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En 2016, les lymphoproliférations malignes [lymphome malin non hodgkinien (LNH) et lymphome de Hodgkin (LH)] restent un problème majeur chez les patients porteurs du VIH (PPVIH) car chaque année de 1 à 6 % de ces patients développent des lymphomes. Ces pathologies apparaissent actuellement comme une des principales causes de mortalité chez les PPVIH.Le virus d’Epstein-Barr (EBV), connu de longue date pour son pouvoir immortalisant des lymphocytes B et les propriétés oncogènes de certaines de ses protéines, apparait comme un cofacteur favorisant plus ou moins important de ces lymphomes chez les PPVIH. Quand le virus est présent dans la cellule tumorale ; on parle de cancer associé à l’EBV.Une des questions toujours en suspens est de savoir si la quantification de l’ADN viral EBV (charge virale) et le profil sérologique EBV dans le sang des PPVIH peuvent aider à mieux comprendre la physiopathologie de ces lymphomes et à mieux prendre en charge les PPVIH qui en souffrent.Ce travail de thèse effectuée en co-tutelle entre l’université de Grenoble-Alpes et l’université médicale d’Irkoutsk vise à contribuer à répondre à cette question.La partie bibliographique de cette thèse synthétise (i) les connaissances actuelles sur l’épidémiologie et la physiopathologie des LH (synthèse non publiée en français) et sur le rôle de l’EBV dans les LNH chez les PPVIH (article publié en russe dans « Siberian Medical Journal » en 2015); (ii) les études publiées sur la mesure de la charge virale et la sérologie EBV chez les PPVIH.La partie expérimentale de cette thèse est constituée de trois articles. Le premier article publié dans Journal of Clinical Microbiology en 2016 rapporte la démonstration que l’utilisation d’un standard international développé par l’OMS peut améliorer la précision de la mesure de la charge virale EBV dans le sang. Le deuxième article en cours d’écriture concerne les résultats préliminaires d’une étude de cohorte mise en place par l’ANRS qui suit des PVVIH atteints de LH. Dans cette étude, l’objectif principal était de savoir si la charge virale et la sérologie EBV lors de la découverte du LH pouvaient constituer des marqueurs pronostiques de cette maladie comme cela été décrit dans des LH survenant chez des patients non infectés par le VIH. Nos résultats préliminaires ne vont pas dans ce sens et ces marqueurs ne semblent donc pas utiles pour une amélioration de la prise en charge des LH chez les PPVIH. Le troisième article publié en russe en 2015 dans « HIV infection and Iimmunosuppressive Disorders » décrit l’épidémiologie des lymphomes chez PPVIH de l’Université Médicale d’Irkoutsk. Il montre une importante augmentation des LNH chez les PPVIH entre 2000 et 2014 liée à une épidémie VIH non contrôlée dans cette région de Russie. / In 2016, malignant lymphoproliférations [non Hodgkin's (NHL) and Hodgkin's lymphomas (HL)] remain a major concern in patients living with HIV (PLHIV), that each year 1-6% of these patients develop lymphomas. Lymphomas are the major cause of mortality in this population.Epstein-Bar Virus (EBV), long known for his immortalizing B cells power and oncogenic properties of some of its proteins, emerges as a cofactor favoring lymphoproliferations, more or less important, depending on the type of lymphoproliferation.One of the outstanding questions is whether the molecular and/or serological characterizations of EBV infection may help to better understand the pathophysiology of these diseases and better manage patients suffering from HIV-associated lymphomas.This dissertation under joint supervision between the University Grenoble Alpes and Irkutsk State Medical University aims to answer this question.The literature review of this thesis summarizes: (i) the role of EBV in LNH development in PLHIV (article published in Russian journal “Siberian Medical Journal” in 2015) and the current knowledge on the epidemiology and pathophysiology of Hodgkin's lymphoma (non published in French); (ii) published studies on the EBV viral load and serological evolutions in PLHIV.The experiments consist of three articles. The first article published in Journal of Clinical Microbiology in 2016, reports the demonstration that the application of international standard EBV developed by WHO can improve the quantification of EBV viral load in whole blood. The second study (in writing for publication) contains preliminary results of French National Agency for Research of HIV and hepatitis cohort study investigating PLHIV suffering from Hodgkin's lymphoma. The study focuses on whether the EBV viral load and serology of newly diagnosed lymphoma could provide prognostic information for this disease, as has been described in HIV-negative patients with HL. Our preliminary results don’t support this hypothesis; than EBV markers don’t can be used for best management of HL in PLHIV. The third study published in Russian Journal “HIV infection and Immunosuppressive disorders” (2015) describes the epidemiology of HIV-associated lymphoma in Irkutsk Oblast. The article shows that non-Hodgkin lymphoma incidence rates in PLHIV during 2007-2014 are probably due to HIV epidemic non-controlled in this Russian region.

