Découvert en 1964, le virus d'Epstein-Barr appartient à la famille des gamma-herpèsvirus. Ce virus à ADN présente une forte prévalence (90% de la population adulte est infectée). Ce fut le premier virus identifié comme associé à des cancers (lymphome de Burkitt et d'Hodgkin, carcinome gastrique et de l'oropharynx). Ce virus a pour spécificité de posséder deux cycles distincts : latent et lytique (production de particules virales). Le facteur de transcription viral EB1 (ou Zebra) est un élément clé lors de l'initiation du cycle lytique et semble une cible importante pour l'élaboration de nouveaux traitements. Une première partie de ce travail concerne la caractérisation d'une protéine cellulaire (l'Ubinucléine) qui interagit et inhibe l'activité de EB1. Cette protéine voyage entre noyaux et jonctions serrées. Elle appartient à la famille des « NACos » (nuclear and adhesion complex components). La fonction de l'Ubinucléine n'est pas connue et sa protéomique quand elle est localisée dans les jonctions serrées, a été réalisée. Des études fonctionnelles montrent que l'Ubinucléine interagit avec plusieurs partenaires cellulaires, emprunte la voie d'endocytose dépendante de la clathrine et que sa localisation cellulaire (nucléaire ou dans les jonctions serrées) est affectée par la PKA. Dans une seconde partie, nous nous sommes intéressés à des molécules inhibitrices du facteur de transcription viral EB1. Après criblage à haut débit de composés chimiques (EMBL-Heidelberg), des tests in-vitro ont permis d'en sélectionner un pour des essais in-vivo. Ce composé chimique inhibe l'activité du facteur de transcription EB1 et bloque précocement la mise en place du cycle lytique dans des cellules de lymphome de Burkitt. Il semble donc intéressant d'améliorer l'efficacité et la spécificité de cette molécule. / Discovered in 1964, Epstein-Barr virus belongs to the Gamma-herpesvirus family. This DNA virus shows an important prevalence (90% of the adult population is infected). It was the first virus identified as associated with cancers (Hodgkin and Burkitt lymphomas, Gastric and oropharyngal carcinomas). It presents two distinct cycles: Latency and Lytic cycle (viral particle production). Viral transcription EB1 (or Zebra) is a key element for lytic cycle initiation and seems to be an important target for future treatment development. A first part of this work concerned the characterization of the cellular protein Ubinuclein that inhibits EB1 activity. This protein travels between nucleus and tight junctions. It belongs to the “NACos” (nuclear and adhesion complex components) protein family. Ubinuclein function is not known and its proteomic was performed when it was localized at tight junction. Next, functional studies showed that Ubinuclein interacts with various cellular partners and goes though the clathrin dependent endocytosis pathway. It's localisation (nuclear or at tight junction) changes with PKA activity. In the second part of this work, we focus on viral transcription factor EB1 inhibitors. After high-throughput screening of compounds (EMBL-Heidelberg), in-vitro assays allowed to select one molecule for in-vivo experiments. This compound inhibits the activity of the transcription factor EB1 and stops early lytic cycle establishment in Burkitt lymphoma cells. Further work needs to be done to increase efficacy and specificity of this molecule.

Submitted by Leonardo Cavalcante (leo.ocavalcante@gmail.com) on 2018-04-25T12:06:32Z No. of bitstreams: 1 Arquivototal.pdf: 2117961 bytes, checksum: 458d516added97ab516b1c8ae4ec5ddd (MD5) / Made available in DSpace on 2018-04-25T12:06:32Z (GMT). No. of bitstreams: 1 Arquivototal.pdf: 2117961 bytes, checksum: 458d516added97ab516b1c8ae4ec5ddd (MD5) Previous issue date: 2017-10-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The microRNAs are of great interest in studies on viral infections. In the context of the Epstein-Barr virus (EBV), they act as important regulators of the expression of viral and cellular genes, facilitating viral persistence and oncogenesis. The present work aims to identify viral microRNAs, their target mRNAs and the essential target proteins specifically for Human gammaherpesvirus 4 tipo 2. First, we used the StructRNAfinder and IntaRNA programs to perform the identification tasks of miRNAs and target mRNAs, respectively. Subsequently, we created a network of proteins from the data obtained from the tool online virusmentha and visualized in the program cytoscape. In addition, we performed a topological analysis of the protein interaction network with the cytoscape tool. We found 55 viral miRNAs and 46 mRNAs and identified 10 proteins that were prominent in the protein interaction network. The BTRF1 and BSFR1 proteins exert a greater control in the protein network, although they are proteins of the integument with unknown functions. Thus, we suggest that the BTRF1 and BSRF1 proteins are potential targets for understanding and controlling EBV infections. / Os microRNAs são de grande interesse em estudos sobre infecções virais. No contexto do vírus Epstein-Barr (EBV), eles atuam como importantes reguladores da expressão de genes virais e celulares, facilitando a persistência viral e a oncogênese. O presente trabalho objetiva identificar microRNAs virais, seus