Based on matching analysis between different EBV strains, we found two nucleotide variations in miR-BART21 and four nucleotide changes in miR-BART22. Interestingly, two nucleotide variations upstream of mature miR-BART22 likely favor its biogenesis by Drosha/DGCR8 processing and we experimentally confirmed this augmentation by in-vitro Drosha digestion, and thus may underline the high and consistent expression of miR-BART22 in NPC tumors. / Infection with the Epstein-Barr virus (EBV) is a strong predisposing factor in the development of nasopharyngeal carcinoma (NPC). Many viral gene products including EBNA1, LMP1 and LMP2 have been implicated in NPC tumorigenesis, although the de novo control of these viral oncoproteins remain largely unclear. / MicroRNAs (miRNAs) are a class of small, non-coding RNAs with a size around 18--24 nucleotides with significant roles in regulating gene expression by either transcriptional silencing or translational suppression. As gene regulators, recent miRNA studies have emphasized the contribution of aberrant miRNA expression in cancer development. The recent discovery of EBV encoded viral miRNAs (ebv-miRNAs) in lymphoid malignancies has prompted us to examine the NPC-associated EBV miRNAs. In this study, we have systematically examined the NPC associated EBV genome for viral-encoded miRNA expression. By constructing small cDNA libraries from a native EBV positive NPC cell line (C666-1) and a xenograft (X2117), we screened about 3000 clones and detected several small EBV fragments, within which two novel ebv-miRNAs in the BARTs region were identified. These two newly identified miRNAs, now named miR-BART21 and miR-BART22, were proven to be abundantly expressed in most NPC samples by both Northern blot and QRT-PCR analysis. / Taken together, this thesis shows that two newly identified EBV-encoded miRNAs are highly expressed in latent EBV infection in NPC. Frequent expression of miR-BART22 can be explained partially by a specific EBV strain that is associated with NPC in our locality. Our findings emphasize the role of miR-BART22 in modulating LMP-2A expression. Because LMP-2A is a potent immunogenic viral antigen that is recognized by the cytotoxic T cells (CTLs), down-modulation of LMP-2A expression by mir-BART22 may permit escape of EBV-infected cells from host immune surveillance. / We attempted to predict the potential viral and cellular targets of miR-BART21 and miR-BART22 by public available computer programs, miRanda and RNAhybrid. A number of potential cellular mRNA targets were suggested, although many failed to be validated by luciferase reporter assay. However, we found a putative miR-BART22 binding site in the LMP2A-3'UTR. Although the LMP-2A transcript is consistently detected in NPC, only 6 out of 26 (23%) primary NPC tumors show weak LMP-2A expression by immunohistochemistry (IHC). The expression levels of miR-BART22 and LMP-2A mRNA have also been determined in eleven of these tumors. Interestingly, the LMP-2A mRNA expression level did not directly correlate with protein expression, and relatively low expression levels of miR-BART22 miRNA were observed in all 3 LMP-2A positive-primary tumors. The suppressive effect of miR-BART22 on LMP-2A was also experimentally validated by a series of dual luciferase reporter assays using reporter constructs containing the putative or mutated recognition site at the LMP-2A 3'UTR. By co-transfection of different amounts of miR-BART22 with the LMP-2A-3'UTR expression vector in reporter assay, we confirmed that miR-BART22 suppressed the LMP-2A protein level in a dose-dependent manner. Furthermore, transfection of miR-BART22 into HEK293 cells that had been stably transfected with pcDNA3.1-LMP-2A, which contains a complete LMP-2A ORF and 3'UTR, readily suppressed levels of the LMP-2A protein. / Lung, Wai Ming Raymond. / Adviser: To Ka Fai. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 197-226). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2008. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 6 uppsatser. - Titel från omslaget.