Após anos de debates, o teste de detecção de ácidos nucleicos (NAT) para HIV e HCV na triagem de sangue foi implementado de forma obrigatória no Brasil em 2013, e HBV, em 2016. Um dos motivos citados sobre o atraso em sua implementação foi o custo elevado que seria adicionado à sorologia e, até o momento, uma ampla avaliação econômica em saúde (AES) a respeito de sua eficiência no país não está disponível. Diversos artigos já demonstraram que a razão incremental de custo-utilidade (ICUR) do NAT em relação à sorologia varia de 0,21 a 8,84 milhões de dólares americanos (US$) para cada QALY ganho. Esta grande variação dá-se, principalmente, por diferenças entre a idade média dos receptores de sangue (RS), incidência/prevalência dos vírus entre os doadores de sangue (DS), custo dos testes e tratamentos médicos, cobertura da vacina contra o HBV e sensibilidade do teste utilizado. Assim, faz-se necessária uma avaliação abrangente desta tecnologia e sua efetividade para o cenário brasileiro. Objetivos: Realizar uma revisão sistemática (RevS) de estudos econômicos completos sobre o uso do NAT para HIV, HCV e/ou HBV no mundo; realizar a AES sobre o NAT sob a perspectiva pública brasileira; caracterizar as doações de sangue em janela imunológica no país. Métodos: Metodologia Cochrane de RevS das bases de dados Medline, Embase, LILACS, CRD, BVS ECO, Google Scholar e IDEAS; questionário aplicado aos bancos de sangue e modelo econômico on-line da International Society of Blood Transfusion (ISBT) para cálculo da ICUR do \"NAT em mini-pool de seis amostras individuais\" (MP6) versus \"testes sorológicos\" (SR) no Brasil. Resultados: Quatorze estudos de dezesseis diferentes países foram avaliados. O NAT apresentou a maior relevância nos países de baixa renda, onde há as maiores prevalências e incidências virais, menores taxas de doadores de repetição (DR) e RS mais jovens. A maioria dos estudos concluiu que o NAT, independente do vírus analisado, não é custo-efetivo. As principais diferenças entre as características dos estudos foram relacionadas aos custos médicos e idade dos RS. O maior desvio dos padrões de uma RevS foram: não incluir o racional para definição dos desfechos e o modelo utilizado e não ter claro o conflito de interesse dos autores; para esta AES, o MP6 versus SR apresentou um ICUR de US$ 231.630,00/QALY, ou seja, 26,2 vezes o PIB per capita nacional) e um ICER de US$ 330.790,00/Ano de vida ganho (AVG). A análise de sensibilidade univariada do modelo demonstrou que somente a taxa de desconto, idade do RS, custo do NAT e epidemiologia dos vírus alteraram de forma significativa o ICUR obtido, variando desde US$ 76.957,00/QALY a US$ 933.311,00/QALY; a maioria dos casos de janela imunológica no Brasil são jovens, média de 29 anos, do sexo masculino, com pelo menos o ensino médio completo e mesmo com a obrigatoriedade do Anti-HBc no Brasil, o NAT-HBV é o que apresentou o maior rendimento. Conclusões: Os jovens, principalmente, ainda buscam os bancos de sangue como locais de testagem após comportamento de risco e é de extrema importância a revisão do custo real e completo do teste NAT no Brasil para ampla abordagem da tecnologia nacional incorporada e, se necessário, revisar a forma e modelo de reembolso da mesma e permitir a defesa do bem-estar da população e do bem público. / After years of discussion, nucleic acid (NAT) testing in the blood screening for HIV and HCV was implemented in Brazil in 2013 and HBV in 2016. One of the reasons cited for the delay in its implementation was the high cost that would be added to serology screening and a comprehensive economic assessment of its efficiency in the country is not yet available. Several articles have already shown that the incremental cost-utility ratio (ICUR) of NAT versus serology ranges from 0.21 to 8.84 million American dollars (US$) for each QALY gained. This large variation is mainly due to differences between the mean age of the blood recipient, viruses\' incidence / prevalence among donor population, cost of medical tests and treatments, HBV vaccine coverage, and sensitivity of the test used. Thus, a comprehensive evaluation of this technology and its effectiveness under the perspective of the Brazilian public health system (SUS) is needed. Objectives: Development of a systematic review (RevS) of complete economic studies about the use of NAT for HIV, HCV and / or HBV in the world. Conduct an economic evaluation of NAT under SUS perspective; characterize Brazilian blood donations in the serology \"window period\". Methods: Cochrane RevS Methodology of the Medline, Embase, LILACS, CRD, CRD ECO, Google Scholar and IDEAS databases; Questionnaire applied to blood banks and online economic model from the International Society of Blood Transfusion (ISBT) to calculate the ICUR for \"NAT in mini-pool of six individual samples\" (MP6) versus \"Serology Tests\" (SR) in Brazil. Results: Fourteen studies from sixteen different countries were assessed. NAT was most relevant in low-income countries, where there are the highest prevalences and viral incidences, lower rates of repeat donors and younger recipients of blood (RS). Most of the studies concluded that NAT, regardless of the virus evaluated, is not cost-effective. Differences in the characteristics of the studies were related to the costs and age of RS. The major deviations from RevS standards were: not including the rationale for selecting the outcomes and the model used and not being clear about the authors\' conflict of interest; MP6 vs SR showed an ICUR of US$ 231.630,00/QALY, 26,2 times Brazilian GND per capita) and an ICER of US$ 330.790,00/Life year gained (AVG). The univariate sensitivity analysis of the model demonstrated that only changes on discount rate, NAT cost, RS age and viruses\' epidemiology significantly altered the ICUR in a range between US$ 76.957,00/QALY and US$ 933.311,00/QALY; Most RS window period cases in Brazil are young, average of 29 years old, male, with at least high school education completed and even with the requirement of Anti-HBc in Brazil, NAT-HBV is the one that presented the highest yield. Conclusions: Young people, mainly, still seek blood banks as testing sites, especially after a risk behavior. It is extremely important to reveal the real and complete cost of the Brazilian NAT to fully evaluate its efficiency and, if needed, reassess its current reimbursement model, allowing the wellbeing defense of the population and public interest.