mRNAs alvos e as proteínas alvos essenciais específicas para o Human gammaherpesvirus 4 tipo 2. Primeiramente, utilizamos os programas StructRNAfinder e o IntaRNA para realizar as tarefas de idenficação de miRNAs e mRNAs alvos, respectivamente. Posteriormente, criamos uma rede de proteínas a partir dos dados obtidos da ferramenta online virusmentha e visualizamos no programa cytoscape. Além disso, realizamos uma análise topológica da rede de interação de proteínas com a ferramenta cytoscape. Encontramos 55 miRNAs virais e 46 mRNAs e identificamos 10 proteínas que se destacavam na rede de interação de proteínas. As proteínas BTRF1 e BSFR1 exercem um maior controle na rede de proteínas, embora sejam proteínas do tegumento com funções desconhecidas. Assim, sugerimos que as proteínas BTRF1 e BSRF1 sejam alvos potenciais para compreender e controlar as infecções por EBV.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The oral hairy leukoplakia (OHL) is a non-malignant epithelial lesions that usually occurs along the side edges of the tongue caused by the Epstein -Barr virus (EBV). This disease is almost exclusively seen in immunocompromised patients, particularly in individuals infected with human immunodeficiency virus (HIV). However, the OHL is related to immunosuppression in general, been described in patients receiving immunosuppressive therapy. The diagnosis of OHL in its clinical or subclinical form, can be done through the detection of EBV cytopathic effects in epithelial cells. Thus, the proposition of this study was to evaluate the oral hairy leukoplakia as a possible marker of immune impairment, through a cytological study in patients undergoing immunosuppressive therapy. 40 patients with autoimmune diseases were evaluated under immunosuppressive treatment, recruited from spontaneous demand of a population of patients regularly treated at the Department of Rheumatology, University Hospital, Federal University of Sergipe. Intraoral history and physical examination were performed, and soon after, two sweeps lateral border of the tongue, one for each side, then the construct two smears on conventional glass slides to be stained by the Papanicolaou technique were performed. Subsequently consulted medical records in order to collect data regarding the diagnosis of the underlying disease, time since diagnosis, type and duration of immunosuppressive therapy and WBC count. Stained by Papanicolaou smears were evaluated for representative nuclear changes cytopathic effect of EBV, which represent the criterion of cytological diagnosis of oral hairy leukoplakia. Most of the examined patients were females (70%) and the overall mean age was 40.5±16.1 years. Subclinical LPO was observed in 52.5% of patients. Thus, the use of shaved bilateral language for cytological diagnosis of subclinical LPO as clinical and laboratory monitoring tool immunosuppressed patients, in order, ultimately, minimize the occurrence of opportunistic diseases through modulation of therapy used is suggested. / A leucoplasia pilosa oral (LPO) é uma lesão epitelial não maligna que ocorre normalmente nas bordas laterais da língua, causada pelo vírus Epstein-Barr (EBV). Esta patologia é quase que exclusivamente vista em pacientes imunocomprometidos, particularmente em indivíduos infectados pelo vírus da imunodeficiência humana (HIV). Contudo, a LPO está relacionada com imunossupressão em geral, sendo descrita em pacientes que receberam terapia imunossupressora. O diagnóstico da LPO, na sua forma clínica ou subclínica, pode ser feito através da detecção dos efeitos citopáticos do EBV nas células epiteliais. Assim, a proposição deste estudo foi avaliar a leucoplasia pilosa oral como um possível marcador de comprometimento imune, através de um estudo citopatológico em pacientes submetidos à terapia imunossupressora. Foram avaliados 40 pacientes portadores de doenças autoimunes, sob tratamento imunossupressor, recrutados a partir de demanda espontânea de uma população de pacientes regularmente atendidos no Serviço de Reumatologia do Hospital Universitário da Universidade Federal de Sergipe. Foram realizados anamnese e exame físico intraoral e, logo após, foram realizadas duas raspagens de borda lateral da língua, uma para cada lado, seguida da confecção de dois esfregaços em lâminas de vidro convencionais para serem corados pela técnica de Papanicolaou. Posteriormente, consultado prontuário médico a fim de colher dados referentes ao diagnóstico da doença de base, tempo de diagnóstico, tipo e tempo de terapia imunossupressora e contagem leucocitária. Os esfregaços corados por Papanicolaou foram avaliados quanto às alterações nucleares representativas do efeito citopático do EBV, que representam o critério de diagnóstico citopatológico da leucoplasia pilosa oral. A maioria dos pacientes examinados pertencia ao gênero feminino (70%) e a média geral de idade encontrada foi de 40,5±16,1 anos. A LPO subclínica foi observada em 52,5% dos pacientes. Assim, sugere-se a utilização do raspado bilateral da língua para diagnóstico citopatológico de LPO subclínica como ferramenta de acompanhamento clínico-laboratorial de pacientes imunossuprimidos, a fim de, em última instância, minimizar a ocorrência de doenças oportunistas através da modulação da terapia empregada.

