de células mono produtoras de IL-10 quando comparado ao ASS. Nossos resultados sugerem que a menor frequência de células Treg CD8+ produtoras de TGF-β em indivíduos infectados pelo HTLV-1 com CCS, pode contribuir para a intensificação da ativação celular e fisiopatologia da doença. / HTLV-1 is the causative agent of leukemia/lymphoma adult T-cell (ATLL), tropical spastic paraparesis / myelopathy associated with HTLV-1 (HAM / TSP) and uveitis. In addition, keratoconjunctivitis sicca (KCS), a multifactorial disease of the tear and of the ocular surface, has been more frequently reported in patients infected with HTLV-1. As for other HTLV-1-associated diseases, KCS has been related to a high proviral load. Regulatory T (Treg) cells are important in maintaining the homeostasis of the immune system. An impairment in the immunoregulation function of Treg may contribute to the inflammatory environment observed in the KCS. This study aimed to evaluate the Treg cells of patients with KCS associated with HTLV-1. Frequency of activated T cells (HLA-DR+) and CD4+ and CD8+ Treg cells (FOXP3+), as well as IL-10 and TGF-β production by Treg were quantified using flow cytometry. Thirty-seven HTLV-1 individuals were included (27 asymptomatic for HAM/TSP with positive diagnosis of ocular manifestation (KCS), 10 with negative diagnosis (ASS - asymptomatic). Seventeen non-infected individuals were included as controls (NI). The frequencies of CD4+ FOXP3+ T cells, CD8+FOXP3+, CD4+HLA-DR+ and CD8+HLA-DR+ were significantly higher in KCS and ASS groups when compared to non-infected individuals. As the production of immunosuppressive cytokines, a higher frequency of CD4+ FOXP3+ double producers of IL-10 and TGF-β in the KCS group was observed when compared to group ASS. Regarding the CD8+FOXP3+ lymphocytes, the KCS group had a higher frequency of mono cells producing IL-10 when compared to the ASS. Our results suggest that the lower frequency of Treg cells CD8+ TGF-β-producing in individuals infected with HTLV-1 with KCS, may contribute to the intensification of cellular activation and pathophysiology of the disease.

Introdução: Vírus Linfotrópico de células T Humanas tipo 1 (HTLV-1) é o agente etiológico da leucemia/linfoma de células T do adulto (ATLL), mielopatia associada ao HTLV-1/paraparesia espástica tropical (HAM/TSP), dermatite infectiva associada ao HTLV-1 (DIH) entre outras doenças, sendo que algumas delas podem ocorrer em associação. Sabe-se que a infecção pelo HTLV-1 pode aumentar o risco de desenvolvimento de doenças de pele. Este estudo avaliou a prevalência das doenças cutâneas em infectados pelo HTLV-1 independente do estado clínico e a relação entre a contagem de células T CD4+ e CD8+, carga proviral inicial de HTLV-1 e linfoproliferação em grupo de indivíduos portadores assintomáticos da infecção pelo HTLV-1 ou com HAM/TSP, com ou sem doenças de pele associada. Método: 193 indivíduos infectados pelo HTLV-1 do ambulatório de HTLV no Instituto de Infectologia Emílio Ribas foram avaliados e submetidos à entrevista, exame dermatológico completo e contagens de células T CD4+ e CD8+; carga proviral de HTLV-1 e linfoproliferação (LPA) no sangue periférico. Resultados: Cento e quarenta e sete pacientes mostraram-se com alguma condição anormal da pele, destes, 116 (79%) apresentaram doença dermatológica associada ao HTLV-1 (DD-HTLV-1) (xerose/ictiose, dermatite seborreica ou dermatite infectiva associada ao HTLV-1), e 21% (n=31) tiveram outros diagnósticos dermatológicos. As DD-HTLV-1 mais prevalentes foram: xerose/ictiose adquirida (49%) e dermatite seborreica (27%). Três pacientes apresentaram DIH de início durante a vida adulta associada à HAM/TSP. Os pacientes com DD-HTLV-1 eram mais velhos (51 vs 47 anos), apresentaram maior prevalência