Les inhibiteurs des histones désacétylase (HDACi) constituent une classe prometteuse de médicaments anticancéreux. Ils peuvent déclencher la voie apoptotique et sont proposés pour le traitement des désordres hématologiques. Cependant, les HDACi qui ciblent les HDAC de classe II, tel que le vorinostat, sont également des agents réactivateurs potentiels de l’EBV, un virus qui infecte de manière latente plus de 90% de la population adulte dans le monde et est associée à de nombreux lymphomes de type B. L’étude de la commutation entre le cycle latent et le cycle lytique de l’EBV est essentielle pour appréhender l’impact des HDACi lors du!traitement des lymphomes B associés à l’EBV (risque du relargage de virions en grande quantité lors des traitements chimio thérapeutiques).Notre étude a porté sur l'effet de vorinostat (25 μM pendant 48h) sur des cellules tumorales B infectées par l’EBV : trois lignées de lymphomes de burkitt (BL2B95.8, BL41B95.8 et P3HR1), trois lignées lymphoblastoides (1602, PRI et RUD) et la lignée B95.8 de marmouset en cycle lytique de l’EBV (contrôle positif). Nous avons mis en évidence que le vorinostat peut induire la réactivation de l’EBV (P3HR1 et B95.8) ou à l’apoptose (BL41B95.8, BL2B95.8, 1602, PRI et RUD) avec une inhibition mutuelle de ces deux processus. Au niveau moléculaire, nous avons pu montrer que le vorinostat active constitutivement et simultanément le facteur de transcription initiateur de la réactivation MEF2D (par déphosphorylation) et la MAP kinase pro-apototique p38 (par phosphorylation) suite à la diminution de l’expression de la MAP kinase phosphatase (MPK1) dont p38 est un substrat. Le pré-traitement avec un inhibiteur de p38 (SB203580) a mis en évidence que cette MAP kinase est à la fois impliquée dans les processus de réactivation de l’EBV et d’’apoptose. Cependant, les lignées cellulaires pour lesquelles l'activation de p38 augmente fortement lors du traitement par le vorinostat, entrent directement en apoptose, sans qu’il puisse y avoir réactivation de l’EBV.Nos résultats suggèrent que le niveau d’activation de la MAP kinase p38 permet de réguler la balance réactivation/apoptose des cellules B infectées par l’EBV lorsqu’elles sont soumises à un agent inducteur de la réactivation, en particulier dans le cas de cellules de lymphomes B traitées par le vorinostat. Ils posent la question de l’utilisation des HDACi lors du traitement des lymphomes associés à l'EBV, avec le risque d’une réactivation virale selon le niveau d’activation intracellulaire de p38 et la nécessité d’utiliser simultanément un anti-viral tel que le ganciclovir. / Histone deacetylase inhibitors (HDAC) are a promising class of anticancer drugs. They can trigger the apoptotic pathway and are available for treatment of blood disorders. However, the HDACi that target HDAC class II, as vorinostat, also are potential reactivators agents of EBV, a virus that infects latently over 90% of the adult population worldwide and is associated with many type B lymphomas the study of switching between the latent cycle and the lytic cycle of EBV is essential to understand the impact of HDACi when treating B-cell lymphoma associated with EBV (salting risk virions in large quantities during the chemotherapeutic treatment).Our study focused on the effect of vorinostat (25 .mu.M for 48) on tumor B cells infected with EBV: three lines of Burkitt lymphoma (BL2B95.8, BL41B95.8 and P3HR1), three lymphoblastoid cell lines (1602 PRI and RUD) and B95.8 marmoset line in the lytic cycle of the EBV (positive control). We have demonstrated that vorinostat can induce EBV reactivation (P3HR1 and B95.8) or apoptosis (BL41B95.8, BL2B95.8, 1602, PRI and RUD) with a mutual inhibition of these two process. At the molecular level, we have shown that the active vorinostat constitutively and simultaneously initiating transcription factor reactivation MEF2D (by dephosphorylation) and MAP kinase p38 pro-apototique (phosphorylation) following the reduction in the expression of the MAP kinase phosphatase (MPK1) p38 which is a substrate. The pre-treatment with a p38 inhibitor (SB203580) showed that MAP kinase is both involved in the process of EBV reactivation and apoptosis. However, cell lines where p38 activation greatly increases during treatment with vorinostat, come directly into apoptosis, without there may EBV reactivation.Our results suggest that the level of activation of p38 MAP kinase helps regulate the balance reactivation / apoptosis of B cell EBV infected when exposed to an inducing agent for the reactivation, in particular in the case of cells B lymphoma treated with vorinostat. They raise the question of the use of HDACi in the treatment of lymphomas associated with EBV, with the risk of viral reactivation by level of intracellular activation of p38 and the need to simultaneously use an antiviral such as ganciclovir.