Orientador: Sandra Cecilia Botelho Costa / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-11T03:22:40Z (GMT). No. of bitstreams: 1 Pasquotto_Juliana_M.pdf: 2465167 bytes, checksum: dcd2cc122494bab62d8ab495927b1f0e (MD5) Previous issue date: 2008 / Resumo: O Vírus Epstein-Barr (EBV) e o Citomegalovírus (HCMV) são membros da família Herpesvírus. São encontrados em aproximadamente 90% dos indivíduos em idade adulta. A infecção ocorre, geralmente, na infância e é assintomática na maioria dos casos, persistindo de forma latente durante toda a vida do indivíduo. A transmissão destes vírus ocorre principalmente pela saliva, sangue e órgãos transplantados. O EBV está relacionado com a mononucleose infecciosa, doença linfoproliferativa (PTLD), leucemia de células pilosas em pacientes com imunodeficiência congênita ou adquirida e doença de Hodgkin. O risco de um paciente transplantado desenvolver linfoma é 28 a 50 vezes maior do que os indivíduos da população geral. Um dos fatores de risco para o desenvolvimento da PTLD são a variedade e intensidade da imunossupressão utilizada no paciente pós-transplante, idade do receptor e sorologia viral (EBV, CMV). Dependendo da idade do receptor, do tipo de transplante e dos fatores de risco, a prevalência da PTLD pode variar de 0.5% a 22%. Em transplantados pediátricos renais a prevalência chega a atingir 37%. A principal medida terapêutica para o controle da PTLD é a diminuição ou mesmo a retirada total da imunossupressão. Portanto a rejeição do enxerto se torna um problema bastante comum, que compromete a qualidade e/ou expectativa de vida dos pacientes. A introdução de testes laboratoriais rápidos e precoces permite aos clínicos detectar a replicação viral do EBV e diagnosticar, consequentemente, a infecção ativa antes do início da doença. Isso proporciona a oportunidade de iniciar o tratamento específico precocemente. Foram estudadas amostras de sangue e soro de 46 pacientes submetidos a transplantes de células hematopoéticas, acompanhados no Serviço de Transplante de Medula Óssea (STMO) do Hospital das Clínicas da UNICAMP/HEMOCENTRO. Trabalhamos no estudo para diagnosticar a infecção ativa e quantificar a carga viral do vírus Epstein-Barr (EBV) em pacientes transplantados de células hematopoéticas. Relacionar infecção ativa do vírus Epstein-Barr com o Citomegalovírus (CMV) e verificar a incidência da Doença linfoproliferativa e a Doença do enxerto contra o hospedeiro (GVHD) nos pacientes estudados. Diagnosticamos infecção ativa pelo EBV em 22 (47,8%) pacientes que apresentaram uma carga viral muito baixa (média de 29 cópias/ul). Co-infecção entre EBV e CMV ocorreu em 15/46 pacientes (32,6%). Doença por CMV ocorreu em 7/46 (15,2%) pacientes no trato gastrintestinal. Todos estes doentes apresentaram infecção ativa pelo CMV e 4/7 (57%) apresentaram infecção ativa pelo EBV. Um destes pacientes foi a óbito por doença por CMV. Verificamos que nenhum dos pacientes apresentou doença linfoproliferativa relacionada ao EBV. Os casos de co-infecção ativa EBV+CMV em relação à ocorrência de GVHD foram estatisticamente significantes (p=0.001). Concluímos que a infecção pelo CMV ainda é a maior causa de morbidade e mortalidade nos pacientes após o transplante. A qPCR é uma ferramenta útil para verificar os pacientes que reativaram pelo EBV após o transplante e pode auxiliar na prevenção da doença linfoproliferativa causada pelo EBV juntamente com a identificação dos fatores de risco associados. Verificamos a ocorrência e significância do GVHD e infecção ativa pelo CMV, mas não observamos esses mesmos resultados comparados ao EBV / Abstract: Epstein-Barr Virus (EBV) and Cytomegalovirus (CMV) are members of herpesvirus family. They are found in approximately 90% of the individuals in adult age. The infection generally occurs subclinicaly during the childhood in the major of the cases persisting in latent form during all the life of the individual. The transmission of these viruses occurs mainly for the saliva, blood and transplanted organs. EBV is related with mononucleose infectious, lynfoproliferative disease (PTLD), leukemia of hair cells in patients with congenital or acquired immunodeficiency and Hodgkin¿s disease. The risk of a transplanted patient to develop linfoma is 28 to 50 % more than other individuals of the general population. One of the risk factor for the development of the PTLD is the variety and intensity of the imunossupression used in the patient after-transplant, age of the recipient and viral serology (EBV, CMV). Depending on the age of the recipient, the type of transplant and the risk factor, the prevalence of the PTLD can vary of 0.5% 22%. In renal pediatric transplantation, the prevalence can arrives to reach 37%. The main therapeutical measure for the control of the PTLD is the reduction or total withdrawal of the imunossupression. Therefore the lost of graft is a common problem, that compromises the quality and/or life expectancy of the patients. The introduction of early and rapid laboratorial tests can permit to the physicians to detect the viral response and detect the active EBV infection before the disease. This provides the chance to initiate the specific treatment. We studied samples of blood and serum of 46 patients submitted to hematopoietic stem cell transplantation at the Bone Marrow Transplant unit of the University Hospital of the UNICAMP/HEMOCENTRO. We worked in the study to diagnosis the active infection and quantify the viral load of the Epstein-Barr virus (EBV) in transplanted patients of hematopoetic stem cells, to relate active EBV infection with active CMV infection and to verify the incidence of the lynfoproliferative disease and graft versus host disease (GVHD) in the studied