Introdução: A população confinada é um segmento exposto a certas situações que aumentam sua vulnerabilidade frente às doenças sexualmente transmissíveis. Infecções pelo HIV, hepatites B e C e sífilis encontram no sistema prisional um ambiente favorável às suas propagações. Estudos em população confinada são cercados de entraves tanto burocráticos como relacionados com a ética e segurança. Assim, assume grande importância um estudo - piloto para, entre outras coisas, identificar pontos prós e contras que possam surgir durante a execução do estudo principal. Objetivo: Desenvolver um estudo piloto para uma pesquisa sobre fatores de risco comportamentais referentes à contaminação pelas infecções pelo HIV, hepatites B e C e sífilis numa população carcerária. Métodos: Estudo epidemiológico transversal. Em julho de 2007, numa amostra de conveniência, 107 reeducandos foram estrevistados usando um questionário padrão e tecnica face-face. O estudo foi aprovado pelo Comitê de Ética em Pesquisa do HCFMUSP. Resultados: Foram realizadas todas as etapas planejadas, a saber: Reuniões na unidade: entre a direção e diversas equipes da unidade prisional; Convite aos participantes: contato com representantes dos reeducandos; Assinatura do TCLE: após formalização do convite à participação do estudo e Aplicação do questionário. Realizadas reuniões sistemáticas para ajuste do questionário. Feita a capacitação de entrevistadores. Elaborado um banco de dados no Microsoft Office para receber os dados por meio de dupla digitação. Foram entrevistados 16,5% da população da unidade. Apresentaram um perfil jovem com média de 31,1 anos de idade. O tempo médio de prisão foi de 18,7 meses. A idade média de início de uso de drogas legais foi de 14,7 e ilegais de 16,6 anos. Após o confinamento, houve redução no consumo de drogas e sem relatado de droga injetável no presídio. 55,1% realizaram tatuagem na prisão. 41,2% relataram ocorrência de DST na vida e 34,0% no ultimo mês, 2,5% referiram serem soropositivos para o HIV. 53,8% mantiveram o numero de relações sexuais após o confinamento e dos 28,6% que faziam uso sistemático de preservativos, 26,3% mantiveram esta freqüência no presídio. Envolvidos com agressões: 78,5% verbais e 65,1% físicas, sendo que 33,6% referiram ameaças de morte. Maconha, álcool e crack foram as drogas envolvidas nestas situações. Discussão: O estudo piloto possibilitou testar o instrumento de pesquisa, sua aplicabilidade e capacidade de identificar fatores de riscos para transmissão das infecções citadas, tanto fora quanto dentro do ambiente prisional. O treinamento dos entrevistadores favoreceu tanto a familiarização com o instrumento, quanto o contato adequado ético e seguro - com os reeducandos. A vivência com esta realidade contribuiu para mapear pontos vulneráveis do planejamento para a execução do estudo principal. Limites do estudo: As análises e a obtenção da sorologia não faziam parte do estudo piloto, postergadas para o estudo principal com amostra adequada. Questionários, quando utilizados como instrumento de coleta, podem apresentar problemas relacionados com as informações obtidas. Muitas delas podem não condizer com a realidade, tanto de forma proposital viés de informação quanto de forma não proposital viés de memória. / Introduction: Confined populations are exposed to circumstances that increase their vulnerability to sexually transmitted infections. HIV, hepatitis B and C, and syphilis, encounter at the prison system an environment favorable to their dissemination. Studies in confined populations are surrounded by bureaucratic, ethical and security barriers. Thus, a pilot study is of great importance -for identify obstacles and opportunities that may arise during the implementation of the main study. Objective: implementation of a pilot study on risk behaviors associated to the dissemination of HIV, hepatitis B and C, and syphilis in an incarcerated population. Methods: Cross Sectional study. In July of 2007, in a convenience sample, 107 prisoners were interviewed, face to face, using a standardized questioner. The study was approved by the Human Subject Committee of the Hospital das Clinicas of the School of Medicine from the University of Sao Paulo. Results: the research protocol was strict followed: institutional meetings of the direction and the different professional teams of the prison system; invitation to participants in close contact with prisoners representatives; signature of the consenting forms after the invitation and before the questionnaire was applied. Meetings were conducted to adjust the questionnaire. Interviewers were trained. A dataset using Microsoft Office was elaborated to allow insertion of the data collected. Subjects represented 16, 5% of the prison population. Participants were young, average of 31, 1 years of age. The length time in prison was 18, 7 months in average. The average of the initiation in the use of legal drugs was 14, 7 and illegal drugs 16, 6 years of age. After the arrestment there was a diminishment of the use of drugs, and no injection of drugs was reported. Tattoo inside of the prison was reported by 55, 1%. STI were reported by 41, 2% in life and by 34% in the last month, and 2, 5% reported to be HIV positive. 53, 8% maintained the same amount of sexual relation that they had outside of the prison. From the 28, 6% that regularly used condoms, 26, 3 regularly used inside of the prison too. Interviewed that were involved in aggression were 78, 5% verbal and 65, 1% physic, and 33, 6% refereed being threatened of dead. Marijuana, Alcohol and crack were the drugs involved in such circumstances. Discussion: The pilot study has tested the instrument of research, its applicability and ability to identify risk factors for transmission of the mentioned infections, both within or outside of the prison. The training of interviewers favored both the familiarity with the instrument, as the appropriate contact secure and ethical - with inmates. The experience with this reality has contributed to map vulnerabilities in the implementation of the main study. Limitations of the study: serology and analysis were not part of the pilot study, therefore postponed for the main study with adequate sample. Questionnaires may present problems with the information obtained. Many of the information may not match the reality; both, information or memory biases could be identified.

