Thèses sur le sujet « Herpesvirus 4 Humano »

Orientador: Estela Kaminagakura
Coorientadora: Patrícia Pimentel de Barros
Banca: Ana Sueli Rodrigues Cavalcant
Banca: Luana Marotta Reis de Vasconcellos
Banca: Lia Mizobe Ono
Banca: Luciana Yamamoto de Almeida
Resumo: A papilomatose laríngea é uma neoplasia benigna causada pelo papilomavírus humano (HPV), sendo os tipos 6 e 11 os mais comuns, e que ocorre em dois grupos etários, juvenil e adulto. A possível coinfecção viral tem sido sugerida em lesões de cabeça e pescoço; nesse sentido, o Epstein Barr vírus (EBV), que também apresenta tropismo por células epiteliais vem sendo estudado neste grupo de lesões. Os objetivos deste estudo foram genotipar os HPVs, investigar a presença de EBV-DNA por PCR e EBV-RNA por hibridização in situ. Além disso, associar a presença de EBV com a imunoexpressão de CD21, os resultados obtidos com a escala laringoscópica de Derkay et al. (1998) e com os dados clinicopatológicos. Oitenta casos de papilomatose laríngea, juvenil (n=36) e adulta (n=44), foram retrospectivamente analisados e subdivididos em grupos de menor e maior severidade, baseando-se na escala de Derkay. Todas as amostras foram HPV posivitas, com 49 casos HPV 6, 26 casos HPV 11, 4 casos HPV 6 e 11, e 1 caso HPV 16. A presença de EBV-DNA foi detectada em 9 amostras, entretanto EBV-RNA não foi não foi identificado em nenhuma amostra. Assim como a presença do EBV-DNA, a imunoexpressão de CD21 não se associou estatisticamente com quaisquer variáveis. A presença de HPV 6 foi mais comum em PLA e, o HPV 11 foi mais comum (p=0,02) e maior em casos de maior severidade (p=0,04), no grupo juvenil. A presença do EBV provavelmente não desempenha papel importante na progressão/severidade desta patologia.
Doutor

Orientador: Sandra Cecilia Botelho Costa
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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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

LIMA, Marcos Antonio Pereira de. Estudo do vírus Epstein-Barr (EBV) em adenocarcinoma gástrico : frequência, associação clínico-histopatológica e relação com a expressão das proteínas BCL-2, BAX e c-MYC. 2006. 147 f. Dissertação (Mestrado em Microbiologia Médica) - Universidade Federal do Ceará. Faculdade de Medicina, Fortaleza, 2006.
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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 GenPoint®. 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 - GenPoint®), 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 [±0.069]) 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 vírus Epstein-Barr (EBV) tem sido associado com a tumorigênese dos adenocarcinomas gástricos, variando entre 1,3-19,3% de acordo com a população estudada. Diversos estudos têm demonstrado importantes evidências do envolvimento do EBV nesse processo, tais como a monoclonalidade do genoma viral e a presença do vírus em quase todas as células tumorais do sitio primário e em células metastáticas. No entanto, os mecanismos utilizados pelo vírus para orquestrar a transformação tumoral, ainda não foram totalmente elucidados. Neste contexto, o presente estudo objetivou investigar a freqüência do EBV e a associação com as proteínas BCL-2, BAX e c-MYC. Para tanto, 100 casos de adenocarcinomas gástricos (67 homens e 33 mulheres), obtidos de dois hospitais de Fortaleza, foram analisados quanto à presença do EBV, detectado através das técnicas de PCR e de hibridação in situ (direcionada ao transcrito viral EBER1) pelo método usual e GenPoint®. Procedeu-se também, estudo imuno-histoquímico das referidas proteínas celulares, através do método da estreptoavidina-biotina-peroxidase. A distribuição por sexo, idade, sítio anatômico do tumor e as análises histopatológicas, de modo geral, reproduziram as tendências da literatura mundial. Pela técnica de hibridação in situ, 8 (8%) casos foram positivos, 6 destes apresentaram marcação difusa e 2 apresentaram marcação focal. Apenas 2 apresentaram linfócitos infectados. De modo geral, o EBV apresentou maior associação com o sexo masculino (87,5% [p=0,265]), com tumores situados na cárdia (37,5% [p=0,549]), de estadiamento avançado (IIIB e IV), do tipo intestinal (87,5% [p=0,136]) e moderadamente diferenciados (75%). Nenhum dos casos EBV-positivos exibiram marcação para BCL-2. Embora as proteínas BAX e c-MYC (nuclear) apresentaram índices de positividade e médias de escores significativos no grupo EBV-positivo, estes foram inferiores aos valores do grupo EBV-negativo, sobretudo a proteína c-MYC nuclear (Teste de Mann-Withney LI p=0,039 e HS p=0,045). A marcação citoplasmática da proteína c-MYC revelou valores de marcação discretamente superiores no grupo EBV-positivo. O balanço entre as proteínas BCL-2 e BAX demonstrou que a maioria dos casos estudados apresentavam tendência à apoptose, mas 62,5% dos casos EBV-positivos exibiram um equilíbrio. Vinte e nove casos (28 negativos e 1 positivo) foram submetidos a outro método de hibridação in situ que emprega o sistema da biotinil-tiramida (GenPoint®),demonstrando resultados idênticos aos obtidos pela técnica convencional. De 61 casos analisados através da técnica de PCR, 35 (57,4%) foram positivos, sendo constatado um baixíssimo índice de concordância (kappa = -0,026 [±0,069]) com a técnica de hibridação in situ. Em 24/35 casos positivos, a deleção de 30pb do gene LMP1 foi investigada, sendo constatada em 37,5% destes. Os resultados obtidos no presente estudo quanto à freqüência do EBV e a correlação com critérios clínico-histopatológicos, reproduziram os achados de estudos realizados em diversas partes do mundo. A correlação com as proteínas sugere que in vivo, o vírus 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 inibição da apoptose. Os resultados da c-MYC citoplasmática apontam para um possível envolvimento do EBV com mecanismos de transporte da membrana nuclear, determinando o acúmulo da proteína no citoplasma. A baixa freqüência de linfócitos infectados indica que os mesmos não são os principais responsáveis pela elevada positividade da técnica de PCR, devendo ser ao menos em parte, decorrente de epitélio normal e/ou pré-neoplásico infectado sugerindo um padrão de latência que não expresse EBER1.

PINTO, Marília Taumaturgo. Estudo comparativo da associação do vírus de epstein-barr com o linfoma de Hodgkin clássico em adulto : estudo imunohistoquimico e por hibridização in situ de casos do Ceará (Brasil) e de diferentes regiões da França. 2003. 80 f. Dissertação (Mestrado em Patologia) - Universidade Federal do Ceará. Faculdade de Medicina, 2003.
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A associação do Linfoma de Hodgkin Clássico (LHc) com o vírus de Epstein-Barr (EBV) tem sido observada em vários países de diferentes condições sócio - econômicas. Recentemente usando-se técnica de Imunohistoquímica (IHQ) e Hibridização in situ (HIS) porções virais foi encontrada exclusivamente nas células características do Linfoma de Hodgkin que são as chamadas células de Reed-Sternberg (RS) e suas variantes chamadas células Hodgkin (H). Empregando estas técnicas nosso trabalho foi realizado de modo a fazer uma análise comparativa de uma amostra de 118 casos de origem do Ceará - Nordeste do Brasil e de diferentes regiões da França, sendo todos os pacientes de faixa etária entre 18 e 64 anos de idade. Com o propósito de convencionar parâmetros para análise comparativa e interpretação de resultados buscou-se alguns trabalhos de pesquisadores cujo objetivo maior era de avaliar o percentual dessa associação nos respectivos países onde observou- se uma escassez de trabalhos comparando dois ou mais países. A prevalência do EBV em lesões nodais de 37 pacientes do nordeste brasileiro com Linfoma de Hodgkin clássico foi comparada com 33 pacientes franceses.Houve predominância em pacientes brasileiros do sexo feminino (51,3%) e em pacientes franceses do sexo masculino (65,3%) sendo a média de idade similar em ambos os grupos (34,8 anos).Dos subtipos histológicos a Esclerose Nodular (EN) esteve presente em 23 casos brasileiros e em 29 franceses e Celularidade Mista (CM) em 11 brasileiros e 4 franceses. Depleção Linfocitária (DL) e não classificados foram raros. O LMP1 (Proteína de Membrana Latente) foi expresso nas células 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 França. A relação entre subtipo histológico e detecção viral foi mais freqüente no subtipo Celularidade Mista. Deduz-se com esses resultados que o EBV tenha uma maior participação na patogênese do LH Clássico nos casos do Ceará que em pacientes oriundos da França.

Orientadores: Manoel Barros Bértolo, Lilian Tereza Lavras Costallat
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: A etiopatogenia da Artrite Reumatoide (AR) envolve fatores genéticos, imunológicos e ambientais que interagem entre si. Os principais fatores de risco estudados são a presença dos alelos do epítopo compartilhado (shared epitope- SE), dos anticorpos anti-peptídeos cíclicos citrulinados (anti-CCP) e do tabagismo. Há evidências que o Epstein-Barr vírus (EBV), ao interagir com esses fatores de risco, possa causar uma resposta imune anômala em indivíduos susceptíveis. Essas interações também podem contribuir para o desenvolvimento da AR. O objetivo principal desse estudo é estabelecer se há uma associação entre o EBV com os alelos do SE, os anticorpos anti-CCP e o tabagismo em pacientes brasileiros com AR. Os objetivos secundários são: avaliar a correlação entre os alelos do SE, os anticorpos anti-CCP e o tabagismo; detectar a exposição ao EBV e quantificar sua carga viral e estimar o risco de cada fator estudado para o desenvolvimento da AR nessa casuística. Nesse estudo caso-controle, incluímos 140 pacientes brasileiros com AR e 143 controles saudáveis; pareados por idade, sexo e etnia. Foi feita uma caracterização clínico-laboratorial da casuística. Foram realizadas a genotipagem para identificar os alelos do SE, a determinação dos anticorpos anti-CCP pelo método de ELISA e coletada a história de tabagismo de todos os sujeitos da pesquisa. Para avaliar a exposição ao EBV, realizamos a dosagem dos anticorpos anti-Epstein-Barr Nuclear Antigen 1 (anti-EBNA1). Para quantificar a carga viral do EBV, realizamos o método quantitativo da reação em cadeia polimerase em tempo real ou real-time PCR. A análise comparativa entre os grupos mostrou uma positividade significativamente maior para os alelos do SE, anticorpos anti-CCP e tabagismo no grupo de pacientes. A análise dos anticorpos anti-EBNA1 mostrou uma positividade alta, tanto em pacientes como em controles, sem diferença significativa. Entretanto, a quantificação da carga viral pela PCR em tempo real mostrou-se muito maior em pacientes do que em controles (p<0.001). As análises associativas dos anticorpos anti-EBNA1 com os outros fatores estudados não se mostraram significativas; assim como as análises associativas da carga viral do EBV pela PCR em tempo real. A positividade do anti-CCP foi maior em pacientes com os alelos do SE que são tabagistas ou ex-tabagistas (p=0.038). Nas análises de regressão logística, a variável com maior risco para o desenvolvimento da AR foi a positividade dos anticorpos anti-CCP. Apesar dos pacientes com AR apresentarem uma carga viral do EBV aumentada, esse estudo não conseguiu associá-la aos demais fatores de risco estudados. Sugerimos que esses achados possam ocorrer devido a um controle deficitário do EBV em pacientes com AR, mas que não está relacionado aos fatores de risco mais conhecidos da doença. A imunidade celular defeituosa dos pacientes com AR dificulta o controle de uma infecção latente do vírus. Portanto, não é possível estabelecer uma relação causal direta entre o EBV e a AR
Abstract: The pathogenesis of rheumatoid arthritis (RA) involves genetic, immunological and environmental factors. The main risk factors are the presence of the shared epitope alleles (shared epitope- SE), anti-cyclic citrullinated peptide antibodies (Anti-CCP) and smoking. There is evidence that the Epstein-Barr virus (EBV), when interacts with these risk factors, may cause an abnormal immune response in susceptible individuals. These interactions may contribute to the development of RA. The main objective of this study is to establish whether there is an association between EBV and alleles of SE, anti-CCP antibodies and smoking in Brazilian patients with RA. Secondary objectives are the assessment of the correlation between alleles of SE, anti-CCP antibodies and smoking; the detection of EBV; the quantification of EBV viral load and the estimation of the likelihood of each analyzed factor for the development of RA in this sample. In this case-control study, we included 140 Brazilian patients with RA and 143 healthy controls; matched for age, sex and ethnicity. We performed a clinical and laboratory characterization of the sample. Genotyping was performed to identify SE alleles, anti-CCP antibodies were examined by ELISA and smoking information was collected from all subjects. To assess the exposure to EBV, we examined anti-Epstein-Barr Nuclear Antigen 1 (anti-EBNA1) antibodies. To quantify the viral load of EBV, we performed the quantitative method of polymerase chain reaction in real time or real-time PCR. The comparative analysis between groups showed a significantly higher positivity for the alleles of SE, anti-CCP antibodies and smoking in patients. The analysis of anti-EBNA1 antibodies showed a high positivity, both in patients and in controls, with no significant difference. However, the quantification of viral load by real-time PCR proved to be much higher in patients than in controls (p <0.001). Associative analysis of anti-EBNA1 antibodies with other factors studied were not significant; as well as the association analyzes of the EBV viral load by PCR in real time. The positivity of anti-CCP antibodies was higher in patients with SE alleles which are smoker or ex-smoker (p = 0.038). In logistic regression analyzes, the variable with higher risk for RA was the positivity of anti-CCP antibodies. Although patients with RA present an increased EBV viral load, this study did not link EBV to the other risk factors studied. We suggest that these findings may be due to a deficient control of EBV in RA patients, which is unrelated to the better-known disease risk factors. Defective cellular immunity of patients with RA complicates the control of latent virus infection. Therefore it is not possible to establish a direct causal relationship between EBV and RA
Doutorado
Clinica Medica
Doutor em Clínica Médica