Notre équipe étudie les mécanismes de résistance à l’apoptose induite par divers agents dans les cellules de lymphomes B infectées ou non par le virus d’Epstein-Barr (EBV). EBV est un virus oncogénique de la famille des gamma-herpès virus qui est associé notamment au lymphome de Burkitt (LB) et aux syndromes lymphoprolifératifs post-transplantation (PTLD). Des résultats précédents ont montré que l’utilisation de la nutline-3, une molécule capable de se fixer sur MDM2, active p53 dans ces cellules tumorales. Cependant cette activation de p53 provoque l’apoptose des cellules B EBV(-) alors que les cellules B EBV(+) en latence III (exprimant toutes les protéines virales dites « de latence ») sont beaucoup plus résistantes. Mon travail de thèse a consisté à étudier les mécanismes impliqués dans cette résistance afin de mettre en place des stratégies thérapeutiques pour la contourner. La première partie de ma thèse a été consacrée à l’étude du rôle de l’autophagie dans la résistance des cellules EBV(+) en latence III à l’apoptose. L’autophagie est un processus de dégradation des protéines qui joue un rôle physiologique complexe impliqué à la fois dans la survie et dans la mort cellulaire. Les travaux effectués ont montré que: 1) l’autophagie est induite en réponse au traitement par la nutline dans les cellules EBV(+) en latence III ; 2) ces cellules expriment fortement la Bécline-1 et présentent une activation constitutive de l’autophagie ; 3) l’autophagie participe à la résistance de ces cellules à l’apoptose. La seconde partie de ma thèse a été consacrée au développement de nouvelles molécules ciblant les protéines anti-apoptotiques de la famille de Bcl-2. En effet, outre Bcl-2 qui est surexprimé dans les cellules EBV(+), les cellules de LB et les PTLD surexpriment aussi Mcl-1, une autre protéine anti-apoptotique. Or il a été montré que cette protéine était fréquemment à l’origine de résistance à des inhibiteurs déjà développés (et en essais cliniques) contre Bcl-2. Le développement de molécules ciblant Mcl-1 s’avère donc utile pour les contrer. Pour cela une collaboration avec une équipe de chimiste (dirigée par Fanny Roussi à l’Institut de Chimie des Substances Naturelles à Gif-sur-Yvette) a été mise en place. Nous avons identifié et étudié les mécanismes d’action de plusieurs molécules inhibitrices potentielles de Mcl-1 et/ou Bcl-xL capables d’induire l’apoptose dans nos deux modèles de lymphomes. / Our team investigates the mechanisms of resistance to apoptosis induced in various B-cell lymphomas including some infected by the Epstein-Barr virus (EBV). EBV is an oncogenic member in the gamma-herpesvirus family. Among other pathologies, it is associated with Burkitt’s lymphoma (BL) and post-transplant lymphoproliferative disorders (PTLD). Previously, our laboratory has found that in these tumor cells, the binding of nutlin-3 to MDM2 results in the activation of p53. However, although p53 activation leads to apoptosis in EBV(-) cells, EBV(+) latency III cells which express all viral « latency » proteins are much more resistant. During this PhD project, I studied the mechanisms involved in this resistance and made attempts to define new therapeutic strategies that would bypass them. First, the role played by autophagy was investigated. This catabolic process which degrades proteins and organelles is physiologically complex as it is involved in both cell survival and cell death. Our work has demonstrated that: 1) autophagy was induced in nutlin-3 treated EBV(+) latency III cells; 2) Beclin-1 was strongly expressed in these cells whose autophagy was constitutively activated; 3) autophagy was involved in the resistance to apoptosis observed in these cells. Second, I turned my efforts to the identification of new molecules targeting anti-apoptotic members of the Bcl-2 family. Like Bcl-2, the antiapoptotic protein Mcl-1 is heavily expressed in LB and PTLD cell lines but in this case, independently of their EBV status and this is a frequent cause for the observed resistance to Bcl-2 inhibitors that are currently tested in clinical trials. Molecules targeting Mcl-1 could thus prove promising to circumvent this resistance. In a collaboration with a Chemistry team supervised by Fanny Roussi at the Institut de Chimie des Substances Naturelles in Gif-sur-Yvette, we have identified the mechanisms of action of potential inhibitors of Mcl-1 and/or Bcl-xL, another anti-apoptotic molecules which induce apoptosis in our two lymphoma models.

Le virus d’Epstein-Barr (EBV) est un gamma-Herpesvirus associé à de nombreux cancers chez l’homme. In vitro, l’infection de lymphocytes B primaires par EBV conduit à leur immortalisation (genèse de lignées lymphoblastoides (LCL)). Dans ces cellules, seules 9 protéines virales (protéines dites de latence) sont exprimées et coopèrent pour stimuler la prolifération des cellules. Afin de comprendre les mécanismes moléculaires par lesquels les 3 protéines de latence de la famille EBNA3 (-3A, -3B et -3C) participent à l’induction et au maintien de la prolifération cellulaire induite par EBV, nous avons réalisé un crible deux-hybrides dans la levure en utilisant EBNA-3A, -3B ou -3C comme appâts. Ce crible nous a permis d’identifier de nombreux nouveaux partenaires particulièrement pertinents au vu de ce que l’on connaît des rôles respectifs des protéines EBNA3. Parmi les nouveaux partenaires de la protéine EBNA-3A se trouve le facteur de transcription Miz-1 qui est connu pour jouer un rôle clef dans l’arrêt du cycle cellulaire en transactivant l’expression de gènes tels CDKN1A, CDKN1C et CDKN2B. Nous avons validé cette interaction par GST-pull down ainsi que par co-immunoprécipitation en cellules humaines. Nous avons ensuite étudié l’effet de la protéine virale EBNA-3A sur l’activation de la transcription induite par Miz-1. Pour cela, nous avons comparé le niveau des transcrits de certains gènes cibles de Miz-1 dans des LCL exprimant ou non EBNA-3A et avons trouvé que certains gènes codant des inhibiteurs du cycle cellulaire sont différemment exprimés en présence d’EBNA-3A. Enfin, nous avons pu montrer que la protéine virale EBNA-3A est capable de réprimer l’activation de la transcription de Miz-1 en inhibant le recrutement de l’une de ses protéines co-activatrices, la protéine NPM. Ces résultats permettent de mieux comprendre les mécanismes par lesquels les protéines EBNA3 et plus largement EBV, dérégulent le cycle cellulaire. / Epstein-Barr Virus (EBV) is a human Herpesvirus that infects over 90% of the world population and is associated with several malignancies. EBV has the unique capacity to activate and to induce growth transformation of resting primary human