INTRODUÇÃO: A infecção pelo vírus da hepatite B (VHB) é responsável por grande parte das doenças hepáticas crônicas em todo o mundo. Em pessoas vivendo com HIV/Aids (PVHA) a infecção pelo VHB tem maior risco de evolução para cirrose e carcinoma hepatocelular. A vacina da hepatite B é importante na prevenção de doença potencialmente grave, particularmente em PVHA, já que ambos os vírus têm as mesmas vias de transmissão e a coinfecção tem uma alta morbidade. Indivíduos imunocompetentes têm uma boa resposta humoral após uma primeira série de vacina da hepatite B e não há recomendações de rotina para doses de reforço. PVHA podem ter uma pior resposta à vacina da hepatite B, quando comparada à resposta em indivíduos imunocompetentes e a duração da imunidade nesses pacientes é desconhecida. OBJETIVOS: Geral: Avaliar os marcadores sorológicos de proteção e infecção pelo VHB em pacientes adultos vivendo com HIV/Aids, vacinados previamente para hepatite B. Específicos: Avaliar a persistência dos anticorpos anti-HBs em PVHA vacinadas previamente para hepatite B e que apresentaram resposta humoral protetora inicial; avaliar a resposta sorológica à revacinação para hepatite B nos pacientes vacinados previamente e que não apresentaram resposta humoral protetora inicial; investigar a presença de marcadores sorológicos de infecção pelo VHB em PVHA vacinadas previamente para hepatite B. MÉTODOS: Estudo observacional de coorte retrospectiva de PVHA vacinadas primariamente para hepatite B entre 2001 e 2002. Marcadores sorológicos de infecção e proteção para o vírus da hepatite B foram investigados nesses pacientes que ainda estavam em acompanhamento no Serviço de Extensão ao Atendimento de Pacientes HIV/Aids (SEAP), da divisão de Clínica de Moléstias Infecciosas e Parasitárias do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo entre 2012 e 2014. RESULTADOS: Uma coorte de 121 PVHA foi analisada quanto à soroconversão e persistência do anti-HBs. A maioria era do sexo feminino (54,5%) com média de idade de 50,1 anos. Destes pacientes, 58 (grupo 1) eram inicialmente respondedores à primeira série da vacina (anti- HBs >=10 mUI/mL) e 63 (grupo 2) eram não respondedores. Após um período mediano de avaliação de 11 anos, nenhum dos pacientes teve evidência sorológica de infecção pelo VHB e 41/58 (70.7%) dos inicialmente respondedores mantinham anti-HBs >= 10 mUI/mL. Maior contagem de células T CD4+ e anti-HBs >= 100 mUI/mL, no momento da primeira série vacinal, estiveram associados à persistência de anti-HBs. Durante o período avaliado, 35/63 (55.6%) dos pacientes inicialmente não respondedores (grupo 2) soroconverteram com sucesso (anti-HBs >= 10 mUI/mL) em resposta a uma ou mais doses de reforço vacinal. Foi associado à soroconversão do anti-HBs o número de doses de reforço recebidas. A partir do momento da soroconversão (anti-HBs >=10 mUI/mL), 70 pacientes não receberam nenhuma dose adicional de vacina de hepatite B (grupo 3). Após um período mediano de 10 anos, 54/70 (77,1%) destes indivíduos mantinham anti- HBs >= 10 mUI/mL. CONCLUSÕES: A avaliação dos marcadores sorológicos para VHB em PVHA vacinadas previamente para hepatite B evidenciou: alta persistência de anti-HBs após um período de 10 a 11 anos; doses adicionais de vacina foram capazes de induzir resposta humoral em indivíduos inicialmente não respondedores; não foram detectados marcadores sorológicos de infecção (HbsAg ou Anti-HBc) após 11 anos da vacinação inicial / BACKGROUND: Hepatitis B Virus (HBV) infection is responsible for great part of chronic hepatic diseases worldwide. In people living with HIV (PLHIV), HBV infection has more risk of progressing to cirrhosis and hepatocarcinoma. Hepatitis B vaccine is important in the prevention of a potentially severe disease, particularly in PLHIV, since both viruses have the same routes of transmission and co-infection has greater morbidity. Immunocompetent individuals have a good humoral response after the first hepatitis B vaccine series and no recommendation is made regarding booster doses. PLHIV may have a poor hepatitis B vaccine response, when compared to immunocompetent and the duration of immunity in these patients is unknown. OBJECTIVES: General: Evaluate serological markers of infection and protection from HBV in PLHIV previously vaccinated for hepatitis B. Specific: Evaluate anti-HBs persistence in PLHIV previously vaccinated for HBV who responded to a primary vaccine series; evaluate response to revaccination for hepatitis B in patients who did not respond to first vaccine series; investigate serological markers of infection from HBV in PLHIV previously vaccinated for hepatitis B. METHODS: Observational retrospective study of a PLHIV cohort primarily vaccinated between 2001 and 2002 for hepatitis B. Serological markers of infection and protection from HBV were investigated in those patients who were still attending the HIV/AIDS Patient Care Extension Service at the Clinical Division of Infectious and Parasitic Diseases attached to Hospital das Clínicas at Faculdade de Medicina at Universidade de São Paulo between 2012 and 2014. RESULTS: A cohort of 121 PLHIV was analyzed for seroconversion and persistence of anti-HBs. The majority were female (54.5%) and mean age 50.1 years. From these patients, 58 (group 1) were initially responders to the first vaccine series (anti- HBs >=10 mIU/mL) and 63 (group 2) were non- responders. After a median period of 11 years, none of the patients had serologic evidence of HBV infection and 41/58 (70.7%) of the initially responders had maintained anti-HBs >=10 mIU/mL. Greater CD4+ cell counts and anti- HBs>= 100mIU/mL at the time of first vaccine series were associated with persistence of anti-HBs. During evaluation period, 35/63 (55.6%) of the initially non-responders (group 2) successfully seroconverted (anti-HBs >=10 mIU/mL) in response to one or more booster doses. Booster doses may be effective in PLHIV. Number of booster doses were associated to seroconversion. Seventy of the 121 patients did not receive any further booster doses of hepatitis B vaccine from the time of their seroconversion (anti-HBs >=10 mIU/mL) (group 3). After 10 years of the seroconversion, 54/70 (77,1%) of these individuals has maintained anti- HBs >= 10 mIU/mL. CONCLUSIONS: Evaluation of serological markers for HBV in PLHIV previously vaccinated for hepatitis B showed: strong persistence of anti-HBs after a period of 10 to 11 years; additional vaccine doses elicited humoral response in initially non-responders; there was no serologic evidence of HBV infection (HbsAg ou Anti-HBc) about 11 years after initial vaccination

慢性乙型肝炎病毒（HBV）感染是肝癌發生的重要因素，其中乙肝病毒X蛋白（HBx）在這一過程起著關鍵作用。研究發現，一些HBV變體和HBx突變具有更高致癌風險，而且這些變體和突變存在地區差異。香港是HBV感染高發地帶，因此本研究目的是從這一地區120個肝癌組織標本中篩查出HBx突變位點。我們用巢式PCR從84.16% (101/120)的標本中提取和擴增了HBx，並進行基因測序。三種HBx突變被檢測出，包括點突變，遠端羧基端截斷和缺失突變。其中點突變位點有39個，特別的是在50%的標本中檢測出A1630G/G1721A 和 A1762T/G1764A雙突變。在31.68% (32/101)的標本中發現遠端羧基端截斷，以及在2.97% (3/101)的標本中檢測出缺失突變。總之，大多數突變集中在HBx轉錄啟動域，表明這些突變在肝癌發生中可能起著重要作用。 / 缺氧誘導因數-1α（HIF-1α）在肝癌的發生和發展中也起著重要作用。研究發現，野生型HBx可以啟動HIF-1α，但是變異型HBx和HIF-1α的關係還沒有研究清楚。我們研究表明HBx轉錄啟動域是必需而且足夠啟動HIF-1α的。在這個區域的突變中，雙突變K130M/V131Z增強HBx對HIF-1α的活性，但遠端羧基端截斷和缺失突變削弱其功能。進一步研究發現，羧基端特別是119-140氨基酸對HBx的穩定和功能非常重要。肝癌標本中，我們也發現HBx和HIF-1α的表達呈正相關。因此，雖然不同的突變對於HBx的功能有不同的影響，但總的來說這些突變可以促進HIF-1α的表達和啟動，進而導致肝癌患者的預後不良。 / 靶向治療在肝癌綜合治療中扮演重要角色。索拉菲尼（Sorafenib）是一種多激酶抑制劑，臨床實驗發現它對晚期肝癌治療有效，但其抑制腫瘤血管生成機制還不完全清楚。我們研究發現Sorafenib明顯而且劑量依賴性地降低HIF-1α的表達和活化，進而抑制血管內皮生長因數（VEGF）的表達。Sorafenib抑制mTOR, ERK, p70S6K, RP-S6, eIF4E和4E-BP1等翻譯起始因數的磷酸化，從而抑制HIF-1α的合成而不影響其降解。體外實驗進一步發現Sorafenib降低HIF-1α和VEGF的表達，從而抑制腫瘤的血管形成和生長。總之，我們的研究表明sorafenib可能通過阻斷mTOR/p70S6K/4E-BP1 和 ERK 信號通路來抑制HIF-1α的合成，從而發揮其抗腫瘤血管生成作用。 / Chronic HBV infection is the leading cause of hepatocellular carcinoma (HCC) and HBx plays a crucial role in the molecular pathogenesis of HBV-related HCC. Previous investigations have indicated that some variations of HBV or mutations of HBx are associated with higher risk of HCC development, whereas the mutations profiles may be disparate in different regions. In the present studies, we thus aim to screen and identify the HBx mutation hotspots in 120 HCC tissues from Hong Kong, a region with HBV hyper-endemic. HBV DNAs were successfully isolated and amplified in 84.16% (101/120) HCC specimens via nest-PCR, and then subjected to gene sequencing. Three types of HBx mutations, including point mutations, distal carboxyl-terminal truncations and deletion mutations, were discovered. Among the point mutations, 39 mutation hotspots were indentified, with two double mutations (A1630G/G1721A and A1762T/G1764A) occurring in approximate 50% of 101 HCC cases. Distal C-terminal truncated mutations were discovered in 31.68% (32/101) of HCC cases, whereas deletion mutations were detected in 2.97% (3/101) of them. Overall, majority of identified mutations were located at the transactivation domain of HBx, suggesting the crucial roles of these mutations in HCC development. / Hypoxia-inducible factor-1α (HIF-1α) also closely involves in the development and progression of HCC. Wild-type HBx has been shown to activate HIF-1α. But the relationship between HBx mutants and activation of HIF-1α has not been fully elucidated. We here revealed that the transactivaiton domain of HBx was necessary and sufficient to activate HIF-1α. Double mutations K130M/V131Z in this domain enhanced the functionality of HBx in upregulating the expression and the activation of HIF-1α, whereas C-terminal truncations and deletion mutations weakened this prosperity of HBx. We further uncovered that the C-terminus, especially the region of amino acids 119-140, was essential for the stability and transactivation of HBx. The positive association between the HBx mutants and HIF-1α was found in the HCC tissue samples. Therefore, although mutations exerted different effects on the functionality of HBx, the overall activity of HBx mutants was suggested to upregulate HIF-1α, whose level is related to poor prognosis of HCC patients. / The therapy targeting a critical molecule in the development of HCC such as HIF-1α may be a potential and effective treatment regimen for HCC patients. Sorafenib, a multikinase inhibitor, has demonstrated promising results for the treatment of advanced HCC in clinical trials, but the mechanism that accounts for the anti-angiogenic efficiency of this agent has not been fully elucidated. We here revealed that sorafenib remarkably and dose-dependently decreased the expression and the transcriptional activity of HIF-1α, and its target gene, vascular endothelial grow factor (VEGF). Further analysis revealed that this reduction of HIF-1α by sorafenib was caused by the inhibition of HIF-1α protein synthesis rather than by the promotion of HIF-1α protein degradation. Moreover, the phosphorylated levels of mTOR, ERK, p70S6K, RP-S6, eIF4E and 4E-BP1 were significantly suppressed by sorafenib. In vivo studies further confirmed the inhibitory effect of sorafenib on the expression of HIF-1α and VEGF proteins, leading to a decrease of tumor vascularisation and growth. Collectively, our data suggest that sorafenib may exhibit anti-angiogenic activity by inhibiting HIF-1α synthesis, which is likely to be achieved through suppressing the phosphorylation of mTOR/p70S6K/4E-BP1 and ERK. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Liu, Liping. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 133-154). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.I / 摘要 --- p.IV / Publications --- p.VI / Acknowledgements --- p.VII / Abbreviations --- p.IX / List of Figures --- p.XI / List of Tables --- p.XIII / Table of Contents --- p.XIV / Chapter Chapter I --- General Introduction --- p.1 / Chapter 1.1 --- Overview of Hepatocellular Carcinoma --- p.1 / Chapter 1.2 --- HBV Infection and HCC Development --- p.6 / Chapter 1.3 --- Overview on Hepatitis B virus X Protein --- p.10 / Chapter 1.4 --- Roles of Hypoxia-inducible Factors in HCC --- p.17 / Chapter 1.5 --- Targeted Therapies and Sorafenib --- p.27 / Chapter Chapter II --- Identification of HBx Mutation Hotspots in HCC Tissues --- p.31 / Chapter 2.1 --- Abstract --- p.31 / Chapter 2.2 --- Introduction --- p.32 / Chapter 2.3 --- Materials and Methods --- p.35 / Chapter 2.4 --- Results --- p.40 / Chapter 2.5 --- Discussion --- p.53 / Chapter Chapter III --- The Relationship between HBx Mutants and HIF-1α --- p.59 / Chapter 3.1 --- Abstract --- p.59 / Chapter 3.2 --- Introduction --- p.60 / Chapter 3.3 --- Materials and Methods --- p.63 / Chapter 3.4 --- Results --- p.70 / Chapter 3.5 --- Discussion --- p.91 / Chapter Chapter IV --- The Effects of Sorafenib on Hypoxia-inducible Factor-1α --- p.96 / Chapter 4.1 --- Abstract --- p.96 / Chapter 4.2 --- Introduction --- p.98 / Chapter 4.3 --- Materials and Methods --- p.101 / Chapter 4.4 --- Results --- p.108 / Chapter 4.5 --- Discussion --- p.124 / Chapter Chapter V --- Conclusion and Future Plans --- p.129 / Chapter 5.1 --- Conclusion --- p.129 / Chapter 5.2 --- Future Plans --- p.131 / References --- p.133

O objetivo principal deste estudo foi avaliar a frequência de cepas do HBV com mutações de resistência aos análogos nucleos(t)ídeos (AN) utilizados no tratamento da hepatite B entre indivíduos cronicamente infectados, não submetidos a tratamento, procedentes de diferentes regiões do Brasil. Além disso, foram avaliadas a presença de mutações que alteram a antigenicidade do HBsAg promovendo escape dos anticorpos anti-HBs; mutações nos genes pré-core/core e a associação dos diferentes subgenótipos com as mutações encontradas e características demográficas e laboratoriais dos pacientes. Foram incluídas 779 amostras de soro de pacientes com infecção crônica pelo HBV e virgens de tratamento com AN ou interferon, as quais foram coletadas no período de 2006 a 2011. Os pacientes eram procedentes dos seguintes estados brasileiros: Pará, Maranhão, Bahia, Minas Gerais, São Paulo, Paraná e Rio Grande do Sul. O DNA do HBV foi extraído das amostras de soro utilizando o Kit QIAamp DNA Blood Mini Kit (Qiagen) e posteriormente foi realizada a amplificação das regiões S/polimerase (S/P) e pré-core/core (PCC) do genoma viral por nested PCR. O fragmento amplificado foi submetido a sequenciamento direto em sequenciador automático de DNA (ABI 3500) e as sequências obtidas foram analisadas para identificação dos genótipos e subgenótipos do HBV, pesquisa de mutações na polimerase, no HBsAg e nos genes pré-core/core. A região S/Pol foi amplificada e sequenciada com sucesso em 702 amostras, as quais foram incluídas para atender aos objetivos deste estudo. Entre as 702 amostras analisadas sete genótipos e 12 subgenótipos do HBV foram identificados. O subgenótipo A1 foi o mais frequente (63,7%, 447/702), seguido pelo HBV/D3 (14,5%, 102/702). Os demais genótipos e subgenótipos encontrados e suas frequências foram as seguintes: A2 (3,3%, 23/702), A3 (0,1%, 1/702), B1 (0,1%, 1/702), B2 (0,1%, 1/702), C2 (0,9%, 6/702), D1 (0,9%, 6/702), D2 (4,6%, 32/702), D4 (5,1%, 36/702), D com subgenótipo não identificado (0,7%, 5/702), E (0,6%, 4/702), F2a (4,6%, 32/702), F4 (0,4%, 3/702), e G (0,4%, 3/702). Cepas do HBV com mutações de resistência (rtS202G, rtM204V/I, rtA194T, rtM250I, rtA181T/S, rtT184S) associadas ou não a mutações compensatórias (rtL80I, rtV173L, rtL180M, rtV207I) foram identificadas em 1,6% (11/702) das amostras analisadas. Cepas com mutações potencialmente associadas com resistência ao adefovir (rtS85A, rtL217R, rtI233V, rtN238T, rtN238D, rtN248H, rtV214A,e rtQ215S) ou ao entecavir (rtS219A) foram identificadas em 7,7% (54/702) e 2,6% (16/702) dos pacientes, respectivamente. Cinquenta e sete (8,5%) amostras apresentaram cepas do HBV com mutações na principal região hidrofílica do HBsAg previamente relacionadas com escape dos anticorpos anti-HBs ou com prejuízo na secreção do HBsAg. Foram feitas análises estatísticas para avaliar a correlação entre os subgenótipos do HBV mais frequentes na casuística (A1, A2, D1, D2, D3, D4 e F2a) e a presença de