O linfoma de Burkitt (LB) é neoplasia linfóide de células B de alto grau que apresenta translocação constante envolvendo o proto-oncogene C-MYC. A associação com o vírus de Epstein-Barr (EBV) varia de acordo com a forma clinicopatológica. O presente estudo tem por objetivo analisar as características clinicopatológicas, imunoistoquímicas, incluindo a expressão do fator de transcrição MUM1/IRF4 e das proteínas p53 e p63, e investigar a associação com infecção pelo Herpesvírus humano 8 (HHV-8) e EBV, através de hibridização in situ e PCR, em 234 casos bem caracterizados de LB no Brasil, provenientes das 5 regiões geográficas em pacientes pediátricos e adultos, incluindo casos associados ao HIV. As características clínicas do LB no Brasil, de maneira geral, foram semelhantes às observadas na forma esporádica do LB ocorrendo nos países desenvolvidos. A infecção pelo EBV foi observada em 52,5% dos casos. A maior associação com EBV foi verificada nas regiões Norte e Nordeste e a menor na região Sul. Através de PCR, demonstrou-se predomínio de EBV do tipo A, sendo exceção a região Centro-Oeste. O fator de transcrição MUM1/IRF4 foi expresso em 39,2% dos tumores e apresentou correlação inversa com infecção pelo EBV. A expressão das proteínas p53 e p63 foi observada em 16,2% e 3,8% dos casos, respectivamente. Não se identificou infecção pelo HHV-8. O LB no Brasil apresenta características clinicopatológicas variáveis entre as regiões geográficas. A associação com infecção pelo EBV é intermediária entre a forma endêmica de LB e a forma esporádica ocorrendo em países desenvolvidos, sendo maior em regiões com indicadores sociais menos favoráveis.
Burkitt lymphoma (BL) is a high grade B cell lymphoma with a consistent translocation involving the proto-oncogene C-MYC. The association with the Epstein-Barr virus (EBV) varies depending on the clinicopathological form. This study aims to analyze the clinicopathologic, immunohistochemical features, including the expression of transcription factor MUM1/IRF4 and p53 and p63 proteins, and investigate the association with infection by human herpesvirus-8 (HHV-8) and EBV, by in situ hybridization and PCR, in 234 well-characterized cases of BL in Brazil from the 5 different geographic regions, in adult and pediatric patients, including HIV associated cases. The clinical characteristics of BL in Brazil, in general, were similar to those observed in the sporadic form of BL occurring in developed countries. EBV infection was seen in 52.5% of cases. The strongest association with EBV was found in the North and Northeast and the lowest in the South. PCR study demonstrated predominance of EBV type A, except in the Central-West region. The transcription factor MUM1/IRF4 was expressed in 39.2% of the tumors and showed inverse correlation with EBV infection. The expression of p53 and p63 proteins was observed in 16.2% and 3.8% of cases, respectively. No evidence of HHV-8 infection was found. The BL in Brazil is clinicopathologic diverse and regionally distinct. The association with EBV infection is intermediate between the endemic form of BL and sporadic form occurring in developed countries and is higher in regions with the less favorable social indicators

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Introdução: O linfoma difuso de grandes celulas B do idoso Epstein-Barr positivo (EBV+DLBCLe) afeta individuos com mais de 50 anos sem imunodefiCiência previa documentada, e apresenta evolucao clinica desfavoravel. Atualmente nao existe um padrao caracteristico da expressao de microRNAs (miRNAs) nesta doenca. Portanto, este estudo tem por objetivo caracterizar um perfil de assinatura para esta nova entidade e explorar miRNAs como biomarcadores e potenciais alvos terapeuticos alternativos para EBV+DLBCLe. Metodos: Setenta e um pacientes com mais de 50 anos e diagnostico de LDGCB foram considerados potenciais candidatos a serem EBV+DLBCLe. A deteccao do EBV (EBER-1) foi realizada por hibridacao in situ. Quatro amostras EBV+ e quatro amostras EBV negativas foram analisadas atraves de PCR quantitativo em tempo real (qPCR). O RNA foi extraido a partir de blocos de parafina e o cDNA inserido em duas plataformas contendo, cada uma, 384 miRNAs humanos. Consideramos miRNAs diferencialmente expressos aqueles que apresentaram expressao com valor acima ou abaixo de 1,5. Resultados: 8,5% dos pacientes com LDGCB foram considerados EBV positivos. A sobrevida global dos pacientes com EBV+DLBCLe foi inferior a dos pacientes EBV negativos (p= 0,0201, teste Log-rank). Foram encontrados 10 miRNAs diferencialmente expressos entre os dois grupos, mas apenas sete alcancaram diferencas estatisticamente significantes a serem validadas em uma coorte multicentrica (29 EBV + DLBCLe versus 65 LDGCB). Confirmamos o aumento de expressao de hsa-miR-126, hsa-miR-146a, hsa-miR-146b, hsa-miR-150 e hsa-miR-222, e tambem a diminuicao de expressao de hsa-miR-151 nos casos EBV+DLBCLe quando casos EBV+ foram comparados com EBV negativos. Utilizando o cutoff de 1,5, o hsa-miR-146b mostrou especificidade de 91,4 %, para identificar os casos EBV+, AUC = 0,8849. Embora o hsa-miR-222 tenha presentado aumento de expressao em menos de 1/3 dos casos, tambem mostrou alta especificidade (98,5%) e valor preditivo positivo (90%), AUC = 0,8180, no grupo EBV + quando comparado com o grupo EBV negativo, com o mesmo cutoff. Conclusoes: O merito do presente estudo foi propor uma assinatura de miRNA para uma doenca recentemente descrita e destacar o hsa-miR-146b e hsa-miR-222 como possiveis biomarcadores e alvos terapeuticos para EBV+DLBCLe. Antagomirs para hsa-miR-146b e hsa-miR-222 (este ultimo em teste em alguns tipos de cancer) tambem poderiam ser utilizados como terapia adjuvante ao R-CHOP nos casos de EBV+DLBCLe, nos quais confirmamos pior prognostico
FAPESP: 2010/17668-6
BV UNIFESP: Teses e dissertações

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
O líquen plano caracteriza-se como uma doença inflamatória crônica mucocutânea relativamente comum na população. Possui etiologia incerta, sendo possivelmente associado a fatores genéticos, psicológicos e infecciosos, dentre os quais o último vem ocupando um maior destaque devido a uma possível correlação com o vírus do papiloma humano (HPV) e com o Epstein-Barr vírus (EBV). O HPV possui alguns tipos considerados oncogênicos associados ao câncer de colo de útero e fortemente associado ao carcinoma espinocelular (CEC) de orofaringe. O EBV pertence à família herpesvirus humano e está relacionado com o carcinoma nasofaríngeo, linfoma de Burkitt e linfoma não-Hodgkin e sua possível relação com o CEC vem sendo estudada. O objetivo deste estudo foi detectar a presença do DNA do HPV e do EBV em amostras de tecido fresco, plasma sanguíneo, saliva e células esfoliadas orais, extraídas de um grupo pareado por sexo e idade de pacientes portadores de líquen plano bucal (LPB) e de um grupo de pacientes sem lesões de LPB, além de correlacionar as variáveis epidemiológicas dos grupos estudados com a presença viral e verificar se as fontes materiais testadas por este estudo são fontes viáveis para detecção do HPV e do EBV. Foram avaliados 24 pacientes portadores de LPB (Grupo caso) e 17 pacientes sem lesões de LPB (Grupo controle). A extração de DNA das amostras foi realizada após confirmar a presença e integridade do DNA. Os resultados obtidos foram submetidos à análise estatística (Teste exato de Fisher e Teste do Qui-Quadrado Mantel-Haenszel, ambos, com nível de significância de 5%). A nPCR foi utilizada para detecção do HPV e do EBV. Obteve-se a positividade viral para o HPV em 41,7% das amostras teciduais, em 12,5% das amostras de células esfoliadas e em nenhuma amostra de plasma sanguíneo e saliva dos pacientes do Grupo
Lichen planus is characterized as a chronic inflammatory mucocutaneous disease relatively common in the population. It has uncertain etiology, possibly associated with genetic, psychological and infectious factors. The infectious factor has excelled due the possible correlation of lichen planus with human papilloma virus (HPV) and Epstein-Barr virus (EBV). HPV has some types considered oncogenic, associated with cervical cancer and strongly associated with squamous cell carcinoma (SCC) of oropharynx. EBV belongs to human herpesvirus family and is associated with nasopharyngeal carcinoma, Burkitt's lymphoma and non-Hodgkin lymphoma. Its possible relation to SCC has been studied. The aim of this study was to detect the presence of the DNA of the HPV and EBV in fresh tissue samples, blood plasma, saliva and oral exfoliated cells extracted from a group of patients with oral lichen planus (OLP) paired by age and gender and from a group of patients without OLP lesions, and also correlate the epidemiological variables of the studied groups with the viral presence and verify that the source materials tested in this study are viable for HPV and EBV detection. It was evaluated 24 patients with OLP (Case group) and 17 patients without OLP lesions (Control group). DNA extraction of samples was performed after confirming the presence and integrity of DNA. The results were subjected to statistical analysis (Fisher's exact test and Mantel-Haenszel 19 chi-square test, both with a significance level of 5%). The nPCR was used to detect the presence of HPV and EBV. Was obtained the viral positivity for HPV in 41.7% of tissue samples and in 12.5% of exfoliated cells samples. No samples of blood plasma and saliva were positive in the Case group. On the other hand, the Control group
FAPESP: 11/05499-8

Dissertação (mestrado) - Universidade Federal de Santa Catarina. Programa de Pós-Graduação em Biotecnologia.
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O Linfoma de Hodgkin (LH) pode ser definido como uma desordem maligna linfoproliferativa. Embora exista uma importante associação desta doença com o vírus Epstein-Barr (EBV), sua etiologia ainda é desconhecida. A freqüência de casos de LH, positivos para o EBV apresenta variação, não só em populações de diferentes condições sócio-econômicas e geográficas, mas também quando relacionado com o subtipo histológico e a faixa etária de desenvolvimento do LH. A baixa freqüência da associação do EBV ao linfoma de Hodgkin clássico (LHC) no adulto jovem tem sugerido, apesar de poucas evidências, o possível envolvimento de um segundo vírus na patogenia da doença. Por todas estas razões, o presente estudo teve por objetivos avaliar a prevalência do EBV em amostras de biópsias de pacientes com LHC, de Florianópolis, Santa Catarina, além de avaliar a prevalência do vírus TT (TTV) nestas mesmas amostras. Um grupo controle composto por 20 amostras de biópsias de tecido com hiperplasia linfóide inespecífica foi incluído no estudo quando o vírus TT foi pesquisado. A detecção do TTV foi realizada através do ensaio de PCR, e a presença do EBV foi avaliada através da técnica de imunoistoquímica (LMP-1) e hibridização in situ (EBER). Foi observada uma prevalência do EBV de 48% (22/46), com predominância em indivíduos do sexo masculino (61,5%). O subtipo histológico esclerose nodular foi o mais incidente (70%) com uma prevalência de 37% para o EBV. Os casos de LHC dos adultos jovens (15-45 anos) demonstraram uma prevalência menor do EBV quando comparado com os outros grupos etários, sendo observado 21% (4/19) de positividade para o EBV. Nos casos de LHC infantis (<15 anos) foi demonstrada uma positividade para o EBV de 64% (9/14). A positividade para o TTV nos pacientes portadores de LHC quando comparada com o grupo controle, não apresentou diferença significativa. Quando avaliada a co-infecção desses vírus nos diferentes grupos etários, observou-se que o grupo composto por adultos jovens, de menor prevalência para o EBV (21%), mostrou uma maior prevalência do TTV (63%). Não foi possível estabelecer qualquer correlação entre as características clínicas ou o prognóstico da doença com a positividade para o EBV e/ou TTV no presente estudo.

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
O vírus de Epstein-Barr (Epstein-Barr virus - EBV) infecta latentemente mais de 90% da população humana. A infecção viral está associada ao desenvolvimento de alguns cânceres, incluindo o carcinoma de nasofaringe. Alguns estudos sugerem a contribuição na evasão imune e progressão dos cânceres causados pelo EBV. Diferentes variantes da proteína latente de membrana 1 (LMP1), principal produto do EBV, são discriminadas, principalmente, por variações nos domínios transmembrana e carboxi-terminais da proteína. O domínio C-terminal de LMP1 é capaz de ativar vias de sinalização intracelular que regulam os fenômenos de migração e invasão celular. Adicionalmente, induz síntese de produtos que degradam a matriz extracelular e favorecem a angiogênese. Até o momento, não se sabe se diferentes variantes de LMP1 apresentam propriedades distintas no que se refere a esses fenômenos do escape imune e progressão tumoral. Assim sendo, no presente estudo avaliamos a influência das variantes de LMP1 sobre a capacidade de migração e invasão celular in vitro e a imunomodulação de HLA-ABC e HLA-DR, CD80, CD83, CD54, CD40 e PD-L1 nas linhagens celulares HEK293T e NP69. Observamos que as variantes Alaskan, Med+, China 1 e China 2 induziram a migração celular da linhagem NP69 de maneira distinta quando comparadas entre si e com a célula sem LMP1. Observamos, também, maior capacidade de invasão celular das células HEK293T-China 2 em comparação à HEK293T. Com relação à expressão de moléculas envolvidas na modulação da resposta imune, observamos que a transfecção de LMP1 aumentou a expressão de CD80 e CD83 apenas nas células NP69-Alaskan, e de CD54 e PD-L1 de maneira similar nas células NP69-LMP1. Conclui-se, portanto, que LMP1 promove migração distinta das células NP69-LMP1 e invasão de células HEK293T-China 2, e modula a expressão de moléculas envolvidas no escape imunológico
The Epstein-Barr virus (EBV) latently infects more than 90% of human adults. Viral infection is associated to the development of some cancers, including nasopharyngeal carcinoma. Oncogenic potential of the virus is usually studied in terms of its capacity to transform the infected cells nevertheless some studies suggest that the EBV infection may also contribute to immune evasion, and cancer progression. Several latent membrane protein 1 (LMP1) variants are described, discriminated mainly by variations in its C-terminal and the transmembrane domains of the protein. LMP1 C-terminal domain activate intracellular signaling pathways that regulated cell migration; moreover, LMP1 upregulate the expression of cell proteins with roles in extracellular matrix remodeling and angiogenesis. Currently it is unknown whether different LMP1 variants possess distinct properties regarding biological phenomena relevant to immune evasion, and cancer progression. Thus, in the present study we evaluated the in vitro migration and invasiveness, and immunomodulation of HLA-ABC, HLA-DR, CD80, CD83, CD54, CD40, and PD-L1 of HEK293T cells, and NP69 cells. We observed that Alaskan, Med+, China 1, and China 2 stimulates distinctly migration of NP69 when compared to cell without LMP1. We also saw that HEK293T-China 2 had a pronounced cellular invasion when compared to HEK293T. In regard of expression of molecules involved in immune response modulation, we observed that LMP1 enhanced expression of CD80, and CD83 in NP69-Alaskan cells; and CD54, and PD-L1 in a similar level in NP69-LMP1 cells. In summary, LMP1 promote distinct cellular migration in NP69LMP1 cells, and HEK293T-China 2 invasion, and is capable of modulate molecules involved in immune evasion