de HAM/TSP (p=0.015), maior carga proviral do HTLV-1 (p=0.009) e linfoproliferação basal de três dias aumentada (p=0.008). As contagens de células T CD4+ e CD8+ não mostraram diferença estatística quando comparados os grupos com ou sem DD-HTLV-1. Quando os pacientes com HAM/TSP foram excluídos da análise, a carga proviral do HTLV-1 continuou mostrando diferença significativa (p=0,021), enquanto LPA, contagem de células T CD4+ e CD8+ não mostraram nenhuma diferença. Conclusões: Houve uma alta prevalência de doenças de pele (76%) entre indivíduos infectados pelo HTLV-1, independente do estado clínico. Carga proviral inicial do HTLV-1 e idade foram maiores em indivíduos com DD-HTLV-1, mas LPA apresentou aumento apenas em indivíduos com DD-HTLV-1 e HAM / TSP / Background: Human T cell lymphotropic virus type 1 (HTLV-1) is the aetiologic agent of adult T-cell leukaemia/lymphoma (ATLL), HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP), infective dermatitis associated with HTLV-1 (IDH) and various other clinical conditions. Several of these diseases can occur in association. HTLV-1 infection can increase the risk of developing skin disorders. This study evaluated the prevalence of skin diseases among HTLV-1 infected and the relation between HTLV-1 proviral load, lymphocyte proliferation and CD4+ and CD8+ T cells count among HTLV-1 infected individuals, regardless of clinical status, with or without associated skin disorders. Methods: A total of 193 HTLV-1-infected subjects from the HTLV outpatient clinic at the Emilio Ribas Institute were studied. Patients underwent an interview, a complete dermatological examination, a lymphocyte proliferation assay (LPA), an assay for HTLV-1 proviral load and CD4+ and CD8+ T cells count. Results: A total of 147patients had an abnormal skin condition; 116 (79%) of these patients also had skin disorder associated with HTLV-1 infection (SD-HTLV-1) (xerosis/ichthyosis or seborrheic dermatitis), and 21% (n=31) of the patients had other dermatological diagnoses. The most prevalent SD-HTLV-1 was xerosis/acquired ichthyosis (49%), followed by seborrheic dermatitis (27%). Three of them had the association of adult onset IDH and HAM/TSP. The patients with SD-HTLV-1 were older (51 vs. 47 years), had a higher prevalence of myelopathy/tropical spastic paraparesis (HAM/TSP) (p=0.015), higher HTLV-1 proviral load (p=0.009) and had an increased 3-days basal LPA compared with patients without SD-HTLV-1 (p=0.008). The T CD4+ and CD8+ cells counts did not show significance when compared to SD-HTLV-1 group or individuals without SD-HTLV-1. When HAM/TSP patients were excluded from the analysis, the HTLV-1 proviral load showed a significant difference (p=0.021), while LPA showed no difference, such as T CD4+ and CD8+ cells counts. Conclusions: There was a high prevalence of skin disorders (76%) among HTLV-1-infected individuals, regardless of clinical status Initial HTLV-1 proviral load and age was higher in SD-HTLV-1 individuals, but the LPA showed an increase only in SD-HTLV-1 subjects with HAM/TSP

O vírus linfotrópico de células T humanas (HTLV-1) foi o primeiro retrovírus descrito e está etiologicamente ligado a duas principais doenças: a leucemia/linfoma de célula T do adulto (ATLL) e a mielopatia associada ao HTLV-1/paraparesia espástica tropical (HAM/TSP). Apenas 0,3 a 5% dos indivíduos infectados desenvolvem essas doenças associadas, enquanto a maioria permanece assintomática. A HAM/TSP é uma manifestação inflamatória do sistema nervoso central e o mecanismo pelo qual o HTLV-1 induz o surgimento de HAM/TSP ainda não está totalmente esclarecido. Atualmente, uma abordagem promissora no entendimento de mecanismos, bem como na fisiopatogênese das infecções virais tem sido a avaliação da função de microRNAs (miRNAs). Há poucos dados na literatura envolvendo estas moléculas na infecção pelo HTLV-1 em linfócitos T CD4+ bem como no estabelecimento da doença HAM/TSP. No presente estudo, foi avaliada a expressão de miRNAs dos linfócitos T CD4+ isolados de portadores sem HAM/TSP (HAC), pacientes HAM/TSP e indivíduos sadios (CT) por meio de PCR em tempo real. A análise do perfil de expressão dos miRNAs nessas células revelou que 56 e 10 miRNAs apresentavamse mais 1,5 vezes aumentados no grupo HAM/TSP e HAC, respectivamente. O miR- 125b-1-1 apresentou expressão significamente maior no grupo HAC e o miR-146a, no grupo HAM/TSP. A análise in silico de predição de alvo demonstrou que o gene IFNG era potencialmente alvo do miR-125b-1-1 e os genes IRAK1 e TRAF6 do miR- 146a. Foi demonstrado que a expressão do IFNG no grupo HAC era 1,3 vezes mais elevado que o grupo CT e 1,8 vezes mais elevado no grupo HAM que no grupo CT. Houve um aumento na expressão de TRAF6 de 15,7 e 1,5 vezes nos grupos HAM/TSP e HAC, respectivamente. Não foi observada diferença na expressão de IRAK1 entre os três grupos. O ensaios de superexpressão do miR-125b-1-1 alterou a expressão do IFNG e do miR-146a alterou a expressão do gene IRAK1 e sua proteína. Os resultados evidenciados neste trabalho ressaltam a importância dos miRNAs na modulação de genes e proteínas importantes durante a infeção pelo HTLV-1. A correlação entre o miR-125b-1-1 e gene IFNG sugere que este miRNA esteja envolvido nos mecanismos de desenvolvimento de HAM/TSP. Além disso, a interação entre o miR-146a e os genes IRAK1 e TRAF6 sugerem que este miRNA esteja relacionado a mecanismos de persistência viral da infecção pelo HTLV-1 em linfócitos T CD4+. / Human T-cell lymphotropic vírus type 1 (HTLV-1) was the first human retrovirus discovered and it is related with two major diseases: adult T cell lymphoma/leukaemia (ATLL) and HTLV-1 -associated myelopathy/tropical spastic paraparesis (HAM/TS). About 0.3 to 5% of infected individuals will develop HTLV-1 related diseases, while the majority will remain life-long asymptomatic carriers of the virus. HAM/TSP is an inflammatory manifestation of central nervous system and the mechanism involved in HAM/TSP development is noy well elucidated. Currently, a promising approach on understanding the mechanisms as well as physiopathogenesis of viral infections has been the evaluation of the role of microRNAs (miRNAs) roles. There are few data involving CD4+ T cells miRNA expression in HTLV-1 infection as well as HAM/TSP establishment. To identify miRNAs differentially expressed in CD4+ T cells among non-infected individuals (CT), asymptomatic (HAC) and HAM/TSP patients we applied quantitative real time PCR. The analysis of miRNA expression profile in these cells showed 56 and 10 miRNAs upregulated 1.5 times in HAM/TSP and HAC groups, respectively. miR- 125b-1-1 was upregulated in HAC group and miR-146a in HAM/TSP. In silico analysis of target prediction showed that IFNG was a potentially miR-125b-1-1 target and IRAK1 and TRAF6 were miR-146a targets. IFNG expression was 1.3 higher in HAC than CT group and 1.8 higher in HAM/TSP than CT group. It was observed that TRAF6 expression was 15.7 and 1.5 times higher in HAM/TSP and HAC groups, respectively. There was no difference of IRAK1 expression among the three groups. Overexpression assays of miR-125b-1-1 altered IFNG expression and overexpression of miR-146a altered IRAK1 gene and protein expression. The results revealed that miRNAs modulate genes and proteins during HTLV-1 infection. miR- 125b-1-1 and IFNG gene correlation suggests that miR-125b-1-1 seems to contribute to HAM/TSP development. Besides, miR-146a and IRAK1 and TRAF6 interaction suggests that miT-146a seems to contribute to HTLV-1 establishment in CD4+ T cells.