mutações nos genes PCC. Dentre as mutações nos genes PCC associadas com redução ou falha na expressão do HBeAg, as mutações A1762T/T1764A estiveram associadas aos subgenótipos A1 e F2a; G1862T e mutações nas posições 1809-1812 ao subgenótipo A1; G1896A e/ou G1899A aos subgenótipos D2, D3 e D4. Mutações associadas com evolução da doença foram detectadas e entre essas as mutações C1766T e T1768A estiveram associadas aos subgenótipos A1 e F2a, e a mutação G1888A foi associada ao subgenótipo A1. As cepas do HBV que circulam nas diferentes regiões brasileiras estudadas apresentam grande variabilidade genética e a distribuição dos genótipos e subgenótipos reflete a formação histórica de cada região e do fluxo migratório mais recente. A frequência de cepas do HBV com mutações de resistência aos AN circulando entre pacientes virgens de tratamento com esses medicamentos nas diferentes regiões do Brasil estudadas é baixa, sendo que o perfil de mutações que confere resistência total à lamivudina e parcial ao entecavir parece ser o mais disseminado. Embora tenham sido detectados casos de infecção com cepas do HBV portando mutações com grande impacto na antigenicidade dessa proteína todas as amostras apresentaram HBsAg detectável. Pacientes com HBeAg negativo foram mais frequentes na casuística estudada, independente do subgenótipo. As mutações encontradas nos genes PCC sugerem que há perfis de mutações diferentes envolvidos na negatividade do HBeAg para cada subgenótipo / The main aim of this study was to evaluate the frequency of HBV strains harboring mutations that confer resistance to nucleos(t)ide analogues (NA) used to hepatitis B treatment among treatment-naïve patients with chronic hepatitis B from different Brazilian region. Furthermore, we evaluated the presence of mutations that alter the antigenicity of HBsAg causing anti-HBs escape; mutations in genes pre-core/core and the association of different subgenotypes with the mutations detected and demographic and laboratory characteristics of the patients. Serum samples from 779 treatment-naïve patients with chronic HBV infection were included in this study. The samples were collected between 2006 to 2011 and the patients were from the following states: Pará, Maranhão, Bahia, Minas Gerais, São Paulo, Paraná and Rio Grande do Sul. HBV DNA was extracted from serum samples using the QIAamp DNA Blood Mini Kit (Qiagen) and amplification of S/polymerase (S/Pol) and pre-core/core (PCC) regions were performed by nested PCR. The amplified PCR products were submitted to sequencing in an automatic DNA sequencer (ABI 3500). The sequences obtained were analyzed to classify HBV genotypes/subgenotypes and to analyze the presence of mutations. S/Pol region was amplified and sequenced successfully from 702 samples, which were included in this study. Among these 702 samples, seven genotypes and 12 subgenotypes have been identified. HBV subgenotype A1 was the most frequent (63.7%, 447/702), followed by HBV/D3 (14.5%; 102/ 702). The remaining genotypes and subgenotypes identified and their frequencies were as follows: A2 (3.3%, 23/702), A3 (0.1%, 1/702), B1 (0.1%, 1/702), B2 (0.1%, 1/702), C2 (0.9%, 6/702), D1 (0.9%, 6/702), D2 (4.6%, 32/702), D4 (5.1%, 36/702), D unclassified subgenotype (0.7%, 5/702), E (0.6%, 4/702), F2a (4.6%, 32/702), F4 (0.4%, 3/702), and G (0.4%, 3/702). HBV strains harboring mutations conferring NA resistance alone (rtS202G, rtM204V/I, rtA194T, rtM250I, rtA181T/S, rtT184S) or combined with compensatory mutations (rtL80I, rtV173L, rtL180M, rtV207I) were identified in 1.6% (11/702) of the patients. Isolates harboring mutations potentially associated with adefovir resistance (rtS85A, rtL217R, rtI233V, rtN238T, rtN238D, rtN248H, rtV214A, and rtQ215S) or entecavir resistance (rtS219A) were identified in 7.7% (54/702) and 2.6% (16/702) of the patients, respectively. HBV with HBsAg mutations previous related with anti-HBs escape or impaired secretion were detected in 8.5% (57/702) of the samples. Statistical analyzes were performed to assess the correlation between the more frequent HBV subgenotypes found in this study (A1, A2, D1, D2, D3, D4 and F2a ) and mutations in PCC genes. Among the mutations found in these genes that were associated with reduction or failure in HBeAg synthesis, A1762T/T1764A mutations were associated to subgenotypes A1 and F2a; G1862T and mutations at positions 1809-1812 to subgenotype A1; G1896A and/or G1899A to subgenotypes D2, D3 and D4. Other mutations associated with disease progression were found: C1766T and T1768A mutations were associated with subgenotypes A1 and F2a, and the G1888A mutation was associated with subgenotype A1. HBV strains circulating in different Brazilian regions studied showed high genetic variability and distribution of genotypes and subgenotypes reflects the population formation history of each region and the occurrence of recent events of migration. The frequency of HBV strains with NA resistance mutations circulating among treatment-naive patients in different regions of Brazil studied is low and the profile of mutations that confer total resistance to lamivudine and partial resistance to entecavir is more widespread. Although some cases of infection have been detected with HBV strains carrying mutations associated with major impact on the antigenicity of this protein, all samples had detectable HBsAg. HBeAg negative cases were more frequent in the studied population, regardless of subgenotype. Different pattern of mutations were found in PCC genes, suggesting that different mechanisms are involved in HBeAg negativity for each subgenotype

O transplante de fígado é definido como a terapêutica de escolha para as doenças hepáticas em estágio terminal. Por muitos anos, o sucesso deste procedimento foi mensurado pelas taxas de mortalidade e a freqüência de complicações nas anastomoses biliares e complicações infecciosas. No entanto, mais recentemente um novo foco de preocupação voltou-se para a avaliação da qualidade de vida no grupo de pacientes transplantados. Os benefícios do transplante hepático sobre a qualidade de vida dos pacientes é um evento já demonstrado previamente em alguns estudos que utilizaram questionários genéricos de avaliação de qualidade de vida. A qualidade de vida em saúde também pode ser acessada através de questionários específicos, como o LDQOL (Liver Disease Quality of Life). Este é um instrumento voltado especificamente para sintomas relacionados às doenças hepáticas, desenvolvido nos Estados Unidos e recentemente traduzido para o Português e adaptado culturalmente para à população brasileira. Objetivo Aplicar este novo instrumento na população de pacientes em lista de espera de transplante de fígado e naqueles submetidos a transplante, reavaliando diferentes aspectos da qualidade de vida destes pacientes e avaliando o impacto da recidiva da doença de base após o transplante hepático. Método: Foram aplicados os questionários LDQOL e SF?36 a 126 pacientes em acompanhamento ambulatorial regular no Serviço de Transplante e Cirurgia do Fígado do Hospital das Clínicas da Universidade de São Paulo, dos quais 65 eram pacientes cirróticos em lista de espera de transplante de fígado e 61 deles eram pacientes submetidos a transplante hepático há pelo menos 6 meses e no máximo há 60 meses. Também foi aplicado o questionário SF-36 a um grupo de doadores de sangue sadios, pareados por sexo e idade e que funcionou como grupo controle. As