Orientador: Glauco Issamu Miyahara
Coorientador: Cáris Maroni Nunes
Banca: Cristiane Fumiko Furuse
Banca: João Paulo de Carli
Resumo: O líquen plano caracteriza-se como uma doença inflamatória crônica mucocutânea relativamente comum na população. Possui etiologia incerta, sendo possivelmente associado a fatores genéticos, psicológicos e infecciosos, dentre os quais o último vem ocupando um maior destaque devido a uma possível correlação com o vírus do papiloma humano (HPV) e com o Epstein-Barr vírus (EBV). O HPV possui alguns tipos considerados oncogênicos associados ao câncer de colo de útero e fortemente associado ao carcinoma espinocelular (CEC) de orofaringe. O EBV pertence à família herpesvirus humano e está relacionado com o carcinoma nasofaríngeo, linfoma de Burkitt e linfoma não-Hodgkin e sua possível relação com o CEC vem sendo estudada. O objetivo deste estudo foi detectar a presença do DNA do HPV e do EBV em amostras de tecido fresco, plasma sanguíneo, saliva e células esfoliadas orais, extraídas de um grupo pareado por sexo e idade de pacientes portadores de líquen plano bucal (LPB) e de um grupo de pacientes sem lesões de LPB, além de correlacionar as variáveis epidemiológicas dos grupos estudados com a presença viral e verificar se as fontes materiais testadas por este estudo são fontes viáveis para detecção do HPV e do EBV. Foram avaliados 24 pacientes portadores de LPB (Grupo caso) e 17 pacientes sem lesões de LPB (Grupo controle). A extração de DNA das amostras foi realizada após confirmar a presença e integridade do DNA. Os resultados obtidos foram submetidos à análise estatística (Teste exato de Fisher e Teste do Qui-Quadrado Mantel-Haenszel, ambos, com nível de significância de 5%). A nPCR foi utilizada para detecção do HPV e do EBV. Obteve-se a positividade viral para o HPV em 41,7% das amostras teciduais, em 12,5% das amostras de células esfoliadas e em nenhuma amostra de plasma sanguíneo e saliva dos pacientes do Grupo
Abstract: Lichen planus is characterized as a chronic inflammatory mucocutaneous disease relatively common in the population. It has uncertain etiology, possibly associated with genetic, psychological and infectious factors. The infectious factor has excelled due the possible correlation of lichen planus with human papilloma virus (HPV) and Epstein-Barr virus (EBV). HPV has some types considered oncogenic, associated with cervical cancer and strongly associated with squamous cell carcinoma (SCC) of oropharynx. EBV belongs to human herpesvirus family and is associated with nasopharyngeal carcinoma, Burkitt's lymphoma and non-Hodgkin lymphoma. Its possible relation to SCC has been studied. The aim of this study was to detect the presence of the DNA of the HPV and EBV in fresh tissue samples, blood plasma, saliva and oral exfoliated cells extracted from a group of patients with oral lichen planus (OLP) paired by age and gender and from a group of patients without OLP lesions, and also correlate the epidemiological variables of the studied groups with the viral presence and verify that the source materials tested in this study are viable for HPV and EBV detection. It was evaluated 24 patients with OLP (Case group) and 17 patients without OLP lesions (Control group). DNA extraction of samples was performed after confirming the presence and integrity of DNA. The results were subjected to statistical analysis (Fisher's exact test and Mantel-Haenszel 19 chi-square test, both with a significance level of 5%). The nPCR was used to detect the presence of HPV and EBV. Was obtained the viral positivity for HPV in 41.7% of tissue samples and in 12.5% of exfoliated cells samples. No samples of blood plasma and saliva were positive in the Case group. On the other hand, the Control group
Mestre

Orientador: Deilson Elgui de Oliveira
Coorientador: Ramon Kaneno
Banca: Flávia Karina Delella
Banca: Bryan Eric Strauss
Resumo: O vírus de Epstein-Barr (Epstein-Barr virus - EBV) infecta latentemente mais de 90% da população humana. A infecção viral está associada ao desenvolvimento de alguns cânceres, incluindo o carcinoma de nasofaringe. Alguns estudos sugerem a contribuição na evasão imune e progressão dos cânceres causados pelo EBV. Diferentes variantes da proteína latente de membrana 1 (LMP1), principal produto do EBV, são discriminadas, principalmente, por variações nos domínios transmembrana e carboxi-terminais da proteína. O domínio C-terminal de LMP1 é capaz de ativar vias de sinalização intracelular que regulam os fenômenos de migração e invasão celular. Adicionalmente, induz síntese de produtos que degradam a matriz extracelular e favorecem a angiogênese. Até o momento, não se sabe se diferentes variantes de LMP1 apresentam propriedades distintas no que se refere a esses fenômenos do escape imune e progressão tumoral. Assim sendo, no presente estudo avaliamos a influência das variantes de LMP1 sobre a capacidade de migração e invasão celular in vitro e a imunomodulação de HLA-ABC e HLA-DR, CD80, CD83, CD54, CD40 e PD-L1 nas linhagens celulares HEK293T e NP69. Observamos que as variantes Alaskan, Med+, China 1 e China 2 induziram a migração celular da linhagem NP69 de maneira distinta quando comparadas entre si e com a célula sem LMP1. Observamos, também, maior capacidade de invasão celular das células HEK293T-China 2 em comparação à HEK293T. Com relação à expressão de moléculas envolvidas na modulação da resposta imune, observamos que a transfecção de LMP1 aumentou a expressão de CD80 e CD83 apenas nas células NP69-Alaskan, e de CD54 e PD-L1 de maneira similar nas células NP69-LMP1. Conclui-se, portanto, que LMP1 promove migração distinta das células NP69-LMP1 e invasão de células HEK293T-China 2, e modula a expressão de moléculas envolvidas no escape imunológico
Abstract: The Epstein-Barr virus (EBV) latently infects more than 90% of human adults. Viral infection is associated to the development of some cancers, including nasopharyngeal carcinoma. Oncogenic potential of the virus is usually studied in terms of its capacity to transform the infected cells nevertheless some studies suggest that the EBV infection may also contribute to immune evasion, and cancer progression. Several latent membrane protein 1 (LMP1) variants are described, discriminated mainly by variations in its C-terminal and the transmembrane domains of the protein. LMP1 C-terminal domain activate intracellular signaling pathways that regulated cell migration; moreover, LMP1 upregulate the expression of cell proteins with roles in extracellular matrix remodeling and angiogenesis. Currently it is unknown whether different LMP1 variants possess distinct properties regarding biological phenomena relevant to immune evasion, and cancer progression. Thus, in the present study we evaluated the in vitro migration and invasiveness, and immunomodulation of HLA-ABC, HLA-DR, CD80, CD83, CD54, CD40, and PD-L1 of HEK293T cells, and NP69 cells. We observed that Alaskan, Med+, China 1, and China 2 stimulates distinctly migration of NP69 when compared to cell without LMP1. We also saw that HEK293T-China 2 had a pronounced cellular invasion when compared to HEK293T. In regard of expression of molecules involved in immune response modulation, we observed that LMP1 enhanced expression of CD80, and CD83 in NP69-Alaskan cells; and CD54, and PD-L1 in a similar level in NP69-LMP1 cells. In summary, LMP1 promote distinct cellular migration in NP69LMP1 cells, and HEK293T-China 2 invasion, and is capable of modulate molecules involved in immune evasion
Mestre

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O Linfoma de Hodgkin clássico (LHC) é caracterizado pela presença de uma pequena população de células grandes mono ou multinucleadas, denominadas células de Hodgkin-Reed-Sternberg (HRS), circundadas por uma grande massa inflamatória de células não neoplásicas. O vírus Epstein-Barr (EBV) está associado ao Linfoma de Hodgkin em cerca de 50% dos casos. O diagnóstico do LH EBV relacionado é possível por meio da identificação de proteínas virais nas células HRS. Os métodos considerados ideais para essa identificação são as reações de imuno-histoquímica utilizando anticorpos antiproteína latente de membrana (LMP1) e hibridação in situ com uma sonda para o RNA viral (EBER). A LMP-1 é considerada um oncogene clássico. Foi demonstrado que a LMP-1 pode controlar a expressão do gene da metaloproteinase 9 (MMP-9), em linhagem de células C33A. A MMP-9 é um membro da família das endopeptidases que facilita a invasão tumoral e metástases pela degradação do estroma extracelular. Objetivos: avaliar se a expressão da MMP-9 está relacionada ao status do EBV no tumor e se houve impacto na sobrevida livre de eventos (SLE) e sobrevida global (SG) em pacientes com LHC. Casuística e Métodos: foram examinados 97 pacientes com LHC. Todos os pacientes foram submetidos a protocolos de tratamentos equivalentes (MOPPABV ou ABVD). O diagnóstico histopatológico foi revisto e o subtipo classificado de acordo com a OMS. Reações de imuno-histoquímica para LMP-1 e MMP-9 e hibridação in situ para EBER foram realizadas. Resultados: A presença do EBV foi identificada em 52,5% dos casos. Houve uma maior prevalência do subtipo histológico celularidade mista em pacientes EBV positivos (P = 0,005). Não houve diferença na positividade do EBV em relação à faixa etária, sexo, estádio da doença ou pela presença de sintomas B. A presença do EBV no LHC não influenciou a SLE (P = 0,38) ou a SG (P = 0,80) com uma mediana de acompanhamento de 71 meses. A expressão da MMP-9 ocorreu em 87,6% dos casos estudados. Não houve diferença de casos positivos e negativos em relação ao status do EBV (P = 0,59). Quando avaliada a intensidade da expressão da MMP-9 nos casos positivos também não observamos correlação com a presença do EBV (P = 0,62). Não houve diferença entre o resultado da MMP-9 e os parâmetros: subtipo histológico, estádio, presença de sintomas B, idade e sexo. Não houve influência da MMP-9 na SLE (P = 0,98) e SG (P = 0,60). Conclusões: Demonstramos que a prevalência do LH relacionado ao EBV na população estudada é de 52,5%, e que a presença do vírus não altera a evolução clínica, SLE e SG de pacientes tratados uniformemente. Concluímos ainda que a MMP-9 é fortemente expressa nas células HRS. Não há correlação entre a expressão de MMP-9 e o status do EBV. Nem a SG nem a SLE foram influenciadas pela expressão dessa enzima.
Clinical and histological features of classical Hodgkin lymphoma (cHL) are primarily due to the effects of cytokines, enzymes and chemokines produced by Hodgkin-Reed-Sternberg (HRS) cells and their surrounding inflammatory cells in response to signals triggered by etiological factors such as Epstein-Barr virus (EBV). Matrix metalloproteinase-9 (MMP-9) has been associated with poorer survival in patients with aggressive non-Hodgkin lymphomas. In EBV-related cancers the expression of viral latent membrane protein 1 (LMP1) correlates with an increased MMP-9 expression. In this study, we evaluated the prognostic relevance of MMP-9 expression and EBV status in HRS cells in patients with cHL in Brazil. Material and Methods: We selected 97 patients with cHL. Patients were included if they had: 1) > 18 years, 2) Undergone similar chemotherapy protocols, 3) Paraffin blocks available for review and for EBV and MMP-9 detection and 4) Clinical, epidemiological and laboratorial parameters available. Results: EBV was detected in 52.5% of all cases. MMP-9 expression positivity was found in 87.6% of all cases. There was no correlation between MMP-9 expression and EBV status. Response to treatment and relapse rate were independent of MMP-9 expression and EBV status. When stratified according to chemotherapy protocol used or disease stage, we still did not find any difference. MMP-9 positivity did not influence overall survival and event free survival. Conclusion: MMP-9 are expressed in the majority of HRS cells and did not correlated with EBV status or survival. The consistent MMP-9 expression in HRS cells makes this enzyme a potential target for therapy.
TEDE
BV UNIFESP: Teses e dissertações

Orientador: Glauco Issamu Miyahara
Coorientador: José Fernando Garcia
Banca: Eder Ricardo Biasoli
Banca: Luiz Alberto Veronese
Resumo: O vírus Epstein-Barr (EBV) faz parte da família herpesvirus humano e está relacionado com doenças benignas e malignas de cabeça e pescoço e sua possível relação com o carcinoma espinocelular (CEC) oral tem sido estudada. O objetivo deste estudo foi detectar a presença do EBV em pacientes com CEC oral e mucosa normal, correlacionando os achados com as variáveis clínico-patológicas, fatores de risco e sobrevida. Foi aplicada a nested-PCR em peças parafinizadas de CEC oral, sendo 20 amostras de assoalho bucal, 25 de língua e 12 de orofaringe. Também foi utilizado grupo controle, sem carcinoma espinocelular, com 19 amostras de mucosa oral normal. O vírus foi encontrado em 10% das amostras de assoalho bucal e 12% de língua, não sendo detectado na orofaringe. Para as amostras de mucosa normal a prevalência foi de 15,79%. Não houve diferença estatisticamente significante entre a presença do vírus e as variáveis: localização anatômica, sexo, idade, tabagismo, etilismo, estadiamento clínico, gradação histológica, esvaziamento cervical e sobrevida. Os resultados sugerem que o EBV não participa isoladamente da carcinogênese oral
Abstract: The Epstein-Barr virus (EBV) is part of human herpesvirus family being associated with benign and malignant diseases head and neck. Its possible relation with oral squamous cell carcinoma (OSCC) has been studied. The aim of this study was to detect the presence of EBV in patients with OSCC and normal mucosa, correlating the findings with clinicopathologic variables, risk factors and patient's survival. Nested PCR was applies in paraffin embedded samples of OSCC being 20 samples of mouth floor, 25 tongue and 12 oropharyngeal. A control group composed of 19 normal oral mucosa samples was used. The virus was found in 10% of mouth floor samples, 12% of tongue not being detected in oropharynx. The prevalence in oral mucosa samples was 15.79%. There was no statistically significant differences between the virus presence and the variables: anatomical localization, gender, age, smoking, alcoholism, clinical stage, histological grade, neck dissection and patient's survival. The results suggest that EBV alone did not have participation in carcinogenesis or oral SCC
Mestre