As características das células estromais mesenquimais multipotentes (MSC) podem ser influenciadas em microambientes inflamatórios. No entanto, o comportamento das MSC frente às infecções virais e a exata contribuição da infecção para disfunção das MSC continuam a ser elucidadas. Neste trabalho, avaliamos o efeito imunossupressor de MSC em linfócitos T infectados pelo HTLV-1 e a susceptibilidade da MSC à infecção por este retrovírus. Os ensaios de co-cultivo utilizando MSC e linhagens de linfócitos infectados pelo HTLV-1 resultaram em diminuição na expressão do gene viral tax e do antígeno p19 do HTLV-1. A redução na expressão do gene tax e da proteína p19 foi relacionada com a maior secreção de IL-6 e aumento na expressão dos genes PGE2, IDO e VCAM-1. Para confirmar a influência da imunorregulação das MSC sobre linfócitos T infectados, comparamos a proliferação de linfócitos T isolados de indivíduos infectados pelo HTLV-1 e indivíduos controles cultivados na presença de MSC. Foi observado que as MSC inibem a linfoproliferação de forma similar em amostras controle e na infecção pelo HTLV-1; e este efeito foi mediado pela expressão de PGE2 e IDO. Além disso, a expressão do gene pol e da proteína p19 do HTLV-1 foi menor após o co-cultivo com MSC, indicando que a imunorregulação pelas MSC também atua nas células infectadas pelo HTLV-1. Em seguida, para investigar as alterações provocadas pelo HTLV-1 nas MSC, realizamos análises morfológicas e ultraestruturais em MSC expostas ao HTLV-1 in vitro. Os resultados revelaram que o HTLV-1 induziu o aparecimento de vesículas intracelulares e a expressão das moléculas de superfície VCAM-1, ICAM-1 e HLA-DR. Os níveis de VCAM-1 e HLA-DR também foram mais expressos em MSC cultivadas na presença de PBMC isoladas de indivíduos HAM/TSP. O HTLV-1 não alterou o processo de diferenciação das MSC em osteócitos e adipócitos. No entanto, o contato direto in vitro das MSC com células infectadas pelo HTLV-1 proporcionou uma eficiente infecção das MSC. As partículas virais isentas de células não foram capazes de causar a infecção em MSC. Por fim, para certificar a existência biológica de MSC infectadas pelo HTLV-1, avaliamos a medula óssea de seis indivíduos acometidos por esta infecção. Foi observado um infiltrado de linfócitos T CD4+ na medula óssea de indivíduos HTLV-1+ e a análise do DNA proviral revelou a presença do provírus integrado nessas células T CD4+. O número de unidades formadoras de colônia fibroblastóide (CFU-F) foi menor em indivíduos infectados pelo HTLV-1 quando comparado com o grupo controle. A expressão dos marcadores de superfície e o potencial de diferenciação in vitro em adipócitos e osteócitos foram similares nas MSC obtidas de indivíduos HTLV-1 e indivíduos controle. Foi demonstrada a presença do DNA proviral e da proteína p19 do HTLV-1 nas MSC isoladas de pacientes HTLV-1+. A comparação do perfil de expressão gênica global entre MSC isoladas de HAM/TSP e indivíduos assintomáticos para o HTLV-1 revelou que os genes da catepsina B e da proteína ribossomal L10 foram diferencialmente expressos. Em conclusão, este trabalho demonstra a importância das MSC na imunomodulação de linfócitos infectados pelo HTLV-1 e que a infecção pelo HTLV-1 altera características biológicas das MSC. / The characteristics of human multipotent mesenchymal stromal cells (MSC) can be influenced by the inflammatory microenvironment. However, the activity of the MSC against viral infections and the exact contribution of the infection to MSC dysfunction remain to be elucidated. We evaluated the immunosuppressive effect of MSC on HTLV-1 infected T lymphocytes and the susceptibility of MSC for this retroviral infection. Assays using co-culture of MSC and HTLV-1+ T lymphocyte lineages resulted in a decrease of tax gene expression and HTLV-1 p19 antigen. The reduction of the tax gene expression and the HTLV-1 p19 were associated with increased IL-6 secretion and higher PGE2, IDO and VCAM-1 gene expression. To confirm if MSC immunoregulation can influence the proliferation of HTLV-1 infected T lymphocytes, we compared the proliferation of HTLV-1+ individuals and healthy individuals cultured in the presence of MSC. It was observed that the