B-lymphocytes, upon their in vitro infection, leading to the establishment of lymphoblastoid cell lines (LCLs). In these cells (called Lymphoblatoid cell lines (LCLs)), nine latent proteins are expressed driving the activation and proliferation of the infected B cells. In order to understand the molecular mechanism by which the EBNA3s latent proteins play a role in growth transformation, we used a large scale two-hybrid yeast screen. Thanks to that screen we identified several cellular partners very interesting in relation to what we know about the EBNA3s functions. One of the proteins identified in this screen is the transcription factor Miz-1, which has a cell growth arrest activity via inhibition of cell-cycle progression and has been shown to activate transcription of target genes including CDKN1A, CDKN1C and CDKN2B. We confirmed the interaction between EBNA-3A and Miz-1 by GST-pull down assay as well as by co-immunoprecipitation in HeLa cells We next investigated the effect of EBNA-3A on Miz-1-dependent regulation by comparing the transcript levels of selected Miz-1 target genes between EBNA-3A positive and negative LCLs by RT-qPCR. Interestingly, several Miz-1 target genes, among which CDKN2B, were found to be differentialy regulated in the presence of EBNA-3A. We found that EBNA-3A inhibits Miz-1 dependant activation by inhibiting the recrutement of the co-activator NPM. Those results bring new insights to the mechanisms by which the EBNA3s, and more largely EBV, regulate the cell cycle.

O Epstein-Barr Vírus (EBV) é um vírus da família Herpes (HHV-4), presente em grande parte da população mundial. É o agente etiológico da mononucleose infecciosa e da leucoplasia pilosa. A leucoplasia pilosa é uma doença epitelial benigna associada ao EBV, caracterizada pela reprodução replicativa do EBV nas células do epitélio oral, e é uma das mais freqüentes lesões oportunistas em pacientes HIV positivos, sendo menos freqüente apenas que a candidíase, com uma prevalência média entre 10 % e 30%. Por ser uma lesão oportunista bucal fortemente relacionada com a infecção pelo HIV e com a imunossupressão, seu diagnóstico é importante, pois pode sugerir o diagnóstico da infecção em pacientes de sorologia desconhecida para o HIV, e auxiliar no estadiamento da doença. Sua detecção e correto diagnóstico são de particular importância por essa condição estar relacionada à capacidade imune do paciente. Além disso, em pacientes já diagnosticados, ela é indicadora da progressão da doença e da eficácia da terapia antirretroviral. O objetivo desse estudo foi avaliar a presença e a quantidade do EBV na saliva e no sangue de pacientes infectados pelo HIV atendidos no CAPE-FOUSP, verificar a presença clínica de leucoplasia pilosa, estabelecendo a possibilidade da existência de vínculo entre a carga viral do EBV, a manifestação clínica da lesão e a carga viral do HIV. Também se buscou estabelecer relação entre o tipo de terapia antirretroviral em uso e a presença de leucoplasia pilosa, bem como estabelecer relação entre a carga viral do EBV na saliva e no sangue. Foram analisadas 20 lesões de leucoplasia pilosa, num total de 94 pacientes avaliados. Foi encontrada uma correlação positiva entre a Carga Viral do EBV no sangue e na saliva (p=0,001). Quanto maior a carga viral no sangue, maior a carga viral na saliva. Foi encontrada associação entre a Carga Viral do EBV na saliva e a presença de Leucoplasia Pilosa (p=0,045). Indivíduos com Leucoplasia Pilosa apresentam maior Carga Viral de EBV na saliva do que indivíduos sem essa lesão. Foi encontrada uma correlação positiva entre a Carga Viral do HIV e a Carga Viral do EBV na saliva (p=0,006) porém não no sangue. Quanto maior a carga viral de HIV, maior a carga viral do EBV na saliva. Foi encontrada correlação positiva entre a Carga Viral do EBV no sangue e as contagens de CD4 mais baixa registrada e a mais atual (p=0,028 e p=0,030 respectivamente). Quanto maior Carga Viral do EBV no sangue, maior a contagem de CD4. Não foi encontrada associação entre o tipo de medicação antirretroviral em uso e presença de lesão de leucoplasia pilosa. / The Epstein-Barr Virus (EBV) is a herpes virus family (HHV-4), is present in great part of the world population. It is the causative agent of infectious mononucleosis and oral hairy leukoplakia. Oral hairy leukoplakia is a benign epithelial disease associated with EBV, which is characterized by the replicative reproduction of EBV in oral epithelial cells, and is one of the most frequent opportunistic lesions in HIV positive patients, only less frequent than candidiasis, with an average prevalence between 10% and 30%. Being an opportunistic oral lesion strongly associated with HIV infection and immunosuppression, its diagnosis is important, because it may suggests the diagnosis of infection in patients of unknown HIV serology, and assist in the staging of the disease. Its detection and correct diagnosis are particularly important because this condition is related to the patient\'s immune capacity. Moreover, in patients already diagnosed, it is indicative of disease progression and effectiveness of antiretroviral therapy. The aim of this study was to evaluate the presence and quantity of EBV in saliva and blood of HIV-infected patients treated at the CAPE-FOUSP, verifying the presence of clinical OHL, establishing the possibility of the existence of a link between viral load and EBV, clinical manifestation of the lesion, and HIV viral load. It was also aimed to establish the relationship between the type of antiretroviral therapy in use and the presence of oral hairy leukoplakia, as well as establish the relationship between viral load of EBV in saliva and blood. We analyzed 20 lesions of oral hairy leukoplakia, a total of 94 patients. Found a positive correlation between viral load of EBV in blood and saliva (p = 0.001). The higher the viral load in blood, the higher the viral load in saliva. Association was found between viral load of EBV in saliva and the presence of oral hairy leukoplakia (p = 0.045).Individuals with oral hairy leukoplakia have a higher viral load of EBV in saliva than those without such injury. Found a positive correlation between viral load and HIV viral load of EBV in saliva (p = 0.006) but not in blood. The higher the viral load of HIV, the higher the viral load of EBV in saliva. A positive correlation was found between viral load of EBV in blood and CD4 counts the lowest recorded and most current (p = 0.028 and p = 0.030 respectively). The higher viral load of EBV in blood, increased CD4 count. No association was found between the type of antiretroviral medications in use and presence of oral hairy leukoplakia lesions.