avaliações, realizadas a partir da formação destes 3 grupos, incluiram a comparação da qualidade de vida entre cirróticos e transplantados, a comparação entre indivíduos saudáveis e portadores de hepatopatia e a influência de outros aspectos como etiologia da doença hepática, escore do MELD, classificação de Child-Pugh, presença de co-morbidades, tempo decorrido do transplante e efeito da recidiva da doença hepática sobre a qualidade de vida do paciente transplantado. O método estatístico aplicado foi o teste de Mann- Whitney. Resultados: As pontuações atingidas para os pacientes em lista de transplante hepático para mensuração da qualidade de vida foram significantemente mais baixas do que aquelas atingidas pelo grupo controle. Na comparação entre cirróticos com MELD <= 15 e cirróticos com MELD > 15, tanto o SF-36 como o LDQOL mostraram pior qualidade de vida nos pacientes com MELD mais elevado. A comparação entre indivíduos antes e depois do transplante evidenciou melhor qualidade de vida no grupo de pacientes transplantados. Isto ficou evidenciado tanto pelo LDQOL como SF-36. No grupo de pacientes pré-transplante, o questionário LDQOL mostrou maior comprometimento da qualidade de vida naqueles pacientes com cirrose por VHC, quando comparado a outras etiologias, enquanto o SF-36 não teve a mesma acurácia em demonstrar esta diferença. Nos pacientes transplantados, a recidiva da hepatite C determina comprometimento da qualidade de vida, quando avaliados pelo LDQOL, mas não pelo SF-36. Conclusões: O uso de um instrumento de medida de qualidade de vida - o LDQOL - pôde demonstrar, com maior acurácia, o seu comprometimento nos pacientes cirróticos, a melhora observada nos pacientes transplantados e o impacto negativo da recidiva da hepatite C no pós-transplante. / Liver transplantation has been established as the standard treatment for patients with end stage liver disease and for many years the outcomes of its procedure has been measured as mortality rates and biliary and infectious complications. More recently a new issue has been raised and many studies have been changing the focus to analyze the health related quality of life among the population of transplanted patients. A positive effect of liver transplantation on health-related quality of life (HRQOL) has been documented in many studies using generic instruments. Health-related quality of life can also be assessed with specific instruments, as the Liver Disease Quality of Life(LDQOL), a questionnaire targeted to symptoms of hepatic disease, that has been recently translated to Portuguese and culturally adapted to Brazilian population. Our aim was to reevaluate different aspects of HRQOL before and after liver transplantation with this new instrument, and determinate the impact of liver disease recurrence after the surgical procedure. Methods: The LDQOL and SF-36 questionnaires were applied to 126 patients at the Service of Transplant and Liver Surgery of Clinical Hospital of University of São Paulo, 65 of them on the transplant waiting list and 61 of them after 6 to 60 months of liver transplantation. It was also analyzed the quality of life, by using the SF-36 questionnaire, of health blood donors, paired by sex and age, to serve as control group. Multiple comparisons were made concerning etiology of cirrhosis, co-morbidities, MELD scores, time elapsed of transplant, recurrence of liver disease after liver transplantation, using the Mann-Whitney test. Results: HRQOL scores for patients waiting for transplantation were significantly lower than those for the control group. Patients with MELD scores > 15 showed worse healthrelated quality of life than patients with MELD scores <= 15, both with SF-36 and LDQOL. When the group of transplanted patients was compared with patients before transplant, an improvement of HRQOL was found in the transplanted group with both SF-36 and LDQOL. Quality of life in pretransplant patients was found to be worse in those with cirrhosis due to hepatitis C than in those with cirrhosis due to other etiologies; the reduction in quality of life was found to be greater using LDQOL than using SF-36. Transplanted patients with recurrence of hepatitis C had worse HRQOL when measured with LDQOL, but not with SF-36. Conclusions: LDQOL, a specific instrument for measuring quality of life is more reliable than SF-36 in showing quality of life impairment. A deterioration in health-related quality of life after recurrence of hepatitis C could be observed with LDQOL.

Hepatitis C virus is a pathogen causing severe hepatic diseases. Though HCV genotype 6a circulated prevalently in Hong Kong, its sequence information is greatly in deficient. The molecular characteristics and epidemiology of HCV 6a were thus extensively investigated in this thesis. The distribution of HCV genotypes in Hong Kong was analyzed from 1055 samples by the Ohno's method. HCV 6a accounted for 23.6% HCV infections in the general population and 58.5% in the injecting drug-users. It is prevalent in Hong Kong, associated with younger age, injecting drug-user status and the male gender. Fourteen independent HCV 6a isolates were sequenced for their full-genomes. They share a sequence homology of 94.5% between each other. HCV 6a had undergone high frequency of recombination. Four (28.5%) of 14 isolates were found having recombination with other strains in different genomic regions. Evolutionary pressure on HCV 6a genomes was analyzed by evaluating dS/dN ratio. NS4A, NS4B and NS3 showed higher dS/dN ratios (16.94-29.30) indicating a purification effect, whilst NS2, E2 and p7 showed lower dS/dN ratios (2.35-7.33) indicating a positive selection effect. This pattern of evolutionary pressure distribution alongside genomic regions was similar to the observations in HCV 1b. However HCV 6a eISDR experienced less extent of positive selection than HCV 1b eISDR (dS/dN = 12.82 vs 4.96) did. Evolutionary history of HCV 6a was inferred by Bayesian coalescent analysis. Twenty-six heterochronic, 513-bp HCV 6a partial-NS5A sequences and 63 HCV 1b sequences were analyzed. The time of exponential growth of HCV 6a in Hong Kong was postulated as during 1986 to 1994, overlaps with the time 1987 to 1997 when the second Vietnamese Boat People influx event occurred. Rooted phylogenetic analysis showed that Vietnamese HCV 6a strains were ancestors of the Hong Kong strains. Hence, a hypothesis was raised that HCV 6a outbreak in Hong Kong may be related to the Vietnamese Boat People influx event. The sequence variations within the eISDR of HCV 6a and HCV 1b were explored for correlation with the outcome of IFN-alpha/ribavirin combination treatment. Twenty-five HCV 6a patients and 37 HCV 1b patients were recruited. Three amino acid variations I2160V, V2256I, and I2292V were significantly correlated with the treatment outcome (P &lt; 0.05) for HCV 6a. Three variations R2260H, V2268I and S2278T (P = 0.023-0.076) were weakly correlated with the outcome for HCV 1b. None of correlated variations located within the previously defined ISDR. These pieces of information can be helpful for predicting the outcome before the commencement of treatment. / Zhou Xiaoming. / "May 2006." / Adviser: Paul K. S. Chan. / Source: Dissertation Abstracts International, Volume: 69-01, Section: B, page: 0207. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 178-186). / 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. / Abstracts in English and Chinese. / School code: 1307.