patients. Active EBV infection detected by Real-Time PCR occurred in 22 patients (47,8%). The viral load found was very low (range 29 copies/ul). Co-infection between EBV and CMV occurred in 15/46 patients (32,6%). CMV disease occurred in 7/46 (15,2%) patients in the gastrointestinal tract. All these patients had active CMV infection and 4/7 patients (57%) had active EBV infection. One of these patients died by CMV disease. No patient presented lymphoproliferative disease related to the EBV. The cases of active EBV and CMV (co-infection) infection in relation to the occurrence of GVHD had been statisticaly significant (p=0.001). We conclude that the active CMV infection is already the most cause of morbidity and mortality of the patients after the transplant. The qPCR is a useful tool to verify the patients who had active EBV infection after the transplant and the identification of the risk factors associates prevention the development of the lymfoproliferative disease caused by the EBV. We verify the occurrence and significance of the GVHD and active CMV infection, but we do not observe these same results related to the EBV / Mestrado / Mestre em Farmacologia

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / The Epstein-Barr virus (EBV) has been related to the tumorigenesis of the gastric carcinomas, varying from 1.3-19.3% according to the studied population. Several studies have demonstrated strong evidences of its relation in this process, such as the monoclonality of the viral genome and its the presence in almost all tumor cells. However, most of the mechanisms used by the virus to control this process are still unknown. In this context, the present study aimed to investigate the frequency of the EBV and the association with the BCL-2, BAX and c-MYC proteins. Therefore, 100 cases of gastric carcinoma (67 males and 33 females), obtained from two hospitals in Fortaleza, were assessed to detect the EBV by PCR and in situ hybridization (aimed to the EBER1 transcript) using the standard method and GenPointÂ. Immunohistochemistry technique was done to evaluate the expression of the referred cellular proteins, by streptavidin-biotin-peroxidase method. The distribution by sex, age, tumor anatomic site and the histopathologic analysis, in general, reproduced the pattern of the world scientific bibliographies. Regarding virus detection by in situ hybridization, 8 (8%) cases were positive, 6 of these had shown diffuse pattern of staining, and 2 had demonstrated focal pattern. From 100 cases, only 2 presented infected lymphocytes. In general, the EBV demonstrated higher association with: males (87.5%[p=0.265]), tumors situated in the cardia (37.5% [p=0.549]), advanced stage (IIIB and IV), intestinal type (87.5%[p=0.136]), and moderately differentiated (75%).There were no EBV-positive cases which exhibited BCL-2 staining. Although the BAX and the c-MYC (nuclear) proteins have demonstrated significant positivity index and scores averages in the EBV-positive group, these were lower than the values of the EBV-negative group, notably the c-MYC nuclear protein (Mann-Withney test LI p=0.039 and HS p=0.045). The cytoplasmic staining of the c-MYC protein revealed slightly higher staining values in the EBV-positive group. The balance between the BCL-2 and BAX proteins demonstrated that the majority of the evaluated cases had exhibited apoptosis-orientation, however 62.3% of the EBV-positive cases exhibited equilibrium between these proteins. Twenty-nine cases (28 negative and 1 positive) were submitted to the biotinyl tyramide system (in situ hybridization method - GenPointÂ), demonstrating the same results obtained by the standard technique. From the 61 cases assessed by PCR, 35 (57.4%) were positive, being verified a low concordance index (kappa = -0.026 [Â0.069]) with the standard in situ hybridization technique. The 30bp deletion of LMP1 gene was investigated in 24 out of 35 positive cases, being verified in 37.5% of these. The results obtained in the present study, concerning the EBV frequency and the correlation with clinic-histopathologic data, reproduced findings of researches done in several world regions. The correlation with the proteins suggests that in vivo the virus is not related to the overexpression of BCL-2 and c-MYC (nuclear) that could act in synergism to promote the tumor development. The suppression of the BAX expression might represent a viral mechanism for apoptosis inhibition. The results of the cytoplasmic c-MYC point to a possible involvement of the EBV with transport mechanisms of the nuclear membrane, resulting in its accumulation in the cytoplasm. The low frequency of infected lymphocytes indicates that they are not the main responsible of the high number of positivity in the PCR technique. It could be, at least in part, due to the infected normal and/or pre-neoplastic epithelium, suggesting a new latency pattern which not express the EBER1. / O vÃrus Epstein-Barr (EBV) tem sido associado com a tumorigÃnese dos adenocarcinomas gÃstricos, variando entre 1,3-19,3% de acordo com a populaÃÃo estudada. Diversos estudos tÃm demonstrado importantes evidÃncias do envolvimento do EBV nesse processo, tais como a monoclonalidade do genoma viral e a presenÃa do vÃrus em quase todas as cÃlulas tumorais do sitio primÃrio e em cÃlulas metastÃticas. No entanto, os mecanismos utilizados pelo vÃrus para orquestrar a transformaÃÃo tumoral, ainda nÃo foram totalmente elucidados. Neste contexto, o presente estudo objetivou investigar a freqÃÃncia do EBV e a associaÃÃo com as proteÃnas BCL-2, BAX e c-MYC. Para tanto, 100 casos de adenocarcinomas gÃstricos (67 homens e 33 mulheres), obtidos de dois hospitais de Fortaleza, foram analisados quanto à presenÃa do