A fim de prevenir a rejeição do órgão transplantado pelo hospedeiro, se faz necessário o uso de drogas imunossupressoras, como a ciclosporina, que pode levar ao aparecimento de efeitos colaterais como hipertensão arterial, nefrotoxicidade, hepatotoxicidade, e hiperplasia gengival medicamentosa (HGM), que pode ser potencializada se bloqueadores de canal de cálcio como a nifedipina forem associados a fim de controlar a pressão arterial. A patogênese da HGM ainda é incerta, entretanto fatores como a presença de cálculo e placa, concentração plasmática da droga, idade e fatores hormonais podem influenciar as características clínicas e o desenvolvimento da HGM. Recentemente, alguns vírus têm sido associados com a HGM. O HPV (Papiloma Vírus Humano) tem sido associado com casos severos de HGM, enquanto que o EBV (Vírus Epstein-Barr) é associado ao aparecimento de desordens linfoproliferativas pós transplante, que se apresentam como HGM. O objetivo deste trabalho foi avaliar a freqüência e o grau da HGM em pacientes transplantados renais (TR), identificar o EBV e HPV na HGM destes pacientes, e correlacionar a HGM, índice de placa, presença de cálculo e presença do EBV e HPV nos pacientes TR. Foram examinados os prontuários de 58 pacientes TR atendidos no CAPEFOUSP, e os dados com relação à medicação imunossupressora em uso e presença ou ausência de HGM foram registrados. Foram contatados 15 pacientes TR, dos quais foram colhidos dados demográficos, história médica, drogas em uso e história dental. No exame intra-oral foram observados o índice de placa, grau da HGM e presença de cálculo. A HGM foi removida e enviada a Disciplina de Patologia Bucal para análise microscópica. Os espécimes removidos foram comparados com um grupo controle composto por 20 casos de hiperplasia gengival inflamatória. Ambos os grupos foram submetidos ao exame de rotina, enfatizando a presença de coilócitos e a análise molecular, com hibridização in situ para o EBV (sondas EBER e Lytic) e HPV (sonda de amplo espectro e tipos 6/11, 16/18 e 31/33 nos casos positivos para a sonda de amplo espectro). 42% dos pacientes apresentaram HGM grau 1, 50% grau 2 e 8% grau 3. Cálculo estava presente em 50% dos pacientes. O índice de placa médio encontrado foi de 72%. Todas as amostras gengivais removidas cirurgicamente apresentaram um quadro histopatológico compatível com HGM. Os coilócitos estavam presentes em 100% dos casos do grupo de estudo e em 80% dos casos do grupo controle. O HPV esteve presente em 20% dos casos do grupo de estudo e em 10% do grupo controle. O EBV estava presente em 100% dos casos do grupo de estudo e em 90% dos casos do grupo controle, para ambas as sondas, entretanto no grupo de estudo foi observada uma expressão maior do EBV, tanto em quantidade de células marcadas, como em áreas mais profundas. Concluímos que a maioria dos pacientes TR apresentou HGM leve a moderada; EBV foi encontrado em todos os pacientes TR, caracterizando uma infecção oportunista, enquanto que o HPV foi encontrado nas mesmas proporções nos pacientes TR e no grupo controle; não foi encontrada correlação entre índice da HGM, índoce de placa, presença de cálculo e presença do EBV e HPV. / In order to prevent graft rejection in organ transplantation, is necessary the use of immunosuppressive drugs, as cyclosporin, that has several side effects, such as high blood pressure, nephrotoxicity, hepatotoxicity and gingival overgrowth (GO), that can be increased if calcium channel blockers, such as nifedipine, are associated in order to control de blood pressure. The pathogenesis of GO is still uncertain, but some factors such as the presence of calculus and plaque, drug plasmatic concentration, age and hormonal factors can influence the clinical aspects and development of GO. Recently some virus have been associated to GO as well. HPV (Human Papilloma Virus) have been associated to severe cases of GO and EBV (Epstein-Barr Virus) have been associated to posttransplantation lymphoproliferative disorders presenting as GO. The aim of this work was to evaluate GO incidence and score in renal transplant patients (RTP), identify EBV and HPV in GO from RTP, and correlate GO, plaque score, presence of calculus, and presence of EBV and HPV in RTP. We reviewed 58 charts from RTP attending to Special Care Dentistry Center (CAPE-FOUSP). Immunosuppressant drugs and presence or absence of GO were registered. 15 RTP were asked to show up in order to be examined. We collected demographic data, medical history, drugs in use and dental history. In intra-oral exam we observed plaque score, GO score and presence of calculus. GO were removed and sent to Oral Pathology Department for microscopic analysis. GO was compared to a control group composed by 20 cases of inflammatory gingival hyperplasia and both groups were submitted to routine exam emphasizing the presence of koilocytes and to molecular analysis with in situ hybridization for EBV (EBER and Lytic probes) and for HPV (wide spectrum probe and 6/11, 16/18, 31/33 types in cases where wide spectrum were positive). 42% of the patients presented GO score 1, 50% score 2 and 8% score 3. Calculus were presented in 50% of the patients. The average of plaque score was 72%.All GO specimens removed had a histopathological exam compatible with drug induced gingival overgrowth. Koilocytes were presented in 100% of study group (SG) and in 80% of control group (CG). HPV were presented in 20% of the SG and in 10% of the CG. EBV was presented in 100% of SG and in 90% of CG, for both probes, but in SG it could be observed in deeper areas of the epithelium and in a more pronounced expression. We concluded that most RTP presented mild to moderate GO, EBV were found in all RTP, characterizing an opportunistic infection, while HPV were found in the same proportions than in the control group and there were no statistical correlation between GO, plaque score, presence of calculus and presence of EBV and HPV.