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Fundação para o Desenvolvimento da UNESP (FUNDUNESP)
O vírus Epstein-Barr (EBV) faz parte da família herpesvirus humano e está relacionado com doenças benignas e malignas de cabeça e pescoço e sua possível relação com o carcinoma espinocelular (CEC) oral tem sido estudada. O objetivo deste estudo foi detectar a presença do EBV em pacientes com CEC oral e mucosa normal, correlacionando os achados com as variáveis clínico-patológicas, fatores de risco e sobrevida. Foi aplicada a nested-PCR em peças parafinizadas de CEC oral, sendo 20 amostras de assoalho bucal, 25 de língua e 12 de orofaringe. Também foi utilizado grupo controle, sem carcinoma espinocelular, com 19 amostras de mucosa oral normal. O vírus foi encontrado em 10% das amostras de assoalho bucal e 12% de língua, não sendo detectado na orofaringe. Para as amostras de mucosa normal a prevalência foi de 15,79%. Não houve diferença estatisticamente significante entre a presença do vírus e as variáveis: localização anatômica, sexo, idade, tabagismo, etilismo, estadiamento clínico, gradação histológica, esvaziamento cervical e sobrevida. Os resultados sugerem que o EBV não participa isoladamente da carcinogênese oral
The Epstein-Barr virus (EBV) is part of human herpesvirus family being associated with benign and malignant diseases head and neck. Its possible relation with oral squamous cell carcinoma (OSCC) has been studied. The aim of this study was to detect the presence of EBV in patients with OSCC and normal mucosa, correlating the findings with clinicopathologic variables, risk factors and patient’s survival. Nested PCR was applies in paraffin embedded samples of OSCC being 20 samples of mouth floor, 25 tongue and 12 oropharyngeal. A control group composed of 19 normal oral mucosa samples was used. The virus was found in 10% of mouth floor samples, 12% of tongue not being detected in oropharynx. The prevalence in oral mucosa samples was 15.79%. There was no statistically significant differences between the virus presence and the variables: anatomical localization, gender, age, smoking, alcoholism, clinical stage, histological grade, neck dissection and patient’s survival. The results suggest that EBV alone did not have participation in carcinogenesis or oral SCC

This thesis focuses on Human herpesvirus 4 and 6, two ubiquitous viruses with a long list of putative disease associations, ranging from malignancies such as lymphomas and carcinomas to multiple sclerosis. To date, the relatively limited genetic data on these organisms hinders the understanding of their variability and their genetic structure at a population level. We here explore wet lab techniques for the production of genetic data in a cost-effective manner in order to reach the order of magnitude that is required to unravel the genetics these viruses. After successfully identifying individuals affected by integrated chromosomally inherited human herpesvirus 6 from public datasets of sequencing data, the sequences of the infecting virus were produced by target enrichment means from the source biological sample. The testing of an in-house target enrichment protocol followed, aiming to target latently infecting virus in human saliva. While the protocol still needs optimization and the aid of alternative techniques to be suitable for cost-effective, large-scale studies, the results were very satisfactory (up to >800-fold enrichment). In parallel, long range PCRs were used to produce human herpesvirus 4 latency genes sequences from one large human healthy saliva panel including populations previously unexplored in terms of human herpesvirus 4 isolates. Thanks to the combination of wet lab techniques and data analysis, the presence of genetic patterns in the two studied viruses is emerging, with human herpesvirus 6 presenting differences in diversity between its two species, as well as signs of geographical patterns possibly in part hidden by recombination events. Different bioinformatics approaches showed instead a stronger geographical stratification in human herpesvirus 4, with regional-driven clades. This information would allow us for a correct study design when addressing the relationship between virus and disease, taking into account the natural variation of the virus, as well as help to pinpoint genetic features that might be determinant for disease triggering or development. The strong geographical patterns presented by the diseases associated to these viruses strengthen the notion of the importance of this investigation and opens an avenue of research focused on disclosing the putative relationship between viruses strain variation and the risk for these virus–associated diseases.

AIMS OF THE STUDY: 1. To perform a retrospective, epidemiological analysis of cases of reactive lymphadenopathy and atypical reactive lymphoid hyperplasia (ARLH) received in the Department of Anatomical Pathology, UCT and GSH, over a 5 year period, in order to determine the number of cases of ARLH, and the frequency of the various subtypes of reactive lymphoid. hyperplasia, so as to provide base-line information for further studies. 2. To set up IN SITU HYBRIDIZATION (ISH) for detection of Epstein-Barr virus (EBV)-encoded RNA's (EBERs) in latently infected cells in selected cases, to determine if virus is present in ARLH. 3. To perform immunohistochemical analysis for the detection of EBY-derived latent membrane protein (LMP) in those cases subjected to ISH.

Epstein-Barr Virus (EBV) has two states of infection, latent and lytic. During the latent state the viral genome remains stable in cells as episomes and replicates with cellular DNA. During the lytic cycle the viral DNA becomes amplified and packaged in newly formed virions. An unsolved problem is whether newly replicated EBV DNA produced upon lytic cycle activation is associated with histones, and if so, whether these histones are acetylated. This question has biological significance as knowing the chromatin structure of genes is important in determining their function and expression profile. Our hypothesis is that newly synthesized EBV lytic DNA is associated with histones and the histone tails are selectively acetylated. To investigate our hypothesis we performed chromatin immunoprecipitation (ChIP) in HH514-16 cells, a Burkitts Lymphoma cell line, during latent and lytic replication. We used quantitative PCR (qPCR) to detect the relative concentration of DNA among the different samples. We tested three different variables: type of inducing agent, duration of treatment, and different regulatory regions in the genome of Epstein-Barr Virus. We found that in cells induced into the lytic cycle with Trichostatin A (TSA), a histone deacetylase inhibitor (HDACi), association of newly replicated EBV DNA with acetylated histone 3 (H3) increased ~ 6-10 fold. This increase in association was greatest 72 hrs after treatment. Furthermore, activation of lytic viral replication in HH514-16 cells using a different inducing agent, Azacytidine (AZC), which is known to function as a DNA methyltransferase inhibitor, increased binding of H3 with viral DNA ~8 fold. However, unlike TSA, AZC increased the acetylation state of histones bound to newly synthesized viral DNA only ~ 2 fold. Changing the regulatory region of the EBV genome analyzed in qPCR did not affect our results. Our results suggest that newly replicated viral DNA is associated with histones, a fraction of which are acetylated. The degree of acetylation likely depends on the agent used to induce the lytic cycle. H3 is highly acetylated when an HDACi is used and less acetylated when AZC is used. Our study provides new insight on the epigenetic profile of newly replicated viral DNA during the lytic cycle. It remains to be determined whether histones are packaged together with viral genomes into virions and whether the chromatin state of virion DNA affects gene expression after the virus enters uninfected cells.

Epstein-Barr virus (EBV) induced infectious mononucleosis (IM) is characterized by the activation and expansion of T lymphocytes and the induction of cytotoxic responses able to mediate the lysis of EBV-uninfected, allogeneic MHC mismatched and EBV-infected autologous target cells. Freshly isolated peripheral blood mononuclear cells (PBMC) were used to examine the nature of these cellular immune responses. Activated lymphocytes, as identified by HLA-DR expression, associated with EBV induced IM were shown to be a heterogeneous population containing significantly elevated cytotoxic/suppressor (CD8+) T cells, helper/inducer (CD4+) T cells and natural killer (NK, CD16+) cells. CD8+ T cells were the primary activated population, representing 24% of the total lymphocyte population and 60-70% of the CD8+ T cell population. The activated CD4+ T cells and natural killer (NK) cells accounted for 7% and 4% of the total lymphocyte population, respectively. Analysis of serum soluble interleukin 2 receptors (IL-2R) and CD8 molecules demonstrated significantly (p<0.001) elevated levels in the sera of IM patients compared with normal controls. These elevated levels of serum IL-2R am CD8 molecules correlated, (r=0. 67 and r=0.82, respectively) with increased percentages of CD8/HLA-DR positive T cells (i.e., activated CD8 T cells). Increased levels of soluble cell surface molecules peaked during the acute phase and normalized as the patients progressed toward convalescence. Individual patients demonstrated strong correlations between the percentage of CD8/HLA-DR positive cells and soluble CD8 levels. The functional significance of the serum IL-2R and CD8 molecules is presently unknown. However, the strong correlative data between serum CD8, and to a lesser extent IL-2R, and CD8 T cell activation suggests that serum CD8 levels may provide a sensitive measure of CD8 T cell activation in systemic infections. The ability of freshly isolated acute IM PBMC to lyse allogeneic, EBV-infected lymphoblastoid cell lines (LCL), demonstrated the ability of acute IM effector cells to lyse MHC mismatched target cells. Effector cells from acute IM patients lysed allogeneic DM-LCL and AF-LCL target cells by 34% (n=7) and 23% (n=6), respectively, compared with 4% (n=5) and 0% (n=5), respectively, for normal controls. MAb-dependent complement depletion of CD3+ or CD8+ cells with anti-CD3 and anti-CD8 mAb decreased the non-MHC restricted cytolysis of LCL by 96% and 89%, respectively. In contrast, complement depletion with NK-cell specific mAbs Leu 11b and NKH-1, resulted in only a slight decrease (<35%) in the lysis of these LCL (46%). Depletion with anti-HLA-DR also significantly (p<0.001) decreased the lysis of LCL. Depletions with anti-CD4 demonstrated no decrease in LCL-lysis. MAbs OKT3 and OKT8 inhibited the non-MHC restricted cytolysis by 87% and 82%, respectively. We interpret these results as evidence that, 1) lysis of allogeneic cells is mediated primarily by CD3+, CD8+, HLA-DR+, cytotoxic T lymphocytes (CTL); and 2) these acute IM cytotoxic T cells utilize the T cell receptor and the CD8 antigen as an accessory molecule. An active role for target cell MHC class I molecules in the recognition and subsequent lysis of target cells is supported by a number of observations: 1) the MHC class I reactive mAbs W6/32 and BBM.1 significantly (p<0.05) inhibited the lysis of 63463-LCL by 65% and 57%, respectively; 2) acute IM effector T cells did not lyse the MHC class I negative Daudi cell line; 3) allogeneic MHC class I matched LCL mediated strong competitive inhibition (72% at 10:1 competitor to target cell ratio) vs 29% competitive inhibition for an allogeneic MHC class I mismatched LCL; and 4) NK-cell depleted effector cells from one patient mediated preferential lysis of the K562 cell line expressing MHC class I. HLA-A2 molecules. We interpret these results as evidence that target cell MHC class I molecules (or associated determinants) are the target antigen(s) for the allogeneic MHC cytotoxic response. The role of EBV in this acute allogeneic response was examined using target cell lines devoid of EBV genome. Acute IM CTL mediated lysis of the allogeneic HSB-2 T cell line (45%), and allogeneic HTLV-I transformed T cell lines (16%). The lysis of the HSB-2 T cell line was inhibited by anti-OKT3 (58% inhibition), W6/32 (53%) and BBM.1 (42%). Similarily, lysis of HTLV-I T cell lines was inhibited by W6/32 (69% inhibition), BBM.1 (69%) and OKT3 (38%). These data demonstrate that EBV antigenic expression is not required for allogeneic recognition and subsequent lysis of these allogeneic target cells. Effector cells from acute IM patients (n=5) were able to lyse their autologous EBV-infected LCL (mean lysis=21%), but were unable to lyse the EBV-uninfected autologous HTLV-I T cell line. These same effectors, however, were able to mediate lysis of both allogeneic B cell lines (21% lysis) and allogeneic T cell lines (8% lysis). These data are consistent with the observations by Strang et al. (1987a), who recently cloned virus specific/MHC-restricted CTL cloned from acute IM PBMC. These virus specific/MHC-restricted T cells presumably mediate the lysis of the autologous EBV-transformed B cell lines but not the autologous EBV-uninfected T cell lines. Whether the CTL which lyse the autologous EBV-transformed LCL are also responsible for the observed allogeneic reactivity was examined with cold target competition using autologous and allogeneic LCL. Lysis of autologous LCL was inhibited only by autologous competitor cells (64% inhibition compared with 24% for allogeneic LCL). Likewise, lysis of the allogeneic LCL was inhibited only by the allogeneic competitor cells (85% inhibition compared with 30% for autologous LCL). These data demonstrated no competition between allogeneic and autologous LCL and therefore support the concept that lysis of autologous LCL and allogeneic target cells is mediated by distinct effector populations. These data help us to understand the unusual immune response observed during acute IM. The strong allogeneic cytotoxic response is thought to represent polyclonal CD8 T cell activities induced by EBV-infected and transformed B cells which circulate in vivo. In addition, a population of CD8 CTL exist which mediate the lysis of autologous EBV-transformed B cells. These CTL likely represent virus-specific/MHC-restricted CTL and presumably play a major role in the control of EBV infections. The role, if any, of the markedly expanded alloreactive CTL population in the elimination of EBV infected and transformed B cells remains to be clarified.