EBV, detectado atravÃs das tÃcnicas de PCR e de hibridaÃÃo in situ (direcionada ao transcrito viral EBER1) pelo mÃtodo usual e GenPointÂ. Procedeu-se tambÃm, estudo imuno-histoquÃmico das referidas proteÃnas celulares, atravÃs do mÃtodo da estreptoavidina-biotina-peroxidase. A distribuiÃÃo por sexo, idade, sÃtio anatÃmico do tumor e as anÃlises histopatolÃgicas, de modo geral, reproduziram as tendÃncias da literatura mundial. Pela tÃcnica de hibridaÃÃo in situ, 8 (8%) casos foram positivos, 6 destes apresentaram marcaÃÃo difusa e 2 apresentaram marcaÃÃo focal. Apenas 2 apresentaram linfÃcitos infectados. De modo geral, o EBV apresentou maior associaÃÃo com o sexo masculino (87,5% [p=0,265]), com tumores situados na cÃrdia (37,5% [p=0,549]), de estadiamento avanÃado (IIIB e IV), do tipo intestinal (87,5% [p=0,136]) e moderadamente diferenciados (75%). Nenhum dos casos EBV-positivos exibiram marcaÃÃo para BCL-2. Embora as proteÃnas BAX e c-MYC (nuclear) apresentaram Ãndices de positividade e mÃdias de escores significativos no grupo EBV-positivo, estes foram inferiores aos valores do grupo EBV-negativo, sobretudo a proteÃna c-MYC nuclear (Teste de Mann-Withney LI p=0,039 e HS p=0,045). A marcaÃÃo citoplasmÃtica da proteÃna c-MYC revelou valores de marcaÃÃo discretamente superiores no grupo EBV-positivo. O balanÃo entre as proteÃnas BCL-2 e BAX demonstrou que a maioria dos casos estudados apresentavam tendÃncia à apoptose, mas 62,5% dos casos EBV-positivos exibiram um equilÃbrio. Vinte e nove casos (28 negativos e 1 positivo) foram submetidos a outro mÃtodo de hibridaÃÃo in situ que emprega o sistema da biotinil-tiramida (GenPointÂ),demonstrando resultados idÃnticos aos obtidos pela tÃcnica convencional. De 61 casos analisados atravÃs da tÃcnica de PCR, 35 (57,4%) foram positivos, sendo constatado um baixÃssimo Ãndice de concordÃncia (kappa = -0,026 [Â0,069]) com a tÃcnica de hibridaÃÃo in situ. Em 24/35 casos positivos, a deleÃÃo de 30pb do gene LMP1 foi investigada, sendo constatada em 37,5% destes. Os resultados obtidos no presente estudo quanto à freqÃÃncia do EBV e a correlaÃÃo com critÃrios clÃnico-histopatolÃgicos, reproduziram os achados de estudos realizados em diversas partes do mundo. A correlaÃÃo com as proteÃnas sugere que in vivo, o vÃrus nÃo esteja relacionado com a expressÃo de BCL-2 e de c-MYC (nuclear), que poderiam atuar em sinergismo favorecendo o desenvolvimento tumoral. A supressÃo da expressÃo de BAX, pode representar um mecanismo viral para inibiÃÃo da apoptose. Os resultados da c-MYC citoplasmÃtica apontam para um possÃvel envolvimento do EBV com mecanismos de transporte da membrana nuclear, determinando o acÃmulo da proteÃna no citoplasma. A baixa freqÃÃncia de linfÃcitos infectados indica que os mesmos nÃo sÃo os principais responsÃveis pela elevada positividade da tÃcnica de PCR, devendo ser ao menos em parte, decorrente de epitÃlio normal e/ou prÃ-neoplÃsico infectado sugerindo um padrÃo de latÃncia que nÃo expresse EBER1.

Les lymphoproliférations survenant après transplantation sont une situation rare mais préoccupante car mettant en jeu la survie des patients. Ces hémopathies ont des caractéristiques épidémiologiques et physio-pathologiques qui les distinguent des lymphomes du sujet immunocompétent. Nous nous sommes intéressé à l’analyse des facteurs de risque associés aux lymphomes post-greffe et à la recherche de facteurs pronostiques de survie des patients par l’analyse détaillée des registres américain et français de patients transplantés rénaux. Le Registre Français des lymphomes survenant après transplantation rénale a permis de recenser tous les cas de syndromes lymphoprolifératifs se développant chez des patients adultes survenant entre le 1er janvier 1998 et le 31 décembre 2007. Tous les centres français de Transplantation rénale ont participé. Nous avons recensé 500 cas de lymphomes sur une période de 10 ans. Les différentes analyses de cette base de données ont donné lieu à la publication d’une analyse intermédiaire sur les 230 premiers cas, à une publication consacrée à l’incidence et aux facteurs de risque de développement des lymphomes surla cohorte complète et à une publication concernant les facteurs pronostiques de décès des patients porteurs d’une lymphoprolifération avec établissement d’un score de risque spécifique de cette population. D’autre part, cette base de données unique au monde représente un support intéressant pour le développement de travaux de recherche: étude de l’origine des cellules tumorales, étude des facteurs de susceptibilité pharmacogénétique au développement des lymphoprolifération post-greffe, étude des microRNA de l’EBV dans les lymphomes post-greffe. / Post transplant lymphoproliferative disorders (PTLD) are a rare but serious complication occurring after kidney transplantation. Features of PTLD are specific and different of those of immunocompetent patients. We studied incidence, risk factors for development of PTLD and prognostic factors of patients with PTLD using two databases: American and French. The French Registry of PTLD enrolled all adult patients with PTLD occurring between the 1st January 1998 and the 31st December 2007 from all transplant centers in France. Five hundred patients were included in the Registry. This enables us to analyse first the incidence and risk factors of PTLD and second the risk factors of kidney recipients’ survival. We constructed a new prognostic score specific of transplant patients. Finally, the French Registry gave us the opportunity to support others research projects: determination of the origin of tumoral cells, analysis of pharmacogenetic factors associated with the risk of developing PTLD, study of EBV microRNA in lymphoproliferations.