O vírus Epstein-Barr (EBV) é um herpes vírus humano que estabelece infecção persistente e está associado com várias doenças, como mononucleose infecciosa, linfomas, carcinoma de nasofaringe e leucoplasia pilosa, afetando principalmente pacientes imunossuprimidos. Leucoplasia pilosa é uma lesão epitelial não maligna associada ao EBV que ocorre principalmente nas bordas laterais de língua. É comum em indivíduos infectados pelo HIV e em pacientes que recebem medicações imunossupressoras. Histopatologicamente, a leucoplasia pilosa é caracterizada por hiperparaqueratose, acantose, células semelhantes a coilócitos ou células balonizantes, e discreto ou nenhum infiltrado inflamatório. As características histopatológicas da lesão não são patognomônicas, sendo necessária a detecção do EBV para o diagnóstico final de acordo com vários autores. O objetivo desse estudo foi verificar a presença do EBV, por meio da técnica de hibridização in situ, em lesões diagnosticadas histológicamente como sugestivas de leucoplasia pilosa e comparar esses resultados com algumas características histopatológicas.Trinta e seis espécimes foram selecionados do Serviço de Patologia Cirúrgica da Disciplina de Patologia Bucal. Todos foram submetidos à reação de hibridização in situ, e 27 casos (75%) foram positivos, confirmando o diagnóstico anterior. Nenhuma das características histológicas analisadas puderam se correlacionar com a hibridização in situ. Pudemos concluir que a análise histopatológica ao H&E não pode substituir a hibridização in situ no diagnóstico final da leucoplasia pilosa / Epstein-Barr virus (EBV) is a human herpesvirus that estabilishes persistent infection and is associated with many diseases, including infectious mononucleosis syndrome, lymphomas, nasopharyngeal carcinoma, and oral hairy leukoplakia, affecting principaly immunocompromised patients. Oral hairy leukoplakia is a non malignant, EBV-associated, epithelial disease that typically occurs on the lateral tongue borders. It is common in individuals with HIV infection and in patients receiving iatrogenic immunossupression. Histologically, hairy leukoplakia is characterized by shaggy hyperparakeratosis, acanthosis, “koilocyte"-like or ballon cells, and a paucity of inflamation. The histologically features of hairy leukoplakia are not patognomonic, and for the many authors definitive diagnosis requires demonstration of EBV. The aim of this study were to verify the presence of EBV, by in situ hibridization, in lesions diagnosed histologically suggestive of hairy leukoplakia and compare this results with histologically features. Thirty six biopsy specimens from lesions histologically suggestive of hairy leukoplakia were selected from the Department of Stomatology’s Oral Pathology Service archives. EBV in situ hibridization was performed on all 36 cases, and 27 cases (75%) were positive, confirmed the diagnose of oral hairy leukoplakia. Histopatologically features did not agree well with EBV in situ hibridization. We concluded that H&E histopathology should not be used as a substitute for in situ hibridization in the definitive diagnosis of hairy leukoplakia

Le risque de LH est augmenté en cas de déficit immunitaire acquis ou inné. Les déficits immunitaires innés associés à un risque accru de LH, sont les DICV (Déficit Immunitaire Commun Variable), XLP (Syndrome lymphoprolifératif lié au chromosome X) et ALPS (Syndrome lymphoprolifératif Autoimmun). L’objectif de notre travail était d’évaluer la prévalence de ces déficits immunitaires chez des enfants atteints de LH. Nous avons reçu, 395 prélèvements de patients atteints de LH au diagnostic. L’âge médian de la population étudiée est de 13 ans, allant de 3 à 18 ans. Le sex-ratio M/F est de 1.1. Il augmente à 3 au dessous de l’âge de 10 ans. Parmi les biopsies (n=84) qui ont été relues, 87% sont de type scléro-nodulaires (SN), 7% à cellularité mixte (CM) et 6% non spécifié. L’EBV est détecté in situ dans 23% des cas de LH. Les patients atteints de LH-EBV+ sont significativement plus jeunes que ceux atteints de LH-EBV- (p=3.10-4). Ce sont plus fréquemment des garçons que des filles (63% ; M/F : 1,7) et fréquemment de sous-type CM (40%). Enfin, ils ont une charge virale EBV significativement plus élevée (p=3.10-3) que les enfants qui ont un LH-EBV-.Parmi les 83 premiers enfants analysés, un immunophénotypage approfondi a montré une diminution de la population lymphocytaire par rapport aux témoins et une lymphopénie B fréquente (31 patients sur 