Heterologous immunity is a mechanism by which immunological memory within an individual, developed in response to a previous infection, plays a role in the immune response to a subsequent unrelated infection. In murine studies, heterologous immunity facilitated by cross-reactive CD8 T-cell responses can mediate either beneficial (protective immunity) or detrimental effects (e.g. enhanced lung and adipose immunopathology and enhanced viral titers) (Selin et al., 1998; Chen et al., 2001; Welsh and Selin, 2002; Nie et al., 2010; Welsh et al., 2010). Protective heterologous immunity results in enhanced clearance of virus during a subsequent infection with an unrelated pathogen. Such is the case when mice are immunized with lymphocytic choriomeningitis virus (LCMV) and subsequently challenged with Pichinde virus (PV) or vaccinia virus (VACV) (Selin et al., 1998). However, heterologous immunity may also mediate enhanced immunopathology as mice immunized with influenza A virus (IAV) and challenged with LCMV show increased viral titers and enhanced lung immunopathology (Chen et al., 2003). The role heterologous immunity plays during infection is not limited to the murine system. In fact, there have now been several reports of enhanced immunopathology due to heterologous immunity during human infections, involving viruses such as IAV, Epstein-Barr Virus (EBV), hepatitis C virus (HCV), and dengue virus (DENV) (Mathew et al., 1998; Wedemeyer et al., 2001; Acierno et al., 2003; Nilges et al., 2003; Clute et al., 2005; Urbani et al., 2005). Interestingly, in all reported cases in humans, heterologous immunity mediated enhanced immunopathology. Upon infection with EBV the clinical presentation can range from asymptomatic to severe, occasionally fatal, acute infectious mononucleosis (AIM) (Crawford et al., 2006b; Luzuriaga and Sullivan, 2010) which is marked by a massive CD8 lymphocytosis. This lympho-proliferative effect in AIM was shown to be partially mediated by reactivation of cross-reactive IAV-M1 58-66 (IAV-GIL) specific CD8 memory T-cells in HLA-A2 patients reacting to the EBV-BMLF1 280 (EBV-GLC) epitope (Clute et al., 2005). Interestingly, EBV infects ~90% of individuals globally by the third decade of life, establishing a life-long infection (Henle et al., 1969). However, it is unknown why 5-10% of adults remain EBV-sero-negative (EBV-SN), despite the fact that the virus infects the vast majority of the population and is actively shed at high titers even during chronic infection (Hadinoto et al., 2009). Here, we show that EBV-SN HLA-A2+ adults possess cross-reactive IAV-GIL/EBV-GLC memory CD8 T-cells that show highly unique properties. These IAV-GIL cross-reactive memory CD8 T-cells preferentially expand and produce cytokines to EBV antigens at high functional avidity. Additionally, they are capable of lysing EBV-infected targets and show the potential to enter the mucosal epithelial tissue, where infection is thought to initiate, by CD103 expression. This protective capacity of these cross-reactive memory CD8 T-cells may be explained by a unique T-cell receptor (TCR) repertoire that differs by both organization and CDR3 usage from that in EBV-seropositive (EBV-SP) donors. The composition of the CD8 T-cell repertoire is a dynamic process that begins during the stochastic positive selection of the T-cell pool during development in the thymus. Thus, upon egress to the periphery a naïve T-cell pool, or repertoire, is formed that is variable even between genetically identical individuals. This T-cell repertoire is not static, as each new infection leaves its mark on the repertoire once again by stochastically selecting and expanding best-fit effectors and memory populations to battle each new infection while at the same time deleting older memory CD8 T-cells to make room for the new memory cells (Selin et al., 1999). These events induce an altered repertoire that is unique to each individual at each infection. It is this dynamic and variable organization of the T-cell repertoire that leads to private specificity even between genetically identical individuals upon infection with the same pathogens and thus a different fate (Kim et al., 2005; Cornberg et al., 2006a; Nie et al., 2010). It is this private specificity of the TCR repertoire that helps explain why individuals with the same epitope specific cross-reactive response, but composed of different cross-reactive T-cell clones, can either develop AIM or never become infected with EBV. Our results suggest that heterologous immunity may protect EBV-SN adults against the establishment of productive EBV infection, and potentially be the first demonstration of protective T-cell heterologous immunity between unrelated pathogens in humans. Our results also suggest that CD8 T-cell immunity can be sterilizing and that an individual’s TCR repertoire ultimately determines their fate during infection. To conclusively show that heterologous immunity is actively protecting EBV-SN adults from the establishment of a productive EBV infection, one would have to deliberately expose an individual to the virus. Clearly, this is not an acceptable risk, and it could endanger the health of an individual. A humanized mouse model could allow one to address this question. However, before we can even attempt to address the question of heterologous immunity mediating protection from EBV infection in humanized mice, we must first determine whether these mice can be infected with, and build an immune response to the two viruses we are studying, EBV and IAV. We show here that these mice can indeed be infected with and also mount an immune response to EBV. Additionally, these mice can also be infected with IAV. However, at this time the immune responses that are made to these viruses in our established humanized mouse model are not substantial enough to fully mimic a human immune response capable of testing our hypothesis of heterologous immunity mediating protection from EBV infection. Although the immune response in these mice to EBV and IAV infection is not suitable for the testing of our model the data are promising, as the humanized mouse model is constantly improving. Hopefully, with constant improvements being made there will be a model that will duplicate a human immune system in its entirety. This thesis will be divided into 5 major chapters. The first chapter will provide an introduction to both general T-cell biology and also to the role of heterologous immunity in viral infection. The second chapter will provide the details of the experimental procedures that were performed to test our hypothesis. The third chapter will describe the main scientific investigation of the role of heterologous immunity in providing natural resistance to infection in human subjects. This chapter will also consist of the data that will be compiled into a manuscript for publication in a peer-reviewed journal. The fourth chapter will consist of work performed pertaining to the establishment of a humanized mouse model of EBV and IAV infection. The establishment of this model is important for us to be able to show causation for protection from EBV infection mediated by heterologous immunity.

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus which causes acute infectious mononucleosis and is etiologically associated with malignant lymphoproliferative disorders including Burkitt's lymphoma, nasopharyngeal carcinoma, B-cell lymphomas in immunocompromised hosts, Hodgkin's disease, T cell lymphomas, and smooth muscle tumors in allograft recipients. The medical significance of EBV is underscored by its potent growth transforming effects on human B-lymphocytes in-vitro and the potentially oncogenic consequences of infection in-vivo. The majority of EBV-associated malignancies occur in the setting of chronic infection and strong virus-specific humoral immunity, suggesting that cellular immunity is primarily responsible for preventing the outgrowth of EBV-transformed B cells in-vivo. Similarly, primary EBV infection in adolescents and adults stimulates an intense cytotoxic-T-lymphocyte (CTL) response which coincides with a marked reduction in the number of infected B cells in the peripheral blood. Evidence of previous EBV infection can be confirmed by the presence of EBV-specific, HLA-restricted memory T cells in the peripheral blood which inhibit the outgrowth of newly EBV-transformed B cells and efficiently lyse established autologous B-lymphoblastoid cell lines. Worldwide, EBV is responsible for substantial morbidity, comparable to measles, mumps and hepatitis virus, for which vaccines exists. Accordingly, the potential public health impact of an EBV vaccine has reinforced our efforts to identify the immunodominant virus-encoded T-cell epitopes which stimulate naive CTL effectors during acute infection and maintain memory CTL surveillance during convalescence. The EBV-encoded antigens against which the memory CTL response is directed have been partially defined, and include most of the EBV latent proteins (EBNA-2, 3a, 3b, 3c, LP, and LMP-l, 2a, 2b) consistently expressed by in-vitro EBV-transformed B lymphocytes (type-III latency). Importantly, all EBV-associated malignancies express EBNA-1, and as yet no EBNA-1-specific memory CTL have been convincingly demonstrated. Additionally, many EBV-specific CTL lines and clones have been described which do not recognize any of the known latent proteins or other EBV protein antigens tested thus far. Thus while much is known about CTL-mediated immunity against EBV, our knowledge of EBV-derived CTL epitopes remains incomplete. In contrast to the EBV-specific memory CTL response, very little is known about the source of viral epitopes recognized during the primary CTL response to EBV. In this regard, acute infectious mononucleosis represents an ideal model system to study virus-specific, cell-mediated immunity. Acute IM is a self-limited illness characterized by the appearance of "atypical" lymphocytes (CD3+/CD8+/HLADR+), including both virus-specific and alloreactive CTL, which undoubtedly contribute to virus elimination and provide CTL precursors for life-long immunity to EBV. Like other herpesvirus, EBV can undergo either lytic or latent cycle replication. During primary EBV infection many lytic cycle genes are expressed which are likely responsible for stimulating the intense cellular immune response associated with acute infectious mononucleosis. During convalescence a minor population of circulating B cells remain latently infected, harbor multiple EBV episomes, and express only EBNA-1 and possibly LMP-2a (type-I latency). Thus, latency type-I infected B cells in-vivo express a much more restricted spectrum of latent proteins and are therefore not subject to elimination by the same virus-specific CTL as are type-III EBV latently infected cells. Accordingly, many mechanisms have been proposed to explain EBV persistence including; restricted expression of EBV latent genes, reduced levels of cellular adhesion molecules, downregulation of MHC class-I molecules, absence of EBNA-1 T-cell-epitopes, and most recently, EBNA-1-mediated inhibition of antigen processing. While these mechanisms may contribute to ineffective T cell surveillance against latency type-I EBV- infected cells, B cells expressing the full spectrum of latent proteins (type-III) also exist transiently in vivoand maintain detectable humoral and CTL responses to most latent proteins. Our first goal was to identify the virus-encoded immunodominant antigens recognized by in-vivoactivated MHC class-I restricted CTL isolated from college students experiencing primary EBV infection, manifested as acute IM. Following a prodromal period of several weeks, newly EBV infected patients present with signs and symptoms of acute IM, including elevated numbers of activated CD8+ T cells in their peripheral blood, many of which, like memory CTL, are EBV-specific and HLA-restricted. In order to address the issue of EBV persistence and the immune control of EBV-induced lymphoproliferation, we also studied the long-term EBV-specific memory CTL response in these same individuals. Blood from acute IM patients and healthy EBV seropositive donors served as a source of peripheral blood lymphocytes to generate bulk CTL cultures and autologous target cells. The infecting strain of EBV was determined for each patient by DNA-PCR amplification of virus from saliva. Lymphocytes were isolated from whole blood by Ficoll-Paque density centrifugation and T- and B-cell enriched populations were obtained by AET-sheep red cell rosette selection. Autologous B cell blasts served as a source of target cells and recombinant vaccinia virus constructs were used to introduce individual EBV latent genes into target cells. Expression of individual EBV genes in target cells was confirmed by both western blot and immunofluorescence. Primary CTL responses to EBV were evaluated in standard 5lCr release assays using freshly isolated, T-cell enriched PBL from acute IM patients as effector cells. EBV-specific memory CTL responses were evaluated with bulk CTL culture generated by in-vitro restimulation with autologous B-LCLs. FACS analyses were routinely performed on bulk cultures of effector CTL populations in order to more clearly characterize their phenotype. Lastly, monoclonal antibody blocking studies and cold target competition assays were performed in order to accurately identify the viral antigen and MHC components responsible for target cell recognition. Our results based upon evaluation of 35 acute IM patients and 32 convalescent patients demonstrate that the virus-specific primary CTL response is broadly directed against the full spectrum of latent proteins, including EBNA1 and the viral coat glycoprotein gp350, while the memoryCTL response, which essentially lacks EBNA1 reactivity, is directed primarily against the EBNA 3 family of proteins (3A, 3B, 3C). Importantly, the immunodominant response by both primary and memory CTL was directed against the EBNA3 proteins. CTL from 7 of the 35 acute IM patients evaluated recognized EBNA1 expressing targets, and in 4 of these 7 patients, EBNA1 was an immunodominant antigen. Similarly, CTL from 7 of 35 acute IM patients recognized gp350 transfected targets, while no gp350-specific memory CTL responses were observed. While the phenotype of in-vivo primed CTL effectors were CD8+/HLA-DR+/CD11b+, the major subpopulation of memory CTL were CD8+/HLA-DR+/CD11b-. The CD11b "memory marker" reached peaked levels on the first sample day for all patients and gradually declined to baseline levels over a period of several months. In contrast, the CD11b marker was quickly shed from in vitropropogated CTL, over a period of 5-10 days. Target cell lysis by in-vivoactivated CTL was almost completely blocked by antibody directed againt [against] class-I molecules (BBM.1), whereas the effect of blocking target cell lysis by anti-CD8 mAb varied between 40-75%. These findings are consistent with an absolute need for class-I restricted antigen presentation, and imply that CD8 was variably required, likely for the lower affinity TCR/ Ag combinations. Cell lysis mediated by in-vitro-restimulated memory CTL was also largely inhibited by anti-class-I mAb, while anti-CD8 mAb was only mild/moderately effective in blocking target cell lysis, in keeping with the concept that memory CTL bear higher avidity TCR which can recognize antigen independent of CD8. Our detection of only one EBNA1-specific memory CTL response among the 32 patients tested supports the theory that latently infected B cells in-vivo, expressing only EBNA1, escape CTL recogition and thus might serve as a reservoir for viral persistence and/or reactivation. The rare ability to detect an EBNA1-specific memory CTL responses remains a relatively unexplained phenomenon and may involve a number of tolerizing mechanisms including the induction of anergy by presentation of EBNA-1 in the absence of costimulation, clonal deletion of low affinity T cells, the absence of dominant T cell epitopes within EBNA1 or a result of the recently described inhibiting properties of EBNA-1 on antigen processing and presentation. Alternatively, the absence of detectable EBNA1-specific memory CTL may be the result of insufficient or inappropriate restimulation of memory CTL in vitro. We addressed this possibility by attempting to selectively restimulate and expand EBNA1-specific CTL from acute IM patients by using EBNA1 expressing B cells blasts as a stimulus. Effector cells generated in this manner killed target cells in an MHC class-I restricted manner but were specific for an unspecified vaccinia antigen. Interestingly, the phenotype of the effector cells was predominantly CD3+/CD4-/CD8-/γδ T cells. In summary, our findings suggest that a multitude of previously unrecognized, EBV-specific CTL are present in the peripheral blood during acute IM, and include EBNA-1-specific CTL. The importance of accurately defining the in-vivo immune response to EBV is underscored by the ever-growing list of EBV associated malignancies. In addition to providing insights into the oncogenesis and potential treatment of NPC, a newly described link between precursor lesions and EBV infection raises the possibility that heightened immunity to EBV or EBV-infected cells may prevent the development of NPC. An obvious expectation would include extension of such knowledge to other EBV associated malignancies such as B and T cell lymphomas, Hodgkin's lymphomas, and smooth muscle tumors. First however, existing gaps in knowledge regarding the immune response to EBV and EBV-associated malignancies must be closed. Details about the viral gene products which are involved in stimulating a broadly protective, virus-specific immune response in a large number of individuals is fundamental to the design of an effective EBV vaccine. Since the presence of activated CD8+ T cells correlates with the rapid decline of EBV infected B cells in the peripheral blood, a concise description of the EBV-specific CTL response in the setting of acute infection will be necessary for the rational design of an effective acute IM vaccine. Increased understanding of viral escape mechanisms is also likely to contribute to therapeutic modalities to treat autoimmune disorders.