Aims: To explore the clinical significance of EBV infection in adult renal transplant recipients when detected in the late post-transplant period. Methods: (1) A prospective observational study recruiting 499 stable adult kidney transplant recipients with serial blood sampling for EBV DNAemia and assessment of clinical outcomes and associated factors. (2) A retrospective analysis of PTLD incidence, timing and outcomes in relation to EBV infection. Results: EBV DNAemia in stable kidney transplant recipients is common, found in 46% of recruited individuals screened over 1 year, with persistent DNAemia seen in 10%. DNAemia prevalence increased significantly with time from transplant (p<0.0001) from 16% within 1 year of transplant to 66% in those transplanted for 20-24 years. High baseline DNA levels predicted persistence of DNAemia. Time adjusted analyses showed significant association of DNAemia with EBV seronegative status and previous PTLD and low DNAemia rates with Mycophenolate Mofetil (MMF) use and lymphopenia. The mechanism did not appear to be directly linked to MMF induced B cell depletion. Chronic high viral load detection was significantly associated with time from transplant, EBV seronegative status at transplant, ciclosporin use and plasma detection of DNA. No significant differences in overall patient survival at 3 years, clinical symptoms or clinical findings such as anaemia, thrombocytopenia or rate of decline in renal function were seen between stable transplant recipients with and without EBV DNAemia. PTLD incidence also increases with time from transplant and was greatest during the 10th-14th post-transplant years. Disease was EBV positive in 68% cases. No statistically significant differences in overall patient survival, or overall disease complete response rates were seen in relation to recipient EBV serostatus or EBV status of PTLD histology. Conclusions: EBV DNAemia prevalence increases with time from transplant but was not associated with worse patient or graft survival or specific symptoms. PTLD incidence including EBV negative disease also increases with time from transplant but response rates and survival were not influenced by EBV serostatus or histological status.

Plasmablastic lymphoma (PBL) was originally described in 1997 as an AIDS associated tumour although cases have been described in individuals not infected with HIV. Due to the high number of people living with HIV in South Africa, a substantial number of cases are diagnosed annually and 45 cases were included in this study. This represented the largest cohort of PBL affecting the oral mucosa published to date. Three main aspects of PBL were investigated: pathological features, viral status and certain genetic characteristics. The results from the genetic studies were the most important and interesting. These included rearrangements of the IGH gene in 63% and MYC- rearrangements in 62% of PBL’s. Seven of 43 cases (16%) showed rearrangement of both the IGH gene alleles, a finding never described before. New genetic findings also included increased CCND1 gene copy numbers in 17/41 (42%) and increased IGH gene copy numbers in 6/41 (15%) of cases. The exact role of MYC-rearrangements in the development of PBL is unclear. Many factors may be responsible for MYC deregulation but in the case of PBL of the oral cavity the possible role of Epstein Barr Virus (EBV) infection was considered. All but one of the patients with known HIV-status (32/45) was HIV positive and I supported the proposal that the diagnosis of PBL should serve as a sign of immunodeficiency, either as diagnostic thereof or as a predictor of a progressive state of immunodeficiency in patients with known HIV/AIDS status. The HIV-negative patient in this study was the only one that presented with an EBV-negative PBL on in situ hybridisation. The clinico-pathological features of the current study therefore strongly suggested an association between EBV, PBL and HIV/AIDS although the exact nature thereof remains uncertain. Routine genetic evaluation of tumours diagnosed as PBL should be introduced, as this may have prognostic and eventually treatment implications in the future. The exact panel of genes to be evaluated with a possible diagnosis of PBL should still be determined but examination of IGH and MYC for rearrangements should be included. This study proved the histomorphological features including the degree of plasmacytic differentiation not to have any diagnostic role although its prognostic value should be determined. The results of the immunohistochemical investigations performed in this study confirmed PBL always to be negative for CD20 but proved PBL not to be a morphological or immunohistochemical diagnosis by any means. In conclusion, it became clear that PBL should never be diagnosed without thorough clinical, systemic, pathological and genetic investigations, especially in the backdrop of HIV/AIDS. No pathologist should make the diagnosis of PBL and no clinician should accept such a diagnosis or decide on the treatment modality for the patient involved unless all other possibilities of systemic plasma cell disease have been excluded. / Thesis (PhD)--University of Pretoria, 2011. / Oral Pathology and Oral Biology / unrestricted

Dans les pays occidentaux, les lymphomes T périphériques et NK représentent environ 10% des lymphomes non-Hodgkiniens. Le lymphome NK/T de type nasal est l'une des entités de présentation extra-ganglionnaire les plus fréquentes, en Asie, et en Amérique Centrale et du Sud. Il survient classiquement dans la sphère nasopharyngée avec une prédilection pour les adultes jeunes. Morphologiquement, la tumeur est souvent angiocentrique avec une invasion de la paroi des vaisseaux par les cellules tumorales d'aspect variable. Ces lymphomes ont le plus souvent une origine NK avec un phénotype CD3+ (cytoplasmique), CD5-, CD56+, CD4-/CD8-, expression des molécules cytotoxiques et absence de réarrangement des gènes des récepteurs T. Le virus d'Epstein-Barr est présent dans la quasi-totalité des cellules tumorales dans sa forme clonage épisomale, avec une latence de type II, suggérant son rôle dans l'oncogenèse. A côté des mutations fréquentes des gènes FAS et TP53 (p53) et des méthylations de TP73 et CDKN2A (p16), des délétions du bras long du chromosome 6q sont fréquemment observées. Très récemment, des méthylations et des mutations des gènes suppresseurs de tumeur PRDM1, ATG5, et AIM1 localisés en 6q21 ont