83 soit 37% des patients). La lymphopénie B était corrélée aux facteurs pronostiques connus du LH. Dans un cas parmi les 31, une baisse des immunoglobulines a été mise en évidence ce qui est évocateur de DICV. Nous avons montré que dans les autres cas, les lymphopénies se corrigeaient à distance de la maladie. La recherche de profil cytokinique associé à ces lymphopénies (TGF, BAFF, IL-7) n’a pas permis de mettre en évidence de mécanisme physiopathologique simple pour expliquer ces lymphopénies. Nous émettons l’hypothèse qu’elles sont liées à l’exposition au contact des cellules tumorales à des signaux favorisant l’apopotose.En ce qui concerne la recherche d’autres déficits immunitaires innés, aucun cas évocateur de XLP n’a été mis en évidence sur la base de la quantification des lymphocytes NKT. Cinq cas parmi les 83 (6%) avaient une expansion de lymphocytes T DN (Lymphocytes TCRαβ CD4-CD8-) dans le sang périphérique. Des dosages de Fas ligand et d’IL-10 plasmatiques ont permis d’exclure un ALPS. Au total, nous n’avons pas pu affirmer de défaut qualitatif des sous-populations lymphocytaires évoquant les déficits immunitaires de type XLP et ALPS. Seule une lymphopénie B avec baisse des IgG est évocatrice de DICV. Nous avons étendu l’analyse à l’ensemble des patients (395patients) avec un contrôle à distance du diagnostic pour ceux qui étaient anormaux. Nous avons identifié 4 patients potentiellement atteints de DICV, 1,5%. Parallèlement, nous avons recherché un déficit de la réponse T anti-EBV par cytomètrie de flux et l’Elispot. L’étude de la réponse T anti-EBV par la cytométrie de flux, a montré une tendance vers une baisse de la production d’IL-2 par les CD4 et les CD8 de patients avec une charge virale EBV élevée en réponse à une stimulation par des peptides EBV en présence de lignées autologues. L’étude de la réponse T anti EBV par la technique d’ELISPOT sur 9 patients n’a pas montré globalement de déficit du contrôle de l’EBV sauf pour une jeune patiente de 10 ans ayant une charge virale EBV très élevée sans réponse T anti-EBV efficace. Les résultats que nous avons obtenus restent à approfondir, ce qui permettra d’enrichir les connaissances actuelles sur cette pathologie. / Hodgkin’s Lymphoma (HL) is one of the most frequent lymphomas occurring in childhood. In young children, there is a high predominance in boys and frequent association with Epstein-Barr Virus (EBV). Cohort studies have shown that patients affected by several immune deficiency syndromes - e.g. X-linked lymphoproliferative syndrome (XLP), functional deficit of Fas/FasL pathway and common variable immunodeficiency (CVID) - are risk factors of HL. We intend to search for qualitative and quantitative immune deficiencies as susceptibility factors to child's HL in a prospective study related to Euronet –PHL C1 protocol. Eighty-three patients at diagnosis of HL have been analysed. Median age of the study population is 13 years, (5-18 years). Gender-ratio M/F is 1.1 with a larger male predominance before the age of 10 (gender-ratio of 3). The search for a defect of NKT population that would be suggesting of XLP was negative in all patients. A moderate expansion of circulating TCRαβ+ double negative cells (DNT) has been detected in 5 patients. This expansion has been further explored in the hypothesis of a defect of Fas/FasL pathway by plasmatic quantification of Fas ligand and Il-10. This led to the exclusionof the diagnosis of ALPS. An unexpected high frequency of B-cell lymphopenia has been detected in 31 out of 83 patients (37%). Peripheral B cell lymphopenia was associated with the following poor prognostic factors: advanced stages (p<0.04), low hemoglobin (p<0.06) and B symptoms (p<0.01). B-cell lymphopenia was not statistically correlated with morphology (subtype, amount of tumor cells and necrosis). Remarkably, B-lymphocytic counts were significantly higher in patients with in situ EBV (<0.05).Only a B lymphopenia with low IgG level suggesting DICV was detected. We extended the analysis to all the 395 patients included in the protocol EURONET, so we identified 4 patients with CVID. These cases will be further explored by molecular analyses. In parallel, the specific T-cells response against EBV was studied by flow cytometry in 15 patients and ELISPOT assay in 9 patients with HL. Flow cytometry , suggested a decrease in production of IL-2 by CD4 T cells in patients with high EBV viral load in response to EBV latent and lytic-cycle peptides and autologous lymphoblatoid cells lines compared to controls or patients with LH-EBV-. The ELISPOT-IFNγ assay was used to determine the frequency of T cells that produced IFNγ in response to peptides. One patient demonstrated inappropriate EBV-specific T-cell IFNγ production (<10 IFNγ secreted T cells and >1,000 EBV copies per 250000 PBMCs). These cases will be further explored by molecular analyses.Our findings confirm the known epidemiological data of HL now mainly associated to NS subtype in children and adolescents and EBV status in HL at this age. We show that peripheral B cell lymphopenia in paediatric and adolescent HL patients is frequent and associated with poor prognosis factors. We confirm the association between CVID and HL.