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / A associaÃÃo do Linfoma de Hodgkin ClÃssico (LHc) com o vÃrus de Epstein-Barr (EBV) tem sido observada em vÃrios paÃses de diferentes condiÃÃes sÃcio - econÃmicas. Recentemente usando-se tÃcnica de ImunohistoquÃmica (IHQ) e HibridizaÃÃo in situ (HIS) porÃÃes virais foi encontrada exclusivamente nas cÃlulas caracterÃsticas do Linfoma de Hodgkin que sÃo as chamadas cÃlulas de Reed-Sternberg (RS) e suas variantes chamadas cÃlulas Hodgkin (H). Empregando estas tÃcnicas nosso trabalho foi realizado de modo a fazer uma anÃlise comparativa de uma amostra de 118 casos de origem do Cearà - Nordeste do Brasil e de diferentes regiÃes da FranÃa, sendo todos os pacientes de faixa etÃria entre 18 e 64 anos de idade. Com o propÃsito de convencionar parÃmetros para anÃlise comparativa e interpretaÃÃo de resultados buscou-se alguns trabalhos de pesquisadores cujo objetivo maior era de avaliar o percentual dessa associaÃÃo nos respectivos paÃses onde observou- se uma escassez de trabalhos comparando dois ou mais paÃses. A prevalÃncia do EBV em lesÃes nodais de 37 pacientes do nordeste brasileiro com Linfoma de Hodgkin clÃssico foi comparada com 33 pacientes franceses.Houve predominÃncia em pacientes brasileiros do sexo feminino (51,3%) e em pacientes franceses do sexo masculino (65,3%) sendo a mÃdia de idade similar em ambos os grupos (34,8 anos).Dos subtipos histolÃgicos a Esclerose Nodular (EN) esteve presente em 23 casos brasileiros e em 29 franceses e Celularidade Mista (CM) em 11 brasileiros e 4 franceses. DepleÃÃo LinfocitÃria (DL) e nÃo classificados foram raros. O LMP1 (ProteÃna de Membrana Latente) foi expresso nas cÃlulas RS em 25 (67,5%) dos casos brasileiros e em 10 (30,3%) dos franceses e o Epstein-Barr encoded RNA (EBER) foi evidente em 75,6% de Brasil e 30,3% da FranÃa. A relaÃÃo entre subtipo histolÃgico e detecÃÃo viral foi mais freqÃente no subtipo Celularidade Mista. Deduz-se com esses resultados que o EBV tenha uma maior participaÃÃo na patogÃnese do LH ClÃssico nos casos do Cearà que em pacientes oriundos da FranÃa.

La cavité buccale constitue une porte d’entrée et un site réservoir majeur pour une diversité de virus. Mis à part l’herpèsvirus simplex, peu d’études virologiques détaillent le rôle des virus pathogènes de la sphère orale dont le virus d’Epstein-Barr. Le consensus est que les inflammations orales surviennent principalement en réaction à des infections bactériennes et fongiques provoquant une dysbiose du biofilm oral. Toutefois, ce modèle n’est pas parfait et il explique mal les récidives observées après décontamination, le découplage entre dysbiose et poussées inflammatoires, la proximité de dents atteintes et saines dans un environnement bactérien similaire. L’hypothèse d'un mécanisme synergique combinant virus et bactéries a été proposée pour la parodontite. Selon ce modèle, l’action complémentaire des 2 types d’agents infectieux sur le microenvironnement immunitaire favoriserait la chronicité inflammatoire et l’évolution de la maladie. Nos travaux se concentrent sur l’étude de l’infection de la cavité orale par EBV. EBV est un virus ubiquitaire infectant l’homme de manière persistante. La cavité orale est le site privilégié pour sa réplication avec une production salivaire quasi-constante. Outre un rôle transformant majeur, l’implication de EBV est aussi suspectée pour plusieurs maladies inflammatoires. Tout d’abord, nous nous sommes intéressés au lichen plan oral (LPO), une maladie auto-immune dont l'étiopathogénie n’est pas clairement établie. Sur des biopsies de patients atteints de LPO (n=99), nous avons démontré par hybridation in situ que le LPO est fréquemment infecté par EBV (74%), notamment les formes cliniques érosives. Nous montrons que le degré d’infiltration des lésions par des cellules infectées par EBV (EBV+) est corrélé avec les paramètres inflammatoires et que les cellules EBV+ infiltrées sont des plasmocytes. Cela apporte un élément nouveau dans la mesure où les plasmocytes sont reconnus comme des cellules immunitaires régulatrices majeures. Nous décrivons des profils cytokiniques différents entre LPO infectés ou non, sans qu’il soit possible à ce stade de pouvoir impliquer directement les plasmocytes. Nous confirmons par microscopie électronique que les plasmocytes hébergent les stades tardifs du cycle d’EBV et produisent des virions dans le LPO, suggérant un mécanisme d’amplification locale de l'infection. Ensuite, nous nous sommes intéressés à la parodontite, maladie inflammatoire chronique commune qui détruit la structure parodontale et provoque le déchaussement dentaire. Cette maladie est clairement identifiée comme facteur aggravant de nombreuses maladies systémiques. Nos études avaient déjà mis en évidence un lien direct entre infection EBV et la sévérité de la parodontite. Mes travaux ont permis de montrer que le parodonte inflammatoire est infiltré par des cellules EBV+ qui semblent être majoritairement des plasmocytes. La présence de plasmocytes producteurs de virus au sein de la lésion inflammatoire pourrait