lymphoproliferative inhibition by MSC in infected lymphocytes was similar to the control cells, and this effect was mediated by the expression of IDO and PGE2 genes. Furthermore, the pol gene and the HTLV-1 p19 protein were less expressed after co-culture assay with MSC, suggesting that the immunoregulation by MSC is effective in HTLV-1 infected T cells. In order to investigate the changes caused by HTLV-1 in MSC, we performed morphological and ultrastructural analysis of MSC exposed to HTLV-1 in vitro. The contact with HTLV-1 induced an increase of the intracellular vesicles, in addition the MSC cell surface molecules VCAM-1, ICAM-1 and HLA-DR were upregulated. The expression levels of VCAM-1 and HLA-DR molecules were increased in MSC cultured in the presence of PBMC isolated from HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) individuals. The MSC differentiation process into osteocytes and adipocytes was not impaired by HTLV-1. In addition, MSCs were efficiently infected by HTLV-1 in vitro due to the direct contact with the HTLV-1-infected cells. However, cell-free virus particles were not capable of causing productive infection. Finally, to ensure the biological function of MSC in HTLV-1 infected patients, we investigated bone marrow (BM) cells from HTLV-1 asymptomatic carriers (HAC) and HAM/TSP individuals. Initially, we observed an infiltration of CD4+ T-cell lymphocytes in BM from HTLV-1 infected individuals and the detection of provirus revealed HTLV-1 integration. The number of colonies of fibroblast progenitor cells (CFU-F) was lower in HTLV-1 infected individuals compared to control. HTLV-1 MSC isolated showed surface molecules expression and differentiation into adipogenic and osteogenic cells similar to control MSC. Proviral DNA and HTLV-1 p19 protein were detected in MSC from HTLV-1 patients. The comparison of global gene expression profiles between MSC isolated from HAM/TSP and HAC individuals revealed that cathepsin B and ribosomal protein L10 were differentially expressed. In conclusion, this study suggests the importance of MSC immunomodulation on HTLV-1 infected T lymphocytes and describe that HTLV-1 infects and alters the biological characteristics of MSC.

L’HTLV-1 est l’agent étiologique de la TSP/HAM et de l’ATL. La TSP/HAM est une méningomyélite chronique d’évolution lente et progressive. La charge provirale HTLV-1 est corrélée à l’évolutivité de la maladie. La protéine virale Tax module la synthèse et la fonction de multiples protéines de la prolifération et de la survie des lymphocytes T infectés. L’expression virale n’est pas toujours détectable in vivo. Une modulation épigénétique par inhibiteurs d’HDAC a été proposée. Des travaux dans le modèle du BLV et in vitro dans l’infection à HTLV-1 ont conforté ce concept et conduit à un essai thérapeutique évaluant le VPA dans le traitement de la TSP/HAM, qui n’a pas montré d’impact sur la charge provirale. Nous nous sommes intéressés à l’effet du VPA sur les lymphocytes T CD4+ en culture, cibles du virus HTLV-1. L’expression du génome viral, surtout la balance entre les protéines Tax et HBZ a été étudiée avec ou sans VPA. Son expression serait corrélée à la charge provirale et à l’évolution de la TSP/HAM. Nous montrons qu’un tiers des cellules CD4+ infectées expriment Tax. Cette proportion augmente sous VPA. La détection de la protéine p19 est augmentée sous VPA. La stimulation par le VPA de la transcription sens du génome viral a été confirmée par quantification des messagers Tax et Gag alors que l’expression d’HBZ se trouve réprimée sous VPA. Les données cinétiques d’expression spontanée d’HBZ montrent l’existence d’un rétrocontrôle négatif exercé par HBZ sur l’expression de Tax et des gènes structuraux. Influer sur l’expression d’HBZ ouvre une voie nouvelle pour le traitement des maladies associées à HTLV-1. / HTLV-1 is the etiological agent of HAM/TSP and ATL. TSP/HAM is a chronic meningomyelitis with a course being slow and progressive. HTLV-1 proviral load correlates with disease progression. Tax protein modulates the synthesis and function of proteins involved in the regulation of infected T-lymphocytes proliferation and survival. Viral expression is not always