INTRODUÇÃO: A infecção pelo vírus da hepatite B (VHB) é responsável por grande parte das doenças hepáticas crônicas em todo o mundo. Em pessoas vivendo com HIV/Aids (PVHA) a infecção pelo VHB tem maior risco de evolução para cirrose e carcinoma hepatocelular. A vacina da hepatite B é importante na prevenção de doença potencialmente grave, particularmente em PVHA, já que ambos os vírus têm as mesmas vias de transmissão e a coinfecção tem uma alta morbidade. Indivíduos imunocompetentes têm uma boa resposta humoral após uma primeira série de vacina da hepatite B e não há recomendações de rotina para doses de reforço. PVHA podem ter uma pior resposta à vacina da hepatite B, quando comparada à resposta em indivíduos imunocompetentes e a duração da imunidade nesses pacientes é desconhecida. OBJETIVOS: Geral: Avaliar os marcadores sorológicos de proteção e infecção pelo VHB em pacientes adultos vivendo com HIV/Aids, vacinados previamente para hepatite B. Específicos: Avaliar a persistência dos anticorpos anti-HBs em PVHA vacinadas previamente para hepatite B e que apresentaram resposta humoral protetora inicial; avaliar a resposta sorológica à revacinação para hepatite B nos pacientes vacinados previamente e que não apresentaram resposta humoral protetora inicial; investigar a presença de marcadores sorológicos de infecção pelo VHB em PVHA vacinadas previamente para hepatite B. MÉTODOS: Estudo observacional de coorte retrospectiva de PVHA vacinadas primariamente para hepatite B entre 2001 e 2002. Marcadores sorológicos de infecção e proteção para o vírus da hepatite B foram investigados nesses pacientes que ainda estavam em acompanhamento no Serviço de Extensão ao Atendimento de Pacientes HIV/Aids (SEAP), da divisão de Clínica de Moléstias Infecciosas e Parasitárias do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo entre 2012 e 2014. RESULTADOS: Uma coorte de 121 PVHA foi analisada quanto à soroconversão e persistência do anti-HBs. A maioria era do sexo feminino (54,5%) com média de idade de 50,1 anos. Destes pacientes, 58 (grupo 1) eram inicialmente respondedores à primeira série da vacina (anti- HBs >=10 mUI/mL) e 63 (grupo 2) eram não respondedores. Após um período mediano de avaliação de 11 anos, nenhum dos pacientes teve evidência sorológica de infecção pelo VHB e 41/58 (70.7%) dos inicialmente respondedores mantinham anti-HBs >= 10 mUI/mL. Maior contagem de células T CD4+ e anti-HBs >= 100 mUI/mL, no momento da primeira série vacinal, estiveram associados à persistência de anti-HBs. Durante o período avaliado, 35/63 (55.6%) dos pacientes inicialmente não respondedores (grupo 2) soroconverteram com sucesso (anti-HBs >= 10 mUI/mL) em resposta a uma ou mais doses de reforço vacinal. Foi associado à soroconversão do anti-HBs o número de doses de reforço recebidas. A partir do momento da soroconversão (anti-HBs >=10 mUI/mL), 70 pacientes não receberam nenhuma dose adicional de vacina de hepatite B (grupo 3). Após um período mediano de 10 anos, 54/70 (77,1%) destes indivíduos mantinham anti- HBs >= 10 mUI/mL. CONCLUSÕES: A avaliação dos marcadores sorológicos para VHB em PVHA vacinadas previamente para hepatite B evidenciou: alta persistência de anti-HBs após um período de 10 a 11 anos; doses adicionais de vacina foram capazes de induzir resposta humoral em indivíduos inicialmente não respondedores; não foram detectados marcadores sorológicos de infecção (HbsAg ou Anti-HBc) após 11 anos da vacinação inicial / BACKGROUND: Hepatitis B Virus (HBV) infection is responsible for great part of chronic hepatic diseases worldwide. In people living with HIV (PLHIV), HBV infection has more risk of progressing to cirrhosis and hepatocarcinoma. Hepatitis B vaccine is important in the prevention of a potentially severe disease, particularly in PLHIV, since both viruses have the same routes of transmission and co-infection has greater morbidity. Immunocompetent individuals have a good humoral response after the first hepatitis B vaccine series and no recommendation is made regarding booster doses. PLHIV may have a poor hepatitis B vaccine response, when compared to immunocompetent and the duration of immunity in these patients is unknown. OBJECTIVES: General: Evaluate serological markers of infection and protection from HBV in PLHIV previously vaccinated for hepatitis B. Specific: Evaluate anti-HBs persistence in PLHIV previously vaccinated for HBV who responded to a primary vaccine series; evaluate response to revaccination for hepatitis B in patients who did not respond to first vaccine series; investigate serological markers of infection from HBV in PLHIV previously vaccinated for hepatitis B. METHODS: Observational retrospective study of a PLHIV cohort primarily vaccinated between 2001 and 2002 for hepatitis B. Serological markers of infection and protection from HBV were investigated in those patients who were still attending the HIV/AIDS Patient Care Extension Service at the Clinical Division of Infectious and Parasitic Diseases attached to Hospital das Clínicas at Faculdade de Medicina at Universidade de São Paulo between 2012 and 2014. RESULTS: A cohort of 121 PLHIV was analyzed for seroconversion and persistence of anti-HBs. The majority were female (54.5%) and mean age 50.1 years. From these patients, 58 (group 1) were initially responders to the first vaccine series (anti- HBs >=10 mIU/mL) and 63 (group 2) were non- responders. After a median period of 11 years, none of the patients had serologic evidence of HBV infection and 41/58 (70.7%) of the initially responders had maintained anti-HBs >=10 mIU/mL. Greater CD4+ cell counts and anti- HBs>= 100mIU/mL at the time of first vaccine series were associated with persistence of anti-HBs. During evaluation period, 35/63 (55.6%) of the initially non-responders (group 2) successfully seroconverted (anti-HBs >=10 mIU/mL) in response to one or more booster doses. Booster doses may be effective in PLHIV. Number of booster doses were associated to seroconversion. Seventy of the 121 patients did not receive any further booster doses of hepatitis B vaccine from the time of their seroconversion (anti-HBs >=10 mIU/mL) (group 3). After 10 years of the seroconversion, 54/70 (77,1%) of these individuals has maintained anti- HBs >= 10 mIU/mL. CONCLUSIONS: Evaluation of serological markers for HBV in PLHIV previously vaccinated for hepatitis B showed: strong persistence of anti-HBs after a period of 10 to 11 years; additional vaccine doses elicited humoral response in initially non-responders; there was no serologic evidence of HBV infection (HbsAg ou Anti-HBc) about 11 years after initial vaccination

INTRODUÇÃO: A patogênese da hepatite C crônica resulta principalmente de mecanismos imuno-mediados com a atuação central das citocinas tanto na lesão hepatocelular como na eliminação e na persistência do vírus da hepatite C (VHC). OBJETIVOS: Investigar a resposta imune adaptativa da hepatite C crônica através da expressão das células inflamatórias no tecido hepático e de citocinas no tecido hepático e no sangue, relacionando-os com dados demográficos, laboratoriais e histológicos. MÉTODOS: Pacientes com hepatite C crônica, virgens de tratamento foram selecionados no ambulatório de Moléstias Infecciosas do Hospital das Clínicas. Foram utilizados dois grupos controles para comparação: de doadores de sangue saudáveis e fragmentos de biópsia hepática de doadores de órgãos. Todos os controles selecionados não tinham evidência de hepatopatia. As seguintes citocinas foram analisadas no sangue pelo método quantitativo de ELISA no sangue dos casos e dos doadores de sangue: interleucina (IL) 1, IL2, IL4, IL6, IL 10, interferon (IFN) , fator de necrose tumoral (TNF) e fator de crescimento e transformação (TGF) . As mesmas citocinas e as populações celulares CD4+, CD8+, CD45RO+, CD57+, CD68+ e S100 foram quantificadas através de método imuno-histoquímico no espaço portal e no lobo hepático dos casos e dos doadores de fígado. Esses dados foram posteriormente associados