expliquer l’infection des épithéliums adjacents. De plus, une étude clinique menée sur une petite cohorte de patients traités pour parodontite met en évidence une corrélation entre la diminution de la charge EBV salivaire et l’amélioration clinique de patients après traitement. Si ce résultat se confirme, il constituerait un argument supplémentaire en faveur d’une contribution de l’infection parodontale par EBV à la globalité de la charge EBV salivaire. En conclusion, nos données mettent en évidence la présence quasi constante d’EBV dans deux types de lésions orales inflammatoires. Cette infection virale contribue à aggraver une situation inflammatoire locale associée ou pas à une dysbiose bactérienne. L’observation majeure concerne la présence, souvent massive, de plasmocytes infectés dont le rôle reste à identifier. Ces observations constituent des avancées significatives qui permettent d’étayer un nouveau modèle de pathogénie orale associant virus et bactéries. / The oral cavity is a major entry point and reservoir site for a variety of viruses. Apart from herpesvirus simplex, few virological studies detail the role of oral pathogenic viruses, including Epstein-Barr virus. The consensus is that oral inflammation occurs mainly in response to bacterial and fungal infections causing dysbiosis of the oral biofilm. However, this model is not perfect and does not explain well the recurrences observed after decontamination, the decoupling between dysbiosis and inflammatory outbreaks, the proximity of affected and healthy teeth in a similar bacterial environment. The hypothesis of a synergistic mechanism combining viruses and bacteria has been proposed for periodontitis. According to this model, the complementary action of the 2 types of infectious agents on the immune microenvironment would promote inflammatory chronicity and disease progression. Our work focuses on the study of oral cavity infection with EBV. EBV is a ubiquitous virus that persistently infects humans. The oral cavity is the preferred site for its replication with almost constant saliva production. In addition to a major transformative role, EBV's involvement is also suspected for several inflammatory diseases. First, we focused on oral plan lichen (OPL), an autoimmune disease whose etiopathogeny is not clearly established. On biopsies of patients with OPL (n=99), we demonstrated by in situ hybridization that OPL is frequently infected with EBV (74%), particularly in erosive clinical forms. We show that the degree of infiltration of lesions by EBV-infected cells (EBV+) is correlated with inflammatory parameters and that the infiltrated EBV+ cells are plasma cells. This brings a new element to the extent that plasma cells are recognized as major regulatory immune cells. We describe different cytokinic profiles between infected and uninfected OPL, although it is not possible at this stage to directly involve plasma cells. We confirm by electron microscopy that plasma cells host the late stages of the EBV cycle and produce virions in the OPL, suggesting a local amplification mechanism of infection. Then, we focused on periodontitis, a common chronic inflammatory disease that destroys the periodontal structure and causes tooth loosening. This disease is clearly identified as an aggravating factor in many systemic diseases. Our studies had already shown a direct link between EBV infection and the severity of periodontitis. My work has shown that the inflammatory periodontium is infiltrated by EBV+ cells, which appear to be predominantly plasma cells. The presence of virus-producing plasma cells within the inflammatory lesion may explain the infection of adjacent epithelia. In addition, a clinical study conducted on a small cohort of patients treated for periodontitis showed a correlation between the decrease in salivary EBV load and the clinical improvement of patients after treatment. If this result is confirmed, it would be an additional argument in favour of a contribution of periodontal EBV infection to the overall salivary EBV burden. In conclusion, our data show the almost constant presence of EBV in two types of inflammatory oral lesions. This viral infection contributes to worsening a local inflammatory situation associated or not with bacterial dysbiosis. The main observation concerns the presence, often massive, of infected plasma cells whose role remains to be identified. These observations represent significant advances that support a new model of oral pathogenesis combining viruses and bacteria.

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.