detectable in vivo. An epigenetic modulation by the use of HDAC inhibitors has been proposed. A study in the BLV model and an in vitro study in a HTLV-1 infection have lead to a therapeutic assay in order to assess VPA in the treatment of HAM/TSP. This assay did not show any impact on the proviral load. We evaluated the impact of VPA treatment on viral genome expression in cultured lymphocytes, especially the balance between Tax and HBZ. A third of provirus-positive CD4+ T cells spontaneously became Tax-positive. The estimation rose up to two-thirds of Tax-positive infected cells when VPA was added. VPA enhanced Gag p19 protein release. VPA treatment enhanced and prolonged Tax mRNA expression, while it blocked HBZ expression. Our data are consistent with the hypothesis of a feed-back loop coordinating Tax and HBZ expression. Modulation of HBZ expression may impact the net outcome of VPA treatment on HTLV-1-infected cell proliferation and survival.

Le virus T lymphotropique humain de type 1 (HTLV-1) est l’agent étiologique d’une forme rare et très agressive de leucémie de l’adulte (ATL). Le processus leucémogène a longtemps été attribué à la seule action de l’oncoprotéine Tax. Cependant, une nouvelle protéine virale, appelée HBZ (HTLV-1 bZIP factor), a été découverte en 2002. Elle est codée par le brin complémentaire du génome proviral et transcrite en antisens à partir du LTR3’. HBZ s’est avéré être un acteur clef de la prolifération et de la transformation des cellules T infectées, et donc du développement de l’ATL. La présente étude propose de nouvelles pistes quant aux mécanismes par lesquels HBZ induit la survie et la prolifération cellulaire. Nous avons montré que la protéine HBZ stimule l’expression de la neurotrophine BDNF et que les cellules de patients ATL surexpriment à la fois BDNF et son récepteur TrkB. De plus, ces patients présentent une concentration sérique anormalement élevée de la forme mature de BDNF, suggérant l’existence d’une boucle autocrine/paracrine BDNF/TrkB. L’activité de cette boucle a été confirmée in vitro et promeut la survie des cellules infectées par HTLV-1. D’autre part, nous avons découvert qu’HBZ dérégule l’expression du suppresseur de tumeur JunD dans les cellules T infectées, et induit celle de l’isoforme potentiellement oncogène ΔJunD. La production de ΔJunD résulterait d’une altération des mécanismes d’initiation de la traduction par HBZ. Nos résultats montrent aussi que ΔJunD promeut la prolifération et la transformation cellulaire en l’absence de sérum. Nous proposons donc que son expression pourrait contribuer à l’évolution des cellules T infectées en cellules leucémiques. / The human T-lymphotropic virus type 1 (HTLV-1) is associated with a rare and aggressive form of adult leukemia (ATL). For a long time, leukemogenesis was thought to mainly result from the action of the Tax oncoprotein. However, a new viral protein, called HBZ (HTLV-1 bZIP factor), was discovered in 2002. It is encoded by the minus strand of the proviral genome and transcribed in antisens from the 3’LTR. Since its discovery, HBZ came out as a key player in proliferation and transformation of infected T cells, thus contributing to ATL development. In this study we provide new leads regarding the mechanisms of HBZ-induced cell survival and proliferation. On one hand, we show that HBZ stimulates the expression of the BDNF neurotrophin and that ATL cells from patients overexpress both BDNF and its high-affinity receptor TrkB. Moreover, sera from patients exhibited abnormal levels of the mature form of BDNF, suggesting the existence of a BDNF/TrkB paracrine/autocrine loop. That loop was confirmed to be activated in vitro and to support the survival of HTLV-1-infected cells. On the other hand, we discovered that HBZ deregulates the expression of the JunD tumor suppressor in infected T cells and induces that of the ∆JunD isoform, which is potentially oncogenic. ∆JunD production would result from alteration of the translational initiation by HBZ. Our results also show that ∆JunD induces proliferation and transformation of serum starved cells. Finally, we hypothesize that HBZ-induced expression of ∆JunD may influence infected T cells to turn leukemic.