Epstein-Barr virus (EBV) is a ubiquitous herpesvirus that establishes a life-long latent infection of B cells. It is usually asymptomatic in healthy individuals; however, individuals with compromised immunity often develop EBV-induced lymphoma. EBV also encodes potential oncogenes that can contribute to tumorigenesis. Therefore, vaccine and immunotherapeutic strategies targeting EBV are desirable. Recent studies have shown that infusion of EBV-specific CD8+T cells can elicit remission of lymphomas arising after administration of immunosuppressive drugs during transplantation, suggesting an important role for T cells in the prevention of EBV-induced malignancy. A better understanding of the cellular immune components involved in the control of EBV will aid in the development of methods to prevent infection and/or treat EBV-associated disease. While EBV infection is usually acquired asymptomatically during childhood, primary infection of adolescents and young adults can result in an illness termed acute infectious mononucleosis (AIM). Because of the characteristic symptoms of the illness, individuals with AIM can be readily identified and diagnosed with acute EBV infection. Thus, primary CD4+ and CD8+ T cell responses against the virus can be evaluated. It has been previously found that there is a marked expansion of lytic EBV protein-specific CD8+ T cells early during AIM, with delayed detection of lower frequencies of latent EBV protein-specific CD8+ T cells. The magnitude and specificity of CD4+T cell responses during AIM has been less well characterized. This thesis dissertation presents data from both functional assays and direct staining experiments documenting the timing, magnitude, and antigen-specificity of CD4+ T cells over the course of primary EBV infection. Lytic and latent protein-specific CD4+ T cells were readily detected by intracellular IFN-γ production at presentation with AIM and declined rapidly thereafter. Blood EBV load was also quantitated and found to decrease over time following AIM. By contrast, CD8+T cell IFN-y responses remained high for several weeks following presentation with AIM. Direct staining of lytic epitope-specific CD4+ T cells during AIM revealed high frequencies of virus-specific cells with low proliferative and IFN-γ-producing potential. Blood EBV load in these patients was persistently high through 6 wk following AIM. These data suggest a relationship between high EBV load during acute infection and impaired EBV-specific CD4+ T cell responses, which are compatible with impaired CD4+ T cell responses reported during high viremia associated with other viral infections. This may represent a mechanism by which persistent viruses, such as EBV, are able to establish a life-long infection in their hosts.

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014
HLH is a rare syndrome which results from an ineffective and uncontrolled immune response with hypercitocinemia and hyperinflammation. It can be classified as primary (FHLH) or secondary (associated with infection, autoimmune diseases, malignancy and immune suppression states). The primary infectious cause of secondary HLH is Epstein-Barr virus and the incidence of this association varies between reported studies and is more prevalent in Asian countries such as Japan. The clinical symptoms and sins appear more frequently in the first years of life and they are fever, hepatoslepnomegaly and cytopenias. The diagnosis and treatment are based on the Histiocyte Society guidelines which were revised in 2004. In this thesis it is presented the clinical case of HLH-EBV in a 14-year old adolescent, which is characterized by fever, hepatosplenomegaly, cytopenias and neurological compromise. The diagnosis was made early and the clinical course and laboratory data evolution was good during the stay at the hospital. This case was compared with other 31 pediatric cases presented in the literature of HLH and EBV infection with or without genetic disease. It was analyzed the clinical and laboratory data and the results of the HLH-2004 protocol. The scarce number of cases did not allow statistical analysis. Despite the limitations of this review, we can conclude that: the diagnosis of this syndrome is difficult, there still is a high mortality rate despite therapy and the role of rituximab is still not well defined.
A HLH é uma síndrome rara, que resulta duma resposta imune ineficaz e não controlada com hipercitocinémia e hiperinflamação. Pode ser classificada como primária (FHLH) ou secundária (associada a infecções, doenças auto-imunes, neoplasias e estados de supressão imunitária). A principal causa infecciosa de HLH secundário é o vírus Epstein-Barr, e a incidência desta associação varia entre os estudos reportados, sendo maior em países asiáticos, nomeadamente no Japão. As manifestações clínicas surgem com mais frequência nos primeiros anos de vida, sendo as mais comuns: febre, hepatoesplenomegália e citopénias. O diagnóstico e tratamento baseiam-se nas recomendações da Histiocyte Society revistas em 2004. Nesta dissertação descreve-se o caso clínico de HLH-EBV numa adolescente de 14 anos, caracterizado por : febre, hepatoesplenomegália, citopénias e compromisso neurológico. O diagnóstico foi feito precocemente, havendo boa evolução clinica e laboratorial durante o internamento com a implementação do protocolo HLH-2004. Comparou-se este caso a 31 casos pediátricos descritos na literatura de HLH e infecção EBV com ou sem doença genética associada. Analisaram-se parâmetros clínicos e laboratoriais no diagnóstico e os resultados da instituição do protocolo de tratamento HLH-2004. O escasso número de casos não permitiu avaliação estatística. Apesar das limitações desta revisão, pode-se concluir que: o diagnóstico desta síndrome é difícil, ainda se verifica uma elevada taxa de mortalidade apesar da instituição da terapêutica e que o papel do rituximab na terapêutica desta síndrome ainda não está bem definido.

1. Study on improving the diagnostic accuracy of treatment-naive nasopharyngeal carcinoma.
2. Study on diagnostic accuracy of EBV-DNA on recurrent nasopharyngeal carcinoma.
3. Studies on EBV-DNA as a screening tool for nasopharyngeal carcinoma. Part 1. To define the detection rate of NPC and the false-positive rate of IgA-VCA in an IgA-VCA-based screening problem, and to define the specificity of IgA-EA in IgA-VCA-positive screenees. Part 2. To define the specificity of EBV-DNA in IgA-VCA-positive screenees. Part 3. To define the sensitivity of IgA-EA, and EBV-DNA in IgA-positive NPC patients.
4. Studies on pre-therapy prognostication of nasopharyngeal carcinoma Study Part 1. Objective. To assess the role of EBV-DNA in pre-therapy prognostication of early-stage NPC.
Background. The specific association between nasopharyngeal carcinoma (NPC) and the Epstein-Barr virus (EBV) had been exploited to develop a spectrum of EBV-antibodies-based blood markers. Among these markers, the Immunoglobulin A antibody against the viral capsid antigen (IgA-VCA) of the EBV has been the most popularly employed marker to assist diagnosis of NPC. There is however a relative paucity of data on the application of blood markers for screening, for detection of relapse, and for prognostification of patient cohorts managed in present-day therapy oncology protocols. Peripheral blood EBV-DNA, measured by quantitative polymerase chain reaction assay, is a newly-developed marker, and represents a prototype model of a nuclei acid-based, as opposed to antibody-based, EBV tumor marker for NPC. The present thesis describes the translation of this basic scientific advance into clinical applications, through several prospective and retrospective studies that address the diagnosis of treatment-naive NPC, the detection of recurrent NPC, the screening of individuals at risk of NPC, the pre-therapy prognostication for NPC to guide for choice of therapy. The role of integration of conventional markers and EBV-DNA in clinical applications was also examined.
Study Part 2. Objectives. To assess whether incorporation of EBV-DNA data to TNM staging improves prognostic discrimination of patients subsets within individual cancer stage, to assess if EBV-DNA is an independent prognostic factor for survival after ontological therapy. (Abstract shortened by UMI.)
Leung Sing-fai.
"February 2005."
Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3695.
Thesis (M.D.)--Chinese University of Hong Kong, 2005.
Includes bibliographical references.
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, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
School code: 1307.

A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in partial fulfilment of the requirements for the degree of Master of Medicine in the branch of Anatomical Pathology Johannesburg, 2016
Introduction: Plasmablastic lymphoma (PBL) is an uncommon variant of aggressive B-cell non-Hodgkin lymphoma that occurs in immune-compromised individuals, most commonly secondary to HIV infection. This tumour classically occurs in the oral cavity but has also been described in a variety of extra-oral locations. This is a clinicopathological study of 45 cases of extra-oral PBL (EPBL). Aim: To define the clinical parameters, histology and immunophenotypic features of extra-oral plasmablastic lymphoma (EPBL) and assess the extent to which Epstein–Barr virus (EBV) is associated with this tumour. Materials and Methods: This retrospective study on archival cases of EPBL included the patients‟ age, gender, race, HIV status where available and the site of tumour presentation. Of 49 archival cases retrieved, 4 were discounted owing to reclassification as diffuse large B-cell lymphoma (1 case), multiple myeloma or extramedullary plasma cell tumour (3 cases). The remaining 45 cases were reviewed histologically and classified according to whether they displayed a pure plasmablastic (PBm) morphology or a plasmablastic morphology with plasmacytic differentiation (PBm+PCd), and assessed immunohistochemically with CD45 (LCA), CD20, CD79a, PAX5, CD138, MUM1, BLIMP1, VS38c, Ki-67, BCL6, CD10, HHV8 and CyclinD1 using standard automated procedures. The presence of EBV was assessed by chromogenic in-situ hybridisation. Ethical clearance was obtained (M10750 and M120993). Results: 27of the 45 cases had a pure plasmablastic morphology. The remaining 18 cases showed plasmacytic differentiation. There was no site predilection according to histological pattern. 60% of the tumours were reported in males and 40% in females and all were black African patients. The anus was the favoured extra-oral site of presentation (13 of 45 cases, 28%), followed by soft tissue (11 of 45 cases, 24%). There was no significant difference in the age of presentation between males (38.5 years) and females (35.4 years). Of the 18 patients of known HIV status, 17 were HIV positive (94%). The immunohistochemical profile of EPBL recapitulated that found for both oral and extra-oral PBL in the literature, except for CD45 (leucocyte common antigen) which signalled positively in a higher percentage of cases. 36 of 42 cases (85.7%) were positive for CD45. The positive membrane signal for CD45 was of variable intensity, between 5 and 100% of tumour cells. EBV was positive by in situ hybridisation in 37 of 40 cases tested (92.5%). Conclusion: EPBL is identical to its oral counterpart in gender and age distribution, HIV status, morphological appearances, immunophenotypic profile and association with EBV. The high association with EBV as assessed by in-situ hybridisation studies mirrors that of oral-based PBL reported in the literature. EPBL should be regarded as the same tumour as that arising within the oral cavity. A peculiarity observed within this case cohort is the high level of expression of CD45 (leucocyte common antigen). This has been reported to be of low or near absent expression in most cases of PBL, as defined by the 2008 WHO Classification of Tumours of the Haematopoietic and Lymphoid Tissues.
MT2016

by Cheung Siu Tim.
Thesis (Ph.D.)--Chinese University of Hong Kong, 1996.
Includes bibliographical references (p. 155-160).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Mode of access: World Wide Web.

Ng Dean Yew, Dennis.
Thesis (M.Phil.)--Chinese University of Hong Kong, 1996.
Includes bibliographical references (leaves 85-98).
Abstract --- p.i
Acknowledgments --- p.iii
Table of contents --- p.iv
List of figures --- p.vii
List of tables --- p.ix
List of abbreviation --- p.x
Chapter Chapter 1 --- Introduction
Chapter 1.1. --- History --- p.1
Chapter 1.2. --- Classification and structure of Epstein-Barr Virus --- p.2
Chapter 1.3. --- Genomic organization of EBV --- p.3
Chapter 1.4. --- Replication cycle of EBV --- p.5
Chapter 1.5. --- EBV latent and lytic cycle proteins --- p.6
Chapter 1.6. --- Clinical diseases associated with EBV Infection --- p.11
Chapter 1.7. --- Association of EBV and NPC --- p.13
Chapter 1.8. --- EBV serological markers in the diagnosis of NPC --- p.13
Chapter 1.9. --- Sources of EBV-specific DNase --- p.15
Chapter 1.10. --- Characteristics of Epstein-Barr virus alkaline DNase --- p.15
Chapter 1.11. --- Aim of the project --- p.18
Chapter Chapter 2 --- Materials & Methods
Chapter 2.1. --- Molecular cloning --- p.19
Chapter 2.1.1. --- Cell culture --- p.19
Chapter 2.1.2. --- mRNA purification --- p.19
Chapter 2.1.3. --- First strand cDNA synthesis --- p.21
Chapter 2.1.4. --- Polymerase chain reaction (PCR) of cDNA --- p.21
Chapter 2.1.5. --- Purification of PCR product after gel electrophoresis --- p.22
Chapter 2.1.6. --- Ligation of PCR amplified DNase gene into pUC18 Sma/BAP vector --- p.23
Chapter 2.1.7. --- Transformation by electroporation --- p.24
Chapter 2.1.7.1. --- Cell preparation --- p.24
Chapter 2.1.7.2. --- Electroporation procedure --- p.25
Chapter 2.2. --- Extraction ofplasmid DNA --- p.28
Chapter 2.2.1. --- Boiling preparation --- p.28
Chapter 2.2.2. --- Plasmid digestion --- p.29
Chapter 2.3. --- Large-scale purification ofplasmid --- p.29
Chapter 2.4. --- Small-scale purification ofplasmid --- p.32
Chapter 2.5. --- DNA sequencing --- p.33
Chapter 2.5.1. --- Annealing of primer to template DNA --- p.33
Chapter 2.5.2. --- Labelling reaction --- p.34
Chapter 2.5.3. --- Sequencing termination reaction --- p.35
Chapter 2.5.4. --- Prepartion of sequencing gel --- p.36
Chapter 2.5.5. --- Autoradiography of sequencing gel --- p.38
Chapter 2.6. --- Epitope mapping --- p.39
Chapter 2.6.1. --- Processing of EBV- specific DNase peptides --- p.39
Chapter Chapter 3 --- Results
Chapter 3.1. --- Molecular cloning --- p.41
Chapter 3.1.1. --- Cell culture --- p.41
Chapter 3.1.2. --- mRNA purification --- p.42
Chapter 3.1.3. --- PCR amplification --- p.42
Chapter 3. 1.4 --- DNA purification of PCR product --- p.42
Chapter 3.1.5. --- Molecular cloning of PCR amplified DNase gene into pUC18 SmaI/BAP vector --- p.44
Chapter 3.1.6. --- Transformation by electroporation --- p.46
Chapter 3.1.7. --- Extraction of plasmid DNA --- p.48
Chapter 3.1.7.1. --- Boiling preparation --- p.48
Chapter 3.1.8. --- Plasmid digestion --- p.51
Chapter 3.2. --- DNA sequencing --- p.51
Chapter 3.2.1. --- Comparison of B95-8 EBV-speicific DNase gene with gene sequence of EBV in GeneBank --- p.50
Chapter 3.2.2. --- Comparison of 5' end of Raji & B95-8 EBV derived EBV-specific DNase gene --- p.57
Chapter 3.2.3. --- Comparison of the 3'end of the Raji and B95-8 denved EBV-specific DNase gene --- p.63
Chapter 3.2.4. --- Amino acid sequence homology between B95-8 & Raji EBV-specific DNase --- p.64
Chapter 3.2.5. --- Amino acid sequence comparison between the 3' end of the B95-8 EBV DNase protein with that of the Raji EBV DNase protein --- p.62
Chapter 3.3. --- Epitope mapping --- p.67
Chapter 3.3.1. --- Amino acid key --- p.67
Chapter 3.3.2. --- Amino acid sequence of peptides --- p.73
Chapter 3.3.2. --- O.D. readings at 492nm of five histologically proven NPC sera --- p.74
Chapter Chapter 4 --- Discussions
Chapter 4.1. --- Overall strategy --- p.75
Chapter 4 2 --- Significance of EBV-specific DNase as marker for NPC --- p.76
Chapter 4.3. --- Characterization of EBV-specific DNase --- p.76
Chapter 4.4. --- Molecular cloning of PCR amplified gene into PUC18 SmaI/BAP vector --- p.77
Chapter 4.4.1. --- Cell culture --- p.77
Chapter 4.4.2. --- PCR amplification --- p.73
Chapter 4.4.3. --- "Blunting,kinasing and ligation of EBV-specific DNase cDNA into pUC18 vector" --- p.78
Chapter 4.4 .4 --- .Transformation by electroporation --- p.80
Chapter 4.4.5. --- Restriction enzyme digestion of pUC18/EBV-DNase plasmid … --- p.81
Chapter 4.5. --- DNA sequencing --- p.81
Chapter 4.6. --- Epitope mapping --- p.83
Reference --- p.85