été retrouvées dans les lignées de lymphome NK/T de type nasal. Nous avons réalisé une analyse combinée du profil d'expression génique et du profil génomique par hybridation comparative sur puces, d'échantillons tumoraux de lymphome NK/T de type nasal (n=9) et de lignées, comparés à celle de lymphocytes NK normaux et de lymphomes T périphériques, sans autre spécificité (PTCL, NOS). Nous avons identifié la signature moléculaire particulière du lymphome NK/T de type nasal caractérisée par un haut niveau des trascrits de marqueurs de cellules NK et de molécules cytotoxiques, notamment de granzyme H dans les lymphomes NK/T de type nasal comparé aux PTCL, NOS. Par immunohistochimie, nous avons validé l'expression "spécifique" de granzyme H par les cellules tumorales du lymphome NK/T de type nasal, qui pourrait constituer un nouveau marqueur de ces lymphomes. Comparé aux cellules NK normales, le lymphome NK/T de type nasal a une signature plus proche des cellules NK activées que des NK au repos et sur-expriment des gènes associés à la biologie vasculaire, des gènes induits par l'EBV, et PDGFRA. Nous avons confirmé l'expression protéique de PDGFRAa et de sa forme phosphorylée, et montré in vitro la sensibilité de la lignée tumorale MEC04 à l'imatinib mesytale. La dérégulation des voies de signalisation AKT, JAK-STAT et NF-kB, suggérée par les analyses bioinformatiques, a été corroborée par la mise en évidence d'une expression nucléaire des formes phosphorylées d'AKT, de STAT3 et de RelA dans les lymphomes NK/T de type nasal. De plus, plusieurs gènes dérégulés dans ces voies moléculaires sont localisés dans des régions altérées de manière récurrente par des gains ou des pertes (AKT3 (1q44), IL6R (1q21.3), CCL2 (17q12), TNFRSF21 (6p12.3)). En plus de l'activation constitutive de STAT3 confirmée par l'expression nucléaire de phospho-STAT3, l'inhibition de croissance et l'augmentation de la mort cellulaire des cellules de la lignée MEC04 résultant de l'inhibition de STAT3 conforte le rôle de STAT3 dan la lymphomagenèse du lymphome NK/T nasal. L'analyse intégrée a également mis en évidence la dérégulation du gène suppresseur de tumeur HACE1 en 6q21, confirmée par RT-PCR quantitative. Bien que les mécanismes exacts conduisant à l'activation de plusieurs voies moléculaires, de même qu'à la dérégultaion de HACE1 ne soient pas déterminés, nos résultats identifient plusieurs voies oncogéniques impliquées dans le lymphome NK/T de type nasal ainsi que de nouveaux biomarqueurs diagnostiques - comme granzyme H - et des cicles thérapeutiques d'intérêt. L'étude en cours du profil d'expression des microARNs pourrait apporter un éclairage sur les mécanismes impliqués dans certaines voies identifiées / In Western countries, mature natural killer (NK)- and T-cell lymphomas account for 15% to 20% of aggressive lymphomas and around 10 % of all non-Hodgkin lymphomas. This number is higher in Asia, with 25% in Japan and 39% in Taiwan. Among those T- and NK-cell lymphomas with primary extranodal presentation, extranodal NK/T-cell lymphoma of nasal type (nasal NKTCL) is one of the most common entities in Asian, Central and South American populations. It classically arises in the nasal region showing a predilection for young adults with male predominance. This tumor morphologically exhibits an angiocentric and angio destructive growth pattern, admixed with polymorphous non-neoplastic infiltrates. Most tumor cells have a cytoplasmic CD3+, CD5-, CD56+, CD4-/CD8- phenotype with expression of cytotoxic granule-associated proteins and without rearrangement of T-cell receptors genes. Killer immunoglobulin-like receptors have been reproted to be expressed in a subset of this lymphoma and its expression might be associated with prognosis. Epstein-Barr virus is present in virtually all neoplastic cells in its clonal episomal form with type II latency program, implying a role in oncogenesis. Although the results were variable between different studies, methylations of TP73 (p73) and CDKN2A (p16) and mutations of FAS and TP53 (p53) were frequently found in nasal NKTCL. Genomic alterations have also been reported in nasal NKTCL with frequent deletion in chromosome 6q. A very recent study also identified both methylations and mutations of three putative tumor suppressor genes PRDM, ATG5, and AIM1 mapping to del6q21 in nasal NKTCL cell times. We performed integrative gene expression profiling and array-based comparative genomic hybridization analyses of nasal NKTCL tumors as well as tumour-derived cell lines, compared to that of normal NK cells and peripheral T-cell lymphomas, not otherwise specified (PTCL, NOS). We identified the distinctive molecular signature of nasal NKTCL with high transcript levels for NK-cell markers ans cytotoxic molecules, especially granzyme H in nasal NKTCL compared to PTCL, NOS. By immunohistochemistry, we validated expression of grnzyme H which appears a novel sensitive biomarker of nasal NKTCL. Compared to normal NK cells, nasal NKTCL tumors were closer to activated than resting cells and overexpressed several genes related to vascular biology, EBV-induced genes and PDGFRA. Notably, we confirmed the expression of PDGFRa and its phosphorylated form at the protein level, and in vitro the MEC04, nasal NKTCL-cell line, was sensitive to imatinib mesylate. Deregulation of the AKT, JAK-STAT and NF-kB pathways suggested by bioinformatical analysis, was corroborated by nuclear expression of phosphorylated AKT, STAT3 and RelA in nasal NKTCL, and several deregulated genes in these pathways mapped to regions of recurrent copy number aberrations (AKT3 (1q44), IL6R (1q21.3), CCL2 (17q12), TNFRSF21 (6p12.3)). In addition to constitutive activation of STAT3 as confirmed by the demonstration of phosphorylated STAT3 in the nuclei of neoplastic nasal NKTCL cells, growth inhibition and cell death of nasal NKTCL cells induced by STAT3 inhibition implied the role of STAT3 in the nasal NK/T-cell lymphomagenesis. Integrative analysis and qRT-PCR analysis also evidenced deregulation of another tumor suppressor HACE1 in the frequently deleed 6q21 region. Although the exact mechanism of activation of several pathways as well as that of HACE1 deregulation remains to be determined, our studies highlight emerging oncogenic pathways in nasal NKTCL and identify novel diagnostic and therapeutic targets. The ongoing investigation of microRNA expression profiling might shed light in a better understanding of the pathogenesis of nasal NKTCL and especially of the activation of oncogenic pathways. Connectivity map analysis may also help to depict other targeted therapies useful to improve the prognosis of this agressive lymphoma