Le lymphome de Burkitt (BL) est due dans 80% des cas à une translocation chromosomique t(8;14)(q24;q32). Cette translocation marque l’évènement initial de la transformation maligne d’une cellule B normale, par délocalisation de l’oncogène CMYC à proximité du locus du gène codant pour la chaîne lourde d’immunoglobuline IGH par le mécanisme de réparation de l’ADN NHEJ durant l’hypermutation somatique (SMH). La probabilité de cette translocation est inversement proportionnelle à la distance qui sépare les loci portés par les deux chromosomes. La translocation (8;14) (q24;q32) qui apparaît durant les étapes de différentiation des lymphocytes B est encore plus importante chez les patients infectés par le virus Epstein-Barr (EBV) et le virus de l’immunodéficience humaine (VIH-1). L’objectif de notre étude est de déterminer les origines possibles de la translocation t(8;14) (q24;q32)dans les lymphocytes B normaux humains. Nous nous sommes intéressés tout d’abord à la dynamique de la localisation nucléaire des loci IGH et CMYC dans les lymphocytes B activés. Nous avons particulièrement étudié l’impact des virus EBV et VIH-1 sur l’organisation des gènes IGH et CMYC.Nous avons utilisé la technique d’hybridation in situ à fluorescence FISH pour la détection de CMYC (8q24) et IGH (14q32). Dans les lymphocytes B naïfs, CMYC est localisé du côté de la périphérie nucléaire, en revanche IGH est central, les deux loci sont complétement distants dans le noyau.L’activation des lymphocytes B induisait une augmentation de la colocalisation IGH-CMYC. La proximité physique entre les deux loci augmente la probabilité de leur translocation durant la SHM et favorise la t(8;14) (q24;q32) dans les lymphocytes B. Nous avons montré que les virus EBV et VIH-1ont un effet important sur la délocalisation IGH-CMYC dans les lymphocytes B. Nous avons aussi déterminé une molécule virale VIH-1 qui intervenait aussi dans la dérégulation de la localisation nucléaire des gènes IGH et CMYC. Nous avons déterminé deux mécanismes différents et indépendants impliqués dans la dynamique des loci IGH et CMYC : le premier mécanisme intervient dans le processus de développement normal des lymphocytes B, et le deuxième mécanisme dépend des virus ainsi que des molécules virales (particulièrement la Tat-HIV-1). / Eighty percent of Burkitt's lymphomas (BL) cases bear translocation t(8;14)(q24;q32). Thistranslocation is the initial event in malignant transformation of normal B-cell and derives from nonhomologousend joining of the oncogene CMYC to the immunoglobulin heavy chain locus IGH duringSomatic Hypermutation (SHM) of IGH. The probability of this translocation is inversely proportionalto the distance between the loci of involved chromosomes. The translocation t(8;14)(q24;q32) occursduring normal development of B-lymphocytes and more probable in patients infected with Epstein-Barr virus (EBV) and the human immunodeficiency virus (HIV-1).The subject of this study was to determine the possible origin of the translocation t(8;14)(q24;q32) inhuman normal B-lymphocytes. We followed the dynamics of the nuclear localization of IGH andCMYC genes in activated B-lymphocytes. We payed particular attention to the impact of EBV andHIV-1 viruses on dynamics of both IGH and CMYC. We applied Fluorescence in situ hybridization(FISH) for detection of CMYC (8q24) and IGH (14q32). In naïve B-cells CMYC is mainly localized inthe periphery of nucleus, whereas IGH is preferentially localized in the nuclear centre, i.e. these lociare distanced by a radius of cell nucleus. Activated B-lymphocytes displayed dramatic increase ofnumber of cells with colocalized IGH and CMYC. Close physical proximity of CMYC to IGH duringSHM amplifies the probability of occurance of translocation t(8;14)(q24;q32) in human Blymphocytes.Interestingly, we observed even more pronounced impact of EBVand HIV-1onproximity of IGH and CMYC. Finaly, among the molecules of HIV-1 we revealed those which possessthe most regulative role on dynamics of both IGH and CMYC. Our results suggest about twoindependent mechanisms of IGH and CMYC dynamics: the first is appropriate for normal developmentof B-lymphocytes and the second depends on virus and viral molecules, such as transactivator of viraltranscription HIV Tat.