às alterações histológicas pela classificação de Ishak. RESULTADOS: Foram selecionados 51 pacientes com hepatite C crônica, 58% do gênero masculino; 66,6% brancos e média de idade de 39 anos (variando de 20 a 59). Foram selecionados 33 doadores de órgãos e 51 doadores de sangue. Comparando com os doadores de sangue, os casos apresentaram maiores níveis séricos de IL2 (p <0,001), IL10 (p <0,001), INF (p 0,018) e TGF (p <0,001). Na análise das biópsias hepáticas, os casos apresentaram maior expressão de LTCD4+ portais (p <0,001), LTCD8+ portais (p <0,001), IL4 lobular (p 0,001), IL10 lobular (p 0,007), INF lobular (p <0,001), TNF portal (p <0,001) e lobular (p <0,001), TGF portal (p <0,001) e lobular (p <0,001) do que os doadores de órgãos. Entre os casos, houve correlações significantes diretas entre os seguintes marcadores e as alterações histológicas: CD4+ portal com a atividade peri portal (p 0,004); CD4+ lobular com a atividade lobular (p 0,017); TGF lobular com a atividade lobular (p 0,016); IL1 portal com atividade peri portal (p 0,009) e IL8 do sangue e fibrose (p 0,036). As populações celulares foram correlacionadas às citocinas no fígado dos casos e houve significância direta entre: CD4+ portal e TNF portal (p 0,004); CD8+ portal e TGF portal (p 0,030); CD57+ portal e IL10 portal (p 0,008); CD57+ lobular com TGF lobular (p 0,040) e IL2 lobular (p 0,048); S100 portal e IL10 portal (p 0,014). Não houve correlação significante entre as citocinas do fígado e as citocinas do sangue nos pacientes com hepatite C crônica. A carga viral do VHC teve correlação indireta com LTCD8+ lobulares (p 0,020), IL2 portal (p 0.049) e lobular (p 0.004). DISCUSSÃO: O comando da resposta imune nesta casuística foi orquestrado pelos linfócitos T CD4+ e CD8+, com predomínio da resposta Th1 e o principal local dos eventos foi o espaço portal. A compartimentalização da resposta imune ao VHC foi evidenciada pela ausência de correlações significantes entre as citocinas do tecido hepático e do sangue nos pacientes com hepatite C crônica. / BACKGROUND: The pathogenesis of chronic hepatitis C results mainly of immunological mechanisms with cytokines playing a central role in hepatocellular necrosis and in the immunopathogenic process involved in viral clearance and persistence. AIM: To investigate immune response to hepatitis C virus (HCV) through expression of inflamatory cells in liver and cytokines in liver and serum, and assess the relationship with demografic, laboratorial and histological features. METHODS: Naïve patients with chronic hepatitis C were selected from Infectious Diseases Division at a University Hospital. Two sets of controls were selected for comparison: healthy blood donors and liver biopsy specimens from liver donors. All controls had no evidence of hepatic disease. The following cytokines were analyzed by quantitative ELISA method in serum of cases and healthy blood donor controls: interleukin (IL) 1, IL2, IL4, IL6, IL10, interferon (IFN) , tumor necrosis factor (TNF) , and transforming growth factor (TGF) . The same cytokines and cellular populations of CD4+ T lymphocytes (TL), CD8+ TL, CD45+, CD57+, CD68+ and S100 were quantified by immunohistochemistry in acinar and portal spaces in liver biopsies of cases and liver donor controls. These data were additionally associated to histological parameters by Ishak Score. RESULTS: Were selected 51 patients with chronic hepatitis C, 58% were males; 66,6% white and the median age was 39 (range 20 to 59) years. Were selected 33 liver donors and 51 blood donors. Compared with heathy blood donor controls, cases showed higher levels of IL2 (p <0.001), IL10 (p <0.001), INF (p 0.018) and TGF (p <0.001). In liver biopsy analyses, cases showed greater expression of the following cell populations and cytokines: portal CD4+ TL (p <0.001), portal CD8+ (p <0.001), acinar IL4 (p 0.001), acinar IL10 (p 0.007), acinar INF (p <0.001), portal TNF (p <0.001), acinar TNF (p <0.001), portal TGF (p <0.001) and acinar TGF (p <0.001). Among cases, significant positive correlations were found between the following markers and Ishak graded patterns: portal CD4+TL and periportal inflammation (p 0.004); acinar CD4+ and focal inflammation (p 0.017); acinar TGF and focal inflammation (p 0.016); portal IL1 and periportal inflammation (p 0.009) and IL8 in blood and fibrosis (p 0.036). The cellular populations were correlated to cytokines in liver of hepatitis C patients and there was significant positive correlation between: portal CD4+ and portal TNF (p 0.004); portal CD8+ TL and portal TGF (p 0,030); portal CD57+ and portal IL10 (p 0,008); acinar CD57+ and acinar TGF (p 0,040) and acinar IL2 (p 0,048); portal S100 and portal IL10 (p 0,014). No significant correlation was found between liver and serum cytokines in cases. Hepatitis C viremia was inversely correlated to acinar CD8+ TL (p 0.020); portal (p 0.049) and acinar IL2 (p 0.004). DISCUSSION: The command of the immune response in this casuistic was orchestrated by CD4+ TL and CD8+ T lymphocytes, with predominance of Th1 answer, and the main site where of the events ocurred was the portal space. The compartimentalization of immune response to HCV was evidenced by the absence of significant correlations between cytokines in hepatic tissue and blood from patients with chronic hepatitis C.

Made available in DSpace on 2015-04-22T22:14:03Z (GMT). No. of bitstreams: 1 Myuki Alfaia Esashika Crispim.pdf: 2246402 bytes, checksum: d9773c605258310c1244020f1e5cb467 (MD5) Previous issue date: 2007-09-20 / Hepatitis B and D are endemic in the Western Brazilian Amazon region , but few studies have been conducted to investigate the genetic variability of both viruses. The hepatitis B virus (HBV) has a high genetic variability, with eight different genotypes defined (A-H). In present classification hepatitis D virus (HDV) is also supposed to present eight different genotypes (I-VIII). The aim of this study was to describe the genotypes of the virus B and D of the Western Brazilian Amazon region. We selected 190 samples of chronic carriers with HBV and 50 of them presented double infection with HDV. The serum samples of HBV were submitted to the genotyping through Polymerase Chain Reaction (PCR), with type-specific primers . In the reactive samples for HDV RNA by RT-PCR was used the genotyping by Restriction Fragment Lenght Polymorphism (RFLP). The genotype A of HBV was detected as the most frequent, in 91 participants (56,5%), following by genotype F, in 41 (25,5%), and genotype D, in 29 (18,0%). In the HDV genotyping, we found only the genotype III. This study showed that the genotypes A, D and F of VHB and the genotype III of HDV represented the predominant genotypes in the Western Brazilian Amazon. / As hepatites B e D são endêmicas na Amazônia Ocidental Brasileira, mas poucos estudos têm buscado investigar a variabilidade genética de ambos os vírus. O vírus da Hepatite B (VHB) tem uma alta variabilidade genética, sendo definidos oito genótipos distintos (A-H). O vírus da hepatite D (VHD), na atual classificação, também é sugerido apresentar oito genótipos (I-VIII). O presente estudo teve o objetivo de descrever os genótipos do vírus B e D na Amazônia Ocidental Brasileira. Selecionamos 190 amostras de portadores crônicos do vírus da hepatite B, sendo que, destas, 50 apresentavam infecção dupla com o VHD. As amostras de soro do VHB foram submetidas a genotipagem por meio da Reação em Cadeia da Polimerase (PCR), com iniciadores tipo específicos. Em amostras reativas para o VHD RNA por RT-PCR foi realizada a genotipagem por Análise do Polimorfismo do Tamanho de Fragmentos de Restrição (RFLP). Foi detectado o genótipo A como o mais freqüente em 91 participantes (56,5%), seguido pelo F em 41(25,5%), e o D em 29 (18,0%). Na genotipagem para o VHD encontramos somente o genótipo III. Este estudo mostrou que os genótipos A, D e F do VHB e o genótipo III do VHD, representam os genótipos predominantes na Amazônia Ocidental Brasileira.