Les proprotéines convertases (PCs) sont des enzymes impliquées de nombreux processus pathologiques. Nous avons étudié deux substrats des PCs: l'apeline et la glycoprotéine B (gB) du virus d'Epstein Barr (EBV), et le rôle de cette maturation protéolytique dans la médiation de leurs fonctions. L'apeline est surexprimée dans plusieurs cancers dont le cancer colorectal (CCR), et nous avons montré que la furine, un membre des PCs, clive l’apeline. Pour déterminer le rôle de ce clivage dans le CCR métastatique, nous avons généré un mutant non clivable (apeline-DM). In vitro, ce mutant inhibe la croissance de cellules cancéreuses du côlonet ne les protège pas de l'apoptose, contrairement à l'apeline sauvage. In vivo, l'apeline-DM diminue drastiquement la croissance tumorale et la formation de métastases hépatiques chez la souris. Les mêmes résultats sont obtenus dans des modèles de souris déficientes pour l’apeline, démontrant l'intérêt d'utiliser l'apeline-DM ou des dérivés comme potentiels agents anticancéreux dans le traitement des CCR métastatiques. La gB du virus EBV, virus impliqué dans certains cancers lymphoïdes et épithéliaux chez l'homme, permet l'entrée du virus dans la cellule lors de l'infection. Nous avons montré que les PCs, et notamment la furine, clivent la gB. In vitro, l'induction de la protéine virale LMP1 augmente l'expression de la furine, qui se traduit par une augmentation de l'infection par EBV. Ces résultats suggèrent l'existence d'une boucle de régulation entre la furine et LMP1 permettant d'améliorer la propagation cellulaire du virus. L'utilisation d'inhibiteurs de l'activité des PCs permettrait donc de bloquer l'infection par EBV. / Proprotein convertases (PCs) are enzymes involved in many pathological processes. We have identified two novel substrates of the PCs: apelin and glycoprotein B (gB) of Epstein Barr Virus (EBV), and studied the role of PC-mediated proteolytic maturation in their functions. Apelin is overexpressed in some cancers including colorectal cancer (CRC), and we demonstrated that furin, one of the PCs member, cleaves apelin in two peptides. To determine the role of apelin cleavage by furin in the metastatic CRC, we generated a non-cleavable mutant (apelin-DM). This mutant inhibited the growth of colon cancer cells in vitro and could not protect against apoptosis, unlike the wild-type apelin. In vivo, apelin-DM drastically reduces tumor growth and the formation of hepatic metastases in mice. These results were confirmed in apelin deficient mouse models thus demonstrating the potential interest of using apelin-DM, or its derivatives, as anticancer agents in the treatment of metastatic CRC. The gB of EBV, a virus involved in some lymphoid and epithelial cancers in humans, is involved in the entry of the virus into the cell during infection. We have shown that PCs, especially furin, cleave gB. In vitro,induction of the LMP1 viral protein increases furin expression, which results in an increase in EBV infection. These results suggest the existence of a regulatory loop between furin and LMP1 to improve the cellular propagation of the virus. The use of inhibitors of PCs activity would thus block EBV infection, a virus against which there is no treatment nowadays.

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.

O Epstein-Barr vírus (EBV) é a única espécie humana pertencente ao gênero Lymphocryptovirus. A transmissão ocorre através da saliva contaminada e geralmente ainda na infância. Nosso estudo analisou 165 amostras clínicas de pacientes, portadores de HIV, em tratamento com antiretrovirais, atendidos no Sistema Hospitalar do Sistema Penitenciário do Estado de São Paulo. Nosso enfoque foi pesquisar o EBV nas células mononucleares do sangue periférico, através das técnicas de PCR, Nested-PCR e seqüenciamento de nucleotídeos. Os resultados obtidos, indicaram que 11,51% (19) das amostras analisadas, apresentaram-se positivas para o EBV. Essas 19 amostras, foram seqüenciadas com primers específicos para a região da EBNA-1 (Epstein Barr Nuclear Antigen 1). As amostras foram alinhadas com o auxílio do DNASTAR. Ao alinharmos as amostras, encontramos uma troca de base (de G para A) em 7 amostras e essa troca não alterou a conformação da proteína EBNA-1. Na análise filogenética de nossas sequências com as depositadas no GenBank, foi possível observar dois grupos, que representam tipo 1 e o tipo 2 do EBV. 100% das amostras estudadas por nós foram identificadas como pertencentes ao grupo que caracteriza o tipo 2. Sendo assim, as 7 amostras que apresentaram a troca sugerem a origem um novo subtipo. / The Epstein-Barr Virus (EBV) is the only species to the genus Lymphocriptovirus that infects humans. One of the possible route for its transmission thought by contamined saliva and usually occurs in the childhood. This study analysed 165 clinical samples from HIV infected patients, treated by HARRT, attended in the Hospitalar System in the Penitentiary System from Sao Paulo State. The aim of this study was to search EBV in peripheral blood mononuclear cells by PCR, Nested-PCR and sequencing analysis. The results showed 11,51% of the analysed samples, positive for EBV. This samples, was sequenced with specifics primers from the EBNA-1 (Epstein Barr Nuclear Antigen 1) region. The samples were aligned by DNASTAR program. The aligned sequences showed the base conversion G to A in seven samples. This conversion caused no alteration in the EBNA-1 protein conformation. In the phylogenetic analysis the studied sequences with the sequences from GenBank was possible to observe two groups represented with type 1 and type 2 from EBV. 100% the samples studied was identified with the group characterized by the type 2 to EBV. So the seven samples showed the conversion, suggesting the origin of the one new subtype.