by Liu Chun Ki, Kevin.
Thesis (M.Phil.)--Chinese University of Hong Kong, 1999.
Includes bibliographical references (leaves 126-142).
Abstracts in English and Chinese.
Abstract --- p.i
Acknowledgements --- p.ii
Table of contents --- p.iii
List of figures --- p.viii
List of tables --- p.x
List of abbreviations --- p.xi
Chapter Chapter 1 --- Introduction Epstein-Barr Virus
Chapter 1.1 --- History --- p.1
Chapter 1.2 --- Classification --- p.2
Chapter 1.3 --- Virus and genome structure --- p.3
Chapter 1.4 --- Epidemiology --- p.6
Chapter 1.4.1 --- Prevalence of infection --- p.6
Chapter 1.4.2 --- Modes of transmission --- p.7
Chapter 1.5 --- Pathogenesis of EBV --- p.7
Chapter 1.5.1 --- "Adsorption, penetration and dissemination" --- p.7
Chapter 1.5.2 --- Lytic infection cycle --- p.8
Chapter 1.5.3 --- Latent infection cycle --- p.9
Chapter 1.5.4 --- Functions of the EBV-specific proteins associated with latent infection cycle proteins --- p.10
Chapter 1.5.4.1 --- EBNA1 --- p.10
Chapter 1.5.4.2 --- EBNA2 --- p.11
Chapter 1.5.4.3 --- "EBNA 3A, 3B and 3C" --- p.11
Chapter 1.5.4.4 --- EBNA LP --- p.12
Chapter 1.5.4.5 --- LMP1 --- p.13
Chapter 1.5.4.6 --- Characteristics of EBV LMP 2 gene --- p.14
Chapter 1.5.4.7 --- Functions of LMP 2A --- p.15
Chapter 1.5.4.8 --- Functions of LMP 2B --- p.18
Chapter 1.6 --- Clinical significance of EBV --- p.20
Chapter 1.6.1 --- Infectious mononucleosis (IM) --- p.20
Chapter 1.6.2 --- Burkitt's lymphoma (BL) --- p.20
Chapter 1.6.3 --- Nasopharyngeal carcinoma (NPC) --- p.21
Chapter 1.6.4 --- Hodgkin's lymphoma (HL) --- p.21
Chapter 1.7 --- Immune response to EBV infection --- p.22
Chapter 1.7.1 --- Humoral immune response --- p.22
Chapter 1.7.2 --- Cellular immune response --- p.22
Chapter 1.8 --- Diagnosis of EBV infection --- p.26
Chapter 1.9 --- Treatment and prevention --- p.27
Chapter 1.10 --- Nasopharygneal Carcinoma (NPC) --- p.28
Chapter 1.10.1 --- Epidemiology --- p.28
Chapter 1.10.2 --- Etiology --- p.28
Chapter 1.10.2.1 --- Environmental factor associated with NPC --- p.30
Chapter 1.10.2.2 --- Genetic factors associated with NPC --- p.31
Chapter 1.10.2.3 --- Association of NPC and EBV --- p.31
Chapter 1.10.3 --- Diagnosis ofNPC --- p.32
Chapter 1.10.4 --- Treatment --- p.33
Chapter 1.11 --- Objective of the project --- p.34
Chapter Chapter 2 --- Materials and Methods
Chapter 2.1 --- EBV-containing cell cultures --- p.35
Chapter 2.2 --- Extraction of total RNA --- p.36
Chapter 2.2.1 --- Cell lysis --- p.36
Chapter 2.2.2 --- Protein digestion --- p.36
Chapter 2.2.3 --- DNA digestion --- p.37
Chapter 2.2.4 --- Elution of total RNA --- p.37
Chapter 2.2.5 --- Purity and electrophoresis analysis of total RNA --- p.38
Chapter 2.3 --- First strand cDNA synthesis --- p.38
Chapter 2.4 --- PCR amplification of LMP 2 cDNA --- p.39
Chapter 2.5 --- Isolation of the PCR amplified LMP 2 cDNA --- p.40
Chapter 2.6 --- Purification of the PCR amplified LMP 2 cDNA --- p.41
Chapter 2.7 --- Confirmation of the PCR amplified cDNA --- p.42
Chapter 2.7.1 --- Nested PCR --- p.42
Chapter 2.7.2 --- Restriction enzyme digestion --- p.44
Chapter 2.8 --- Ligation of insert LMP 2 cDNA with vector --- p.45
Chapter 2.9 --- Transformation of competent cells JM109 --- p.45
Chapter 2.10 --- Screening of the recombinant clones --- p.47
Chapter 2.11 --- Small scale purification of plasmid DNA --- p.47
Chapter 2.12 --- Determination of the size of the insert DNA --- p.48
Chapter 2.13 --- DNA sequencing --- p.48
Chapter 2.13.1 --- The cycle sequencing reaction --- p.48
Chapter 2.13.2 --- Preparation of the acrylamide gel and TBE buffer --- p.51
Chapter 2.13.3 --- Running conditions of the electrophoresis --- p.52
Chapter 2.13.4 --- "Processing, editing and exporting the sequences" --- p.52
Chapter 2.14 --- Data analysis --- p.53
Chapter 2.14.1 --- Sequence analysis --- p.53
Chapter 2.14.2 --- Amino acid analysis --- p.53
Chapter 2.14.3 --- Protein secondary structure analysis --- p.53
Chapter 2.14.4 --- Hydrophobicity analysis --- p.54
Chapter 2.14.5 --- Isoelectric point analysis --- p.54
Chapter Chapter 3 --- Results
Chapter 3.1 --- Cell Cultures --- p.55
Chapter 3.2 --- Extraction of total RNA --- p.56
Chapter 3.3 --- PCR amplification --- p.61
Chapter 3.4 --- Isolation of PCR amplified LMP 2 cDNA --- p.62
Chapter 3.5 --- Confirmation of the PCR amplified cDNA --- p.66
Chapter 3.5.1 --- Nested PCR --- p.66
Chapter 3.5.2 --- Restriction enzyme digestion --- p.71
Chapter 3.6 --- Transformation and screening --- p.77
Chapter 3.7 --- Extraction of plasmid DNA and its digestion with restriction enzyme --- p.78
Chapter 3.8 --- DNA sequencing --- p.83
Chapter 3.8.1 --- DNA sequence comparison --- p.84
Chapter 3.9 --- Amino acid sequence homology --- p.89
Chapter 3.9.1 --- Amino acid sequence comparison --- p.90
Chapter 3.10 --- Hydrophobicity analysis --- p.92
Chapter 3.10.1 --- Comparison of hydrophobicity of B95-8 derived LMP2 with GeneBank --- p.93
Chapter 3.10.2 --- Comparison of hydrophobicity of CB 14022-derived LMP2 with GeneBank --- p.95
Chapter 3.10.3 --- Comparison of hydrophobicity of Raji-derived LMP2 with GeneBank --- p.97
Chapter 3.11 --- Protein secondary structure analysis --- p.100
Chapter 3.11.1 --- Comparison of secondary structure of B95-8-derived LMP2 with GeneBank --- p.100
Chapter 3.11.2 --- Comparison of secondary structure of CB 14022-derived LMP2 with GeneBank --- p.100
Chapter 3.11.3 --- Comparison of secondary structure of Raji-derived LMP2 with GeneBank --- p.101
Chapter 3.12 --- Isoelectric point analysis --- p.103
Chapter Chapter 4 --- Discussions
Chapter 4.1 --- Overall strategy for the cloning and sequencing of EBV LMP 2 gene --- p.106
Chapter 4.2 --- Implications of the results obtained in sequencing --- p.107
Chapter 4.3 --- Results interpretation --- p.108
Chapter 4.3.1 --- Cell culture --- p.108
Chapter 4.3.2 --- Extraction of total RNA --- p.108
Chapter 4.3.3 --- PCR amplification --- p.109
Chapter 4.3.4 --- Confirmation of the PCR amplified cDNAs using nested PCR --- p.109
Chapter 4.3.5 --- Confirmation of the PCR amplified cDNAs using restriction enzyme digestion --- p.110
Chapter 4.3.6 --- Ligation of EBV LMP 2 cDNA to pGEM-T Easy Vector --- p.111
Chapter 4.3.7 --- Transformation and screening --- p.114
Chapter 4.3.8 --- Extraction of plasmid DNA and digestion with restriction enzyme --- p.115
Chapter 4.4 --- DNA sequencing and sequence homology --- p.116
Chapter 4.5 --- Amino acid sequence homology --- p.117
Chapter 4.6 --- Hydrophobicity analysis --- p.119
Chapter 4.7 --- Protein secondary structure analysis --- p.120
Chapter 4.8 --- Isoelectric point analysis --- p.122
Chapter 4.9 --- Summary of results --- p.122
Chapter 4.10 --- Conclusions --- p.124
References --- p.126

by Shik Yuen Lo.
Thesis (M.Phil.)--Chinese University of Hong Kong, 1995.
Includes bibliographical references (leaves 102-124).
Abstract
List of Illustrations
List of Tables
Acknowledgements
Chapter 1. --- Introduction --- p.1
Chapter 2. --- Literature Review
Chapter 2.1 --- Anatomy of the Human Nasopharynx --- p.4
Chapter 2.2 --- Histology of the Human Nasopharynx --- p.6
Chapter 2.3 --- Intra-epithelial Lesions of the Nasopharyngeal Epithelium --- p.10
Chapter A. --- Hyperplasia
Chapter B. --- Metaplasia
Chapter C. --- Koilocytes
Chapter D. --- Nasopharyngeal intra-epithelial neoplasia
Chapter 2.4 --- Nasopharyngeal Carcinoma --- p.19
Chapter A. --- Histopathological classification of NPG
Chapter B. --- Epidemiology
Chapter C. --- Etiological factors
Chapter 2.5 --- Epstein-Barr Virus and Nasopharyngeal Carcinoma --- p.27
Chapter A. --- Serological
Chapter B. --- EBV genome in NPC
Chapter C. --- EBV encoded latent gene products
Chapter 2.6 --- Cancer Genes in Nasopharyngeal Carcinoma --- p.28
Chapter A. --- "Tumours suppressor Gene, p53"
Chapter B. --- "Oncogenes, c-myc, ras and bcl-2"
Chapter 2.7 --- Immunohistochemical methods --- p.33
Chapter A. --- Avidin-Biotin Complex method (ABC)
Chapter B. --- Alkaline phosphotase Anti-alkaline phosphotase method (APAAP)
Chapter C. --- Unmasking of antigens
Chapter 2.8 --- Techniques in ISH --- p.40
Chapter 3. --- Material and Methods --- p.42
Chapter 3.1 --- Tissue Samples --- p.42
Chapter A. --- "Samples for ras, c-myc and p53 studies"
Chapter B. --- Samples for LMP-1 study
Chapter C. --- Samples for bcl-2 study
Chapter D. --- Samples for EBER-RNAs study
Chapter 3.2 --- Monoclonal Antibodies --- p.47
Chapter 3.3 --- Tissue Processing --- p.49
Chapter A. --- Tissue processing for formalin fixed tissue
Chapter B. --- Tissue processing for frozen section
Chapter 3.4 --- IHC Techniques --- p.50
Chapter A. --- Pretreatment of Laboratory Wares
Chapter B. --- Determination of optimum dilution and incubation time for p53antibody
Chapter C. --- Determination of optimum dilution and incubation time for bcl-2 and LMP-1 antibodies
Chapter D. --- Determination of optimum dilution and incubation time for c-myc and ras
Chapter E. --- "Detection of p53, c-myc and ras by ABC method"
Chapter F. --- Detection of bcl-2 and LMP-1 by APAAP method
Chapter 3.6 --- ISH --- p.57
Chapter A. --- Pretreatment of laboratory wares
Chapter B. --- FITC conjugated EBER oligonucleotide probe
Chapter C. --- Determination of PK dilution for paraffin section
Chapter D. --- Determination of PK dilution for frozen section
Chapter E. --- Determination of the choice of fixative for frozen section
Chapter F. --- Detection of EBER-RNAs by ISH method
Chapter 3.7 --- Statistical analysis --- p.62
Chapter A. --- p53
Chapter B. --- c-myc and ras
Chapter 4. --- Results --- p.63
Chapter A. --- ras
Chapter B. --- c-myc
Chapter C. --- p53
Chapter D. --- LMP-1
Chapter E. --- Bcl-2
Chapter F. --- EBER-RNAs
Chapter 5. --- Discussion --- p.86
Chapter 6. --- Conclusion and Summary --- p.97
Appendix --- p.99
Reference --- p.102