Différentes observations montrent que la protéine INT6 humaine possède une activité suppresseur de tumeurs. Il a été démontré que chez l’homme le gène int6 était sous-exprimé dans environ 30% des cancers du poumon non à petits cellules et que cette sous-expression était un facteur de mauvais pronostic. Des expériences de criblage double hybride avec INT6 comme appât ont identifié une protéine nommée SLIP1 (SLBP Interacting Protein 1). Un effet de SLIP1 sur la traduction des ARN messager des histones a été montré. Les travaux que j’ai menés indiquent qu’INT6 en interagissant avec SLIP intervient dans le contrôle de la stabilité et de la traduction des ARNs codant pour les histones. Un knockdown d’INT6 provoque une baise des niveaux des histones endogènes sans avoir un effet au niveau d’ARN. Mes études, en révélant un nouveau mécanisme de dans lequel INT6 joue un rôle direct, permettent ainsi de faire le lien entre – d’une part – les fonctions connues de cette protéine dans la traduction et son contrôle et – d’autre part – les effets oncogéniques connus de son altération. Par ailleurs, l’étude de la fonction d’INT6 dans les cellules humaines réalisée par ARN interférence montre une inhibition de la dégradation des ARNm possédant un codon stop prématuré par la voie du Nonsense Mediated mRNA Decay (NMD). Nous avons étudié son action par rapport aux ARNs HTLV-1. Nous avons observé une stabilisation significative des cibles de NMD. Ceci démontre que la protéine Tax interfère avec cette voie de dégradation des ARN d’une part en empêchant l’interaction entre UPF1 et INT6 et d’autre part en interagissant lui-même avec la protéine UPF1 phosphorylée. En agissant sur le NMD, Tax intervient à un niveau post transcriptionel qui pourrait avantager la réplication virale et aussi permettre la tolérance cellulaire aux mutations liées à l'effet mutagénique établi de Tax. / Several observations show that the human protein INT6 has a tumour suppressor activity.It has been demonstrated that in humans the expression of the int6 gene is reduced in about 30% of non-small cell lung cancers and that this under-expression is linked with a bad prognosis. Yeast two hybrid screening experiments using INT6 as bait have led to the identication ofa protein named SLIP1 (SLBP Interacting Protein 1). An effect of SLIP1 on the translation of histone messenger RNAs has been shown. My work indicates that INT6 – through its interaction with SLIP1 – plays a role in the controlof the stability and translation of histone mRNAs. INT6 knockdown induces a reduction of the level of endogenous histones without affecting that of their mRNAs. My studies, by revealing a new histone translation mechanism in which INT6 plays a direct role, establish a connection between – on one side – the known functions of this protein in translation and its control and – on the other – the known oncogenic effects of its alteration. The study of the function of INT6 in human cells by RNA interference resulted in inhibition of the degradation of mRNAs containing a premature termination codon by the Nonsense Mediated mRNA Decay pathway (NMD). Since we identified the HTLV-1 protein Tax as an interactor of INT6, and since some features of HTLV-1 mRNAs suggest that they may be potential NMD targets, we have studied the effect of Tax on the NMD pathway.We have observed that Tax significantly stabilizes NMD targets.We have demonstrated that the interference of Tax with the NMD pathway is mediated through – first – the perturbation of the interaction between INT6 and the NMD factor UPF1 and – second – through direct interaction between Tax and phosphorylated UPF1.Through its effect on NMD, Tax may favor viral replication at a post-transcriptional level as well as enable cellular tolerance for some mutations resulting from the established mutagenic activity of Tax.

