A infecção pelo vírus da hepatite C acomete cerca de 180 milhões de pessoas em todo mundo. Trata-se de doença com pouca manifestação clinica, onde cerca de 75% a 85% dos casos evolui para cronificação e aproximadamente 15% para hepatocarcinoma. Entre os fatores de risco mais conhecidos estão a realização de transfusões de sangue e hemoderivados anterior a 1993, uso de drogas endovenosas e relações sexuais desprotegidas. Este trabalho tem por objetivo estimar a prevalência de sorologia positiva para hepatite C e seus possíveis fatores de risco no distrito de Botafogo, município de Bebedouro, SP, onde a elevada freqüência de casos de hepatite chamou a atenção dos seus próprios moradores. Da população de 1318 habitantes, 353 foram sorteados para participar da pesquisa, sendo submetidos a questionário padronizado e coleta de sangue. Infecção pelo vírus da hepatite C foi pesquisada através de exames imunoenzimáticos e de PCR, no Instituto Oswaldo Cruz (Fiocruz), e por teste imunocromatográfico, no Laboratório de Sorologia do Hospital da Clínicas de Ribeirão Preto-USP . A prevalência encontrada foi de 8,8% (IC95%: 5,8 11,7). As variáveis que mostraram associação na análise univariada foram submetidas a um procedimento multivariado por aplicação do modelo de log binomial. As variáveis preditoras independentes de infecção pela hepatite C foram sexo masculino, tempo de residência acima de trinta anos e uso de medicações parenterais com material esterilizado por técnica de fervura. Uma possível explicação para a elevada prevalência nessa população reside na possibilidade de disseminação do vírus a partir de um antigo morador, que exercia informalmente atividades ligadas ao atendimento à saúde, especialmente aplicação de injeções, numa época anterior ao uso de seringas descartáveis. / Approximately 180 million people worldwide are infected by the hepatitis C virus. It is an illness with little clinical manifestation where about 75% - 85% of the cases evolve to chronification and about 15% to hepatocarcinoma. Among the bestknown risk factors are blood and blood by-product transfusions prior to 1993, use of intravenous drugs and unprotected sexual relations. This study has the objective of estimating the prevalence of positive serology for hepatitis C and its possible risk factors in the district of Botafogo, municipality of Bebedouro, São Paulo, where the high frequency of hepatitis cases caught the attention of the population itself. Out of a population of 1318 inhabitants, 353 were selected to participate in the research, being submitted to a standard questionnaire and blood collection. Hepatitis C infection was researched through immunoenzimatic and PCR exams at the Oswaldo Cruz Institute (FIOCRUZ), and by immunochromatographic tests at the Serology Laboratory of the Hospital das Clinicas in Ribeirão Preto USP. The prevalence found was of 8,8% (CI95%: 5,8 11,7). The variables that demonstrated an association in the univariate analysis were submitted to a multivariate procedure through the application of the binomial log model. The independent predictors for hepatitis C infection were male sex, local residence time over thirty years and use of parenteral medication with material sterilized through boiling technique. A possible explanation for the high prevalence in this population lies in the possibility of dissemination of the virus from an older inhabitant who informally exercised medical activities, especially the application of injections in a period before there was use of dischargeable syringes.

by Frankie Tat-kwong Lau. / Bibliography: leaves 63-70 / Thesis (M.Ph.)--Chinese University of Hong Kong, 1985