Xu Xuequn.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2004.
Includes bibliographical references (leaves 100-125).
Abstracts in English and Chinese.
Chapter Chapter 1 --- Introduction --- p.1
Chapter 1.1 --- Epstein-Barr (EBV) Virus --- p.1
Chapter 1.1.1 --- Virus Structure and Genome Structure --- p.1
Chapter 1.1.2 --- Virus Types --- p.2
Chapter 1.2 --- EBV Infection and malignancies --- p.3
Chapter 1.2.1 --- In Vitro Infection --- p.3
Chapter 1.2.2 --- Infection in the Natural Host --- p.8
Chapter 1.2.3 --- Malignancies Associated with EBV --- p.11
Chapter 1.3 --- T Cell-Mediated Immune Response to EBV --- p.16
Chapter 1.3.1 --- The Pathway of Cell-Mediated Immune Response in Viral Infection --- p.16
Chapter 1.3.2 --- Cell-Mediated Immune Response to EBV --- p.18
Chapter 1.3.3 --- The Feature of CTLs Response to EBV --- p.20
Chapter 1.4 --- CTLs to EBV Relevant MalignancieśؤApplications and Challenges --- p.21
Chapter 1.5 --- HLA Polymorphisms and Strategy of Epitope-Based CTLs Therapy --- p.24
Chapter 1.6 --- The Effect of HLA Polymorphism on EBV-Specific CTL Epitope Choice in Southern Chinese --- p.27
Chapter 1.7 --- ELISPOT Assay 226}0ؤ Detection of CTLs Response --- p.32
Chapter 1.8 --- Aim of This Study --- p.37
Chapter Chapter 2: --- Material and Methods: --- p.39
Chapter 2.1 --- Peptides --- p.39
Chapter 2.2 --- PBMCs Preparations --- p.43
Chapter 2.3 --- PBMC Counting and Cells Dilution --- p.43
Chapter 2.4 --- Elispot Assay --- p.44
Chapter 2.5 --- Counting the Spots --- p.45
Chapter 2.6 --- Spots Forming Cells (SFC/106) and Positive Standard --- p.46
Chapter Chapter 3: --- Results --- p.47
Chapter 3.1 --- Validation of ELISPOT assay methodology --- p.47
Chapter 3.2 --- CTLs Response to Each Epitope in the Population --- p.55
Chapter 3.2.1 --- Positive Response to A11 Restricted and Mutant Epitopes in the Population --- p.55
Chapter 3.2.2 --- Positive Frequencies of A2 Restricted Epitopes in the Population --- p.63
Chapter 3.2.3 --- Positive Frequencies of Other HLA Allele Restriction Peptides --- p.70
Chapter 3.3 --- CTLs Response Frequencies Categorized by Proteins --- p.74
Chapter 3.3.1 --- "CTLs Response to LMP1, LMP2, EBNA1 Epitopes" --- p.74
Chapter 3.3.2 --- "CTLs Response to EBNA2, EBNA-LP Epitopes, EBNA3 Epitopes" --- p.75
Chapter 3.3.3 --- CTLs Response to LYTIC Epitopes --- p.79
Chapter 3.4 --- Summary --- p.80
Chapter Chapter 4: --- Discussion --- p.82
Chapter 4.1 --- Discussion of A11 Restricted Epitopes --- p.82
Chapter 4.2 --- Discussion of A2 Restricted Epitopes --- p.86
Chapter 4.3 --- Discussion of Other HLA Restricted Epitopes --- p.89
Chapter 4.4 --- "Discussion ofLMPl, LMP2, EBNA1 Epitopes" --- p.92
Chapter 4.5 --- "Discussion of EBNA2, EBNA3, and EBNA-LP epitopes" --- p.96
Chapter 4.6 --- Discussion of LYTIC Epitopes --- p.96
Chapter 4.7 --- Discussion of Summary --- p.98
Chapter Chapter 5 --- Conclusion --- p.99
Chapter 6 --- Reference --- p.100
Chapter 7 --- Appendix --- p.126
Chapter 7.1 --- "Appendix 1, raw data of Elispot assay on CTLs response to EBV relevant epitopes m Hong Kong donors" --- p.126
Chapter 7.2 --- "Appendix 2, frequencies from highest cell number wells of the peptides (SFC/106)" --- p.126
Chapter 7.3 --- "Appendix 3, typical Elispot assay figure " --- p.126

Chow Chit.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2003.
Includes bibliographical references (leaves 130-156).
Abstracts in English and Chinese.
Acknowledgements --- p.i
Abstract --- p.ii
Table of Contents --- p.vii
List of Tables --- p.xi
List of Figures --- p.xii
List of Abbreviations --- p.xiv
Chapter Chapter 1: --- Introduction --- p.1
Chapter 1.1 --- Malignant Lymphoma --- p.1
Chapter 1.2 --- Non-Hodgkin's Lymphoma --- p.1
Chapter 1.3 --- NK/T Cell Lymphoma --- p.2
Chapter 1.3.1 --- General Features of NK/T Cell Lymphoma --- p.2
Chapter 1.3.2 --- Histology of NK/T Cell Lymphoma --- p.3
Chapter 1.3.3 --- Subtypes NK/T Cell Lymphoma --- p.4
Chapter 1.3.4 --- Overview of NK/T Cell Lymphoma Cell Lines --- p.5
Chapter 1.3.4.1 --- NK/T Cell Lymphoma Cell Lines-NK-92 and SNK-6 --- p.6
Chapter 1.3.5 --- NK/T Cell Lymphoma and Interleukins --- p.8
Chapter 1.4 --- Interleukin-2 and Interleukin-15 --- p.9
Chapter 1.5 --- IL-2 and IL-15 Receptor --- p.10
Chapter 1.6 --- Cellular Signaling Pathways Regulated by IL-2 and IL-15 --- p.11
Chapter 1.6.1 --- JAK/STAT Pathway --- p.12
Chapter 1.6.2 --- PI3K/Akt Pathway --- p.14
Chapter 1.7 --- Epstein-Barr Virus: an Oncogenic Virus --- p.20
Chapter 1.7.1 --- Overview of EBV --- p.20
Chapter 1.7.2 --- Epidemiology --- p.20
Chapter 1.7.3 --- Life Cycle of EBV --- p.21
Chapter 1.7.4 --- Latency Infection of EBV --- p.21
Chapter 1.7.5 --- Role of EBV latent genes in oncogenesis --- p.23
Chapter 1.7.5.1 --- EBER1 and 2 --- p.23
Chapter 1.7.5.2 --- EBNAs --- p.24
Chapter 1.7.5.3 --- LMPs --- p.25
Chapter 1.7.6 --- Lytic Cycle of EBV --- p.26
Chapter 1.7.7 --- Signaling Pathways and EBV --- p.27
Chapter Chapter 2: --- Aim of Study --- p.29
Chapter Chapter 3: --- Materials and Methods --- p.31
Chapter 3.1 --- IL-2 and IL-15 on NK/T Cell Lymphoma Cell Lines and patients --- p.31
Chapter 3.1.1 --- Cell Lines Maintenance --- p.31
Chapter 3.1.2 --- Patients --- p.32
Chapter 3.2 --- "Assays of IL-2, IL-15 and IFN-γ in culture supernatants and patient sera" --- p.32
Chapter 3.2.1 --- IL-2 ELISA --- p.32
Chapter 3.2.2 --- IL-15 ELISA --- p.33
Chapter 3.2.3 --- IFN-γ ELISA --- p.34
Chapter 3.3 --- Effect of IL-2 and IL-15 on NK/T Cell Lymphoma Cell Lines --- p.35
Chapter 3.3.1 --- Cell Growth and Viability Determination --- p.35
Chapter 3.3.2 --- Apoptosis Assays on Interleukin-starved NK-92 cells --- p.35
Chapter 3.3.2.1 --- DNA Laddering Analysis --- p.36
Chapter 3.3.2.2 --- Cell Cycle and Apoptosis Determination by PI Staining --- p.37
Chapter 3.3.2.3 --- Caspase 3 Activity Assay --- p.37
Chapter 3.4 --- PI3K/Akt Pathway Study --- p.39
Chapter 3.4.1 --- Determination of AKT1 Gene Amplification by Real-Time Quantitative PCR --- p.39
Chapter 3.4.1.1 --- DNA Extraction for Real-Time Quantitative PCR --- p.39
Chapter 3.4.1.2 --- AKT1 Real-Time Quantitative PCR --- p.40
Chapter 3.4.2 --- Determination of Akt Expression --- p.41
Chapter 3.4.2.1 --- Normal NK Cell Purification from Buffy Coat --- p.41
Chapter 3.4.2.2 --- Determination of the Purity of Extracted NK Cells --- p.43
Chapter 3.4.2.3 --- Interleukin Treatment of Normal NK Cells and NK-92 --- p.43
Chapter 3.4.2.4 --- Protein Extraction and Western Blot Analysis --- p.43
Chapter 3.4.3 --- Study of PI3 K/Akt pathway using PI3K inhibitor --- p.47
Chapter 3.4.3.1 --- Cell Growth and Viability Assay --- p.47
Chapter 3.4.3.2 --- Apoptosis Assay by DNA Laddering and Pi-Staining on NK-92 cells --- p.48
Chapter 3.4.3.3 --- Determination of activated Akt after LY294002 and Wortmannin treatment --- p.48
Chapter 3.5 --- Effect of IL-2 and IL-15 on the JAK/STAT pathway and PDK/Akt pathway of NK/T Cell Lymphoma Cell Lines --- p.49
Chapter 3.5.1 --- Cell Treatment --- p.49
Chapter 3.5.2 --- Study of JAK/STAT and PI3K/Akt Pathways by Western Blotting --- p.49
Chapter 3.5.3 --- "Assays of IL-2, IL-15 and IFN-γ in the NK-92 Cell Culture Medium by ELISA" --- p.50
Chapter 3.5.4 --- Determination of EBV Status after IL-2 and IL-15 Treatment --- p.51
Chapter 3.5.4.1 --- RNA Extraction --- p.51
Chapter 3.5.4.2 --- Reverse-transcriptase Reaction --- p.52
Chapter 3.5.4.3 --- PCR for EBV-related Genes --- p.53
Chapter 3.5.4.4 --- EBER-ISH --- p.54
Chapter 3.5.4.5 --- Real-time Quantitative PCR for EBER1 --- p.56
Chapter 3.5.4.6 --- Western Blot for LMP1 --- p.56
Chapter 3.6 --- Statistical Analysis --- p.57
Chapter Chapter 4: --- Results --- p.59
Chapter 4.1.1 --- "IL-2, IL-15 and IFN-γ Levels in the Serum of Patients with NK/T Cell Lymphoma" --- p.59
Chapter 4.1.2 --- IL-2 and IL-15 Level in Culture Supernatant of NK-92 --- p.59
Chapter 4.1.3 --- IFN-γ induction in supernatant of NK-92 --- p.60
Chapter 4.2 --- Effect of IL-2 and IL-15 on NK/T Cell Lymphoma Cell Lines --- p.61
Chapter 4.2.1 --- Cell Growth and Viability --- p.61
Chapter 4.2.2 --- Apoptosis Study of Interleukin-starved NK-92 --- p.62
Chapter 4.2.2.1 --- DNA fragmentation and Cell Cycle studies --- p.62
Chapter 4.2.2.2 --- Caspase 3 Activity in NK-92 --- p.62
Chapter 4.3 --- Akt in NK-92 --- p.63
Chapter 4.3.1 --- Confirmation ofAKTl Amplification in NK-92 --- p.63
Chapter 4.3.2 --- Akt Protein Quantification in NK-92 cells --- p.63
Chapter 4.3.3 --- Activated Akt and STAT proteins in IL-2 or IL-15 stimulated NK-92 and normal NK cells --- p.64
Chapter 4.4. --- PI3K/Akt Pathway --- p.64
Chapter 4.4.1 --- Phosphorylation of Components of the PI3K/Akt Pathway --- p.64
Chapter 4.4.2. --- Role of PI3K/Akt Pathway in NK/T Cell Lymphoma Cell Lines --- p.65
Chapter 4.4.2.1 --- Cell Growth and Viability Studies --- p.65
Chapter 4.4.2.2 --- Apoptosis and Cell Cycle Arrest Induction by LY294002 in NK-92 --- p.66
Chapter 4.4.2.3 --- Confirmation of the effect of LY294002 on the PDK/Akt pathway in NK-92 cells --- p.67
Chapter 4.5 --- Phosphorylation of STAT family proteins --- p.67
Chapter 4.6 --- Regulation of EBV-related genes in NK-92 --- p.68
Chapter Chapter 5: --- Discussion --- p.71
Chapter 5.1 --- Cytokine level in patient serum --- p.71
Chapter 5.2 --- Source of IL-2 and IL-15 for NK-92 cells --- p.72
Chapter 5.3 --- Induction of IFN-γ in NK-92 --- p.73
Chapter 5.4 --- Role of IL-2 and IL-15 on NK/T cell lymphoma cell lines --- p.75
Chapter 5.4.1 --- Cell Growth and Viability Maintenance by IL-2 and IL-15 --- p.75
Chapter 5.4.2 --- Apoptosis induced by interleukin-starving in NK-92 --- p.75
Chapter 5.5 --- Aberrant activation of signaling pathways by IL-2 or IL-15 in NK-92 --- p.77
Chapter 5.5.1 --- Hypersensitivity of NK-92 cells to IL-2 or IL-15 --- p.77
Chapter 5.5.2 --- PI3K/Akt pathway in NK/T cell lymphoma cell lines --- p.78
Chapter 5.5.2.1 --- Confirmation of AKT1 amplification --- p.78
Chapter 5.5.2.2 --- Constitutive activation of Akt in NK/T cell lymphoma cell lines --- p.79
Chapter 5.5.2.3 --- Role of PI3K/Akt in NK/T cell lymphoma cell lines --- p.80
Chapter 5.5.2.4 --- IL-2 and IL-15 induce differential sensitivity of NK-92 to LY294002 --- p.81
Chapter 5.5.2.5 --- NK/T cell lymphoma cell lines are wortmannin-insensitive --- p.82
Chapter 5.6 --- Jak/STAT pathway in NK/T cell lymphoma cell lines --- p.83
Chapter 5.6.1 --- STAT3 and STAT5 were activated by both IL-2 and IL-15 --- p.83
Chapter 5.6.2 --- STAT6 was activated in NK/T cell lymphoma cell lines --- p.84
Chapter 5.6.3 --- "Differential regulation of STAT 1, STAT3 (Ser-727) and STAT6 in NK/T cell lymphoma cell lines" --- p.85
Chapter 5.6 --- EBV gene regulation in NK-92 --- p.87
Chapter Chapter 6: --- Conclusion --- p.91
Tables --- p.93
Figures --- p.102
Reference --- p.128