Duas patologias principais estão associadas ao HTLV-1: a leucemia/linfoma de células T do adulto (ATLL) e a mielopatia associada ao HTLV-1/paraparesia espástica tropical (HAM/TSP). O mecanismo pelo qual ocorre o desenvolvimento destas doenças em apenas 1 a 5% dos indivíduos infectados é desconhecido. Sabe-se que diversos fatores estão associados ao desenvolvimento destas patologias, como carga proviral, características celulares, genéticas e imunológicas do hospedeiro. No entanto, tais mecanismos não foram totalmente elucidados e novas abordagens de estudos são necessárias para a compreensão da patogênese viral. Nesse sentido, vários estudos relatam o papel dos exossomos no desenvolvimento de infecções virais como HIV, EBV e HCV através do transporte de componentes incluindo proteínas, RNAm e miRNA. A possibilidade da transferência do conteúdo viral por essas vesículas para células não infectadas na ausência de vírus é um mecanismo intrigante. Dessa forma, esse trabalho teve como objetivo isolar os exossomos diretamente do soro de indivíduos infectados pelo HTLV-1 e comparar o conteúdo dessas vesículas quanto à expressão dos genes virais tax e HBZ entre os grupos assintomático e sintomático. Para tanto, os exossomos foram isolados por precipitação polimérica (Exoquick(TM)) e caracterizados por Western Blot (expressão dos marcadores CD9, CD81, Alix e citocromo c) e Análise de Rastreamento de Nanopartículas (tamanho e concentração). Além disso, o conteúdo dos exossomos foi analisado por PCR em tempo real para quantificação dos genes virais regulatórios tax e HBZ. Por fim, no intuito de estabelecer o papel dos exossomos como biomarcadores para o desenvolvimento da HAM/TSP, os níveis de expressão destes genes foram associados à carga proviral do HTLV-1. Os transcritos de RNAm de tax e HBZ foram detectados apenas em exossomos isolados do soro dos indivíduos sintomáticos, com ausência nos assintomáticos. Além disso, a expressão de tax e HBZ nos exossomos dos indivíduos infectados pelo HTLV-1 foi correlacionada positivamente com a CPV. Dessa forma, os resultados sugerem que os exossomos podem desempenhar importante papel na patogênese da mielopatia, através do transporte de genes virais de uma célula infectada para não-infectada. No entanto, mais estudos que relacionam o papel dos exossomos na infecção pelo HTLV-1 são necessários. / Two major pathologies has been associated with HTLV-1 infection: adult T- cell leukemia/lymphoma (ATLL) and HTLV-1 associated myelopathy/ tropical spastic paraparesis (HAM / TSP). The mechanism which is responsible for the appearance of these diseases in only 1 to 5% of the infected individuals is unknown. Is known that several factors has been related to the development of these diseases, such as proviral load and cellular, genetic and immunological characteristics of the host. However, these mechanisms have not been fully elucidated and new approaches are necessary to understand the viral pathogenesis. Thus, several studies have reported the involvement of exosomes in the development of the HIV, HCV, and EBV viral infections carrying components including proteins, mRNA and miRNA. The possibility of transfer of viral content via these vesicles to uninfected cells in the absence of virus is an intriguing mechanism. Therefore, this proposal aimed to isolate exosomes from serum of HTLV-1 infected patients and compare the content of these vesicles regarding viral gene expression of tax and HBZ between asymptomatic and symptomatic group. For this, the exosomes were isolated by polymeric precipitation (Exoquick(TM)) and characterized by Western Blot (expression of markers CD9, CD81, and Alix) and Nanoparticle tracking Analysis (size and concentration). Furthermore, the exosomes content were analyzed by real-time PCR for the quantification of tax and HBZ regulatory viral genes. Finally, to establish the role of exosomes like biomarkers to HAM/TSP development, levels of these genes were associated with proviral load of HTLV-1. Viral mRNA transcripts including tax and HBZ were found only in exosomes isolated from serum of symptomatic individuals and absence in assymptomatic. Furthermore, tax and HBZ expression in exosomes from HTLV-1 infected individuals were positive correlated with proviral load. Thus, the results suggests that exosomes can able to develop important role in myelopathy pathogenesis carrying viral genes of infected cell to uninfected cell. However, more studies that relate the role of exosomes in HTLV-1 are needed