Nach aktuellem Kenntnisstand zur Pathogenese der Multiplen Sklerose (MS) wird der Entzündungsprozeß im Zentralnervensystem (ZNS) durch autoreaktive, Myelin-spezifische T-Zellen aufrechterhalten, wobei virale Infektionen bekannte Auslöser klinischer Schübe darstellen. Wir zeigten, dass eine Infektion mit dem Epstein-Barr Virus (EBV) eine notwendige aber nicht hinreichende Voraussetzung für die Entwicklung einer MS darstellt, deren potentielle Bedeutung sich aus der Korrelation zwischen Reaktivierung einer latenten EBV-Infektion mit Krankheitsaktivität bei MS-Patienten ergibt, die eine Aktivierung autoreaktiver T-Zellen im Rahmen der antiviralen Immunantwort nahe legt. Als biologische Marker der Krankheitsaktivität identifizierten wir die Hochregulation der ß2-Kette des Interleukin-12 Rezeptors sowie des Chemokinrezeptors CCR5 in peripheren Blutlymphozyten von MS-Patienten. Ferner konnten wir erstmals direkt nachweisen, dass die Aktivierung und klonale Expansion Myelin-spezifischer T-Zellen in peripherem Blut direkt mit Krankheitsaktivität korreliert. Der Wirkmechanismus der immunmodulatorischen Therapie der MS mit Interferon (IFN)-ß ist weitestgehend unverstanden. Unverstanden ist auch, warum die Therapie nur in einer Subpopulation von Patienten ihre Wirksamkeit entfaltet. Mit dem Molekül TRAIL gelang uns die Identifizierung des ersten klinischen Response Markers einer IFN-ß Therapie bei MS, der zugleich als prädiktiver Marker eine prognostische Aussage über den individuellen Behandlungserfolg erlaubt. Auf Grund seiner funktionellen Relevanz für den Therapieerfolg einer IFN-ß Behandlung stellt TRAIL zudem ein vielversprechendes Zielmolekül für neue therapeutische Strategien bei der MS dar. / Multiple sclerosis (MS) is considered a T-cell mediated demyelinating disorder of the central nervous system (CNS). Furthermore, it is well established that viral infections might trigger disease relapses. We demonstrate that infection with the Epstein-Barr virus (EBV) represents a necessary but not alone sufficient prerequisite for developing MS. The potential significance of EBV infection in MS arises from the correlation between viral reactivation and disease activity, indicating an activation of autoreactive T cells in the course of EBV reactivation and rendering EBV one important trigger of MS relapses. As biological markers for monitoring disease activity, we identified expression and upregulation of the chemokine receptor CCR5 and of the interleukin-12 receptor b2-subunit on potential effector cells in the peripheral blood of MS patients. In addition, we demonstrate for the first time that activation and clonal expansion of autoreactive T cells in the peripheral blood is clearly correlated with MS activity. Despite the well documented efficacy of interferon-b (IFN-b) therapy in a subgroup of patients, it remains unclear how IFN-b alters the clinical course of MS. We identified TRAIL as the first clinical response marker of IFN-b therapy in MS that might even be used as prognostic marker of therapy response in individual patients. Furthermore, our data imply that the immunoregulatory potential of TRAIL represents a novel and promising target for future therapeutic strategies in MS.