Acute viral infections induce CD8 memory T cells that play an important role in the protection of the host upon re-infection with the same pathogen. These virus epitope-specific memory CD8 T cells develop complex TCR repertoires that are specific for that epitope. As individuals age virus-specific immunity appears to wane. Older people have difficulty controlling infection with common viruses such as influenza A (IAV), a RNA virus which causes recurrent infections due to a high rate of genetic mutation, and Epstein Barr virus (EBV), a DNA virus which persists in B cells for life in the 95% of people that become acutely infected. Many factors may contribute to this waning immunity including changes in virus-specific TCR repertoires. We hypothesize that epitope-specific memory CD8 TCR repertoires to these two common viruses change with increasing age and that CD8 T cell cross-reactivity may be one of the mechanisms mediating these changes. To address this hypothesis in our first study, we compared epitope-specific CD8 memory TRBV repertoires directly ex vivo for these two common human viruses. In cross-sectional and longitudinal studies of EBV seropositive, HLA-A2+, young (18-22 years), middle age (25-59 years), and older (>60 years) donors, we demonstrated that CD8 memory TCR repertoires to three immunodominant epitopes, known to have cross-reactive responses, IAV-M158-66, EBV-BM280-288, and EBV-BR109-117 co-evolve as individuals age. Cross-sectional studies showed that IAV-M1-and both EBV-specific repertoires narrowed their TRBV usage by middle-age. In fact, narrowing of EBV-BM and EBV-BR-specific TRBV usage correlated with increasing age. Although narrowing of IAV-M1-specific TRBV did not directly correlate with increasing age there was clear evidence that the TRBV usage was changing with age. The dominant TRBV19 usage appeared to become bimodal in the older age group and interestingly TRBV30 usage did directly correlate with age. For the EBV epitope-specific responses there was preferential usage of particular TRBV and changes in the hierarchy of TRBV usage in the different age groups. Longitudinal studies tracking 3 donors over 10-15 years (middle age to older) showed that there were changes in the TCR repertoire of IAV-M1, EBV-BM and -BR-specific responses over time. In two of the donors who experienced acute IAV infection there was evidence these repertoire changes may be influenced by TCR cross-reactivity, which is enhanced during acute IAV infection. The results of this first ex vivo study are consistent with our hypothesis. They suggest that virus-specific TCR repertoires change over time as an individual ages leading to narrowing of the repertoire and may co-evolve in the presence of CD8 T cell cross-reactivity. To further test our hypothesis in a second study we compared CD8 memory TRAV and TRBV repertoires to the three immunodominant epitopes IAV-M1, EBV-BM, and EBV-BR in the two extreme age groups, young donors (YSP) (18-22 years) and older donors (OSP) (>60 years) using the same donors as in the first study. Since these three epitopes are known to generate cross-reactive CD8 T cell responses and humans during their lifetime are frequently infected with both viruses at the same time these studies were also designed to more closely examine if TCR cross-reactivity could contribute to changes in TCR repertoire with increasing age. We examined the differences in both TRAV and TRBV in these two age groups by monoclonal antibody (mAb) staining and by deep sequencing and single cell sequencing in tetramer positive sorted cells from short-term cultures. Our initial studies showed that there were strong correlations in TRBV usage between short-term cultured and ex vivo antigen-specific responses; functional differences as well as differences in TRBV usage and diversity as measured by mAb staining particularly for the EBV epitope-specific responses between YSP and OSP donors. The TCR deep sequencing data also showed significant differences in TRBV usage between YSP and OSP. However, there were many more differences in TRAV and TRAJ usage than TRBV between the age groups suggesting that TRAV may play a greater role in evolution of the TCR repertoire. With increasing age, there was a preferential selection or retention of TCR for all three epitopes that have features in their CDR3a and b that increase their ease of generation, such as greater usage of convergent recombinant amino acids, and increase cross-reactive potential, such as multiple glycines. YSP and OSP differed in the patterns of TRAV/AJ and TRBV/BJ pairings and usage of dominant CDR3 motifs in all three epitope responses. Both YSP and OSP had cross-reactive responses between these 3 epitopes which were unique and differed from the cognate responses. Analyses of single cell sequencing data suggested that unique combinations of TRAV and TRBV are occurring, where one chain has features consistent with interaction with antigen one and the other chain had features consistent with interaction with antigen two. Interestingly, both the deep sequencing and single cell data show an increased tendency for the classic IAV-M1 specific clone BV19-IRSS-BJ2.7/AV27-CAGGGSQGNLIF-AJ42 to appear among the cross-reactive clones, suggesting that the dominance of this highly public clone may relate to its cross-reactivity with EBV. These results suggest that although OSP and YSP retain some of the classic TCR features for each epitope the TCR repertoire is gradually changing with age retaining TCR that are cross-reactive between these two common human viruses that we are exposed to frequently, one with recurrent infections and the other a persistent virus which frequently reactivates. These results are highly supportive our hypothesis and their importance in relation to viral immune-pathogenesis and potential novel immunotherapies will be discussed. These studies further emphasize the complexity and potential importance of human virus-specific T cell responses and TCR repertoires as people age and the need for a better understanding of TCR cross-reactivity between different viruses. For instance, at the present time these studies are highly relevant to better understanding the immune-pathogenesis observed during the COVID19 pandemic.