Orientador: Domingues Alves Meira / Banca: Alexandrina Sartori / Banca: Ricardo Sobhie Diaz / Banca: Fernando Lopes Gonçalves Júnior / Resumo: A aids e a hepatite C crônica são infecções caracterizadas por importante processo inflamatório contínuo, regulado por uma complexa interação entre citocinas. A persistência da atividade inflamatória crônica está intimamente relacionada com a progressão da patogênese da aids, bem como na indução de fibrose na hepatite C. Com o objetivo de avaliar o padrão de citocinas na infecção pelo HIV e na hepatite C crônica, as citocinas IL-2, IL-4, IL-10, TNF-α, INF-γ, TGF-β foram dosadas por Elisa e RT-PCR em cinco grupos: pacientes coinfectados pelo HIV/VHC (n=22), monoinfectados pelo HIV com supressão virológica pelo tratamento, e sem supressão virológica (n=17), monoinfectados pelo VHC (n=22) e um grupo controle composto por indivíduos doadores de sangue (n=10). IL-4 e IL-10 estiveram aumentadas consistentemente nos quatro grupos de estudo, determinando predomínio do perfil Th-2. INF-γ, TNF-α e TGF-β estiveram aumentados apenas nos grupos com infecção pelo VHC, com ou sem coinfecção pelo HIV. No grupo de monoinfectados pelo HIV com supressão virológica, a IL-2 dosada por RT-RCR esteve aumentada, porém os níveis séricos dosados por Elisa estavam normais. A alta produção de citocinas pró-inflamatórias INF-γ, TNF-α e TGF-β nos dois grupos de pacientes com infecção pelo VHC refletem o processo progressivo de acúmulo de inflamação e fibrose hepática. Já o predomínio de IL-4 e IL-10 em todos os grupos, citocinas ligadas ao perfil Th-2, demonstram a incapacidade de produção de uma resposta citotóxica Th-1, perpetuando a infecção e a inflamação crônica, mesmo naqueles indivíduos com supressão virológica pelo tratamento. Além de drogas antivirais, novos tratamentos imunomoduladores têm sido propostos para a erradicação viral, ou a interrupção das lesões causadas pelo estado inflamatório crônico... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Both AIDS and chronic hepatitis C (HCV) are characterized by continuous inflammatory process, regulated by a complex interaction between cytokines. The persistence of chronic inflammatory activity is closely related to the progression of the pathogenesis of AIDS, as well as the induction of fibrosis in HCV. In order to analyze the role of cytokines in HIV/HCV coinfection and the fibrosis progression, IL-2, IL-4, IL-10, TNF- α, INF-γ, TGF-β were measured by ELISA and RT -PCR in five groups: HIV/HCV coinfected patients (n = 22), HCV monoinfected patients (n = 22), HIV monoinfected patients with and without virological suppression (n = 17) and a control group composed by blood donors (n = 10). Hepatic biopsy and METAVIR classification were performed in all HCV patients (n=44). The baseline characteristics (sex, age and race) of all groups were similar. No correlations were found between cytokines and hepatic fibrosis. IL-4 and IL-10 were consistently increased in the four study groups, findings associated to a Th-2 profile. INF- γ, TNF-α and TGF-β were increased only in groups with HCV infection. In the group of HIV monoinfected patients with virological suppression, IL-2 measured by RT-RCR was increased, but serum levels measured by ELISA were normal. The high production of proinflammatory cytokines INF-γ, TNF-α and TGF-β in two groups of patients with HCV infection reflect the gradual process of inflammation and liver fibrosis. The predominance of IL-4 and IL-10 in all study groups demonstrates an inability to promote a cytotoxic Th-1 response. Even in HIV monoinfected patients with virological suppression with increased IL-2 expression, Th-2 cytokines were the predominant, perpetuating the chronic inflammation. In addition to antiviral drugs, new immunomodulatory treatments have been proposed... (Complete abstract click electronic access below) / Doutor

Chan Ming Lok. / Thesis submitted in: December 1999. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 93-108). / Abstracts in English and Chinese. / Ackowledgments --- p.i / Abstract in English --- p.ii / Abstract in Chinese --- p.iii / List of Abbreviations --- p.iv / List of Tables --- p.v / List of Figures --- p.vi / Chapter Chapter 1 --- Introduction and Objectives / Chapter 1.1 --- Hepatocellular Carcinoma --- p.1 / Chapter 1.1.1 --- Epidemiology --- p.1 / Chapter 1.1.2 --- Geographical Distribution --- p.1 / Chapter 1.1.3 --- Sex and Age --- p.1 / Chapter 1.1.4 --- Etiology --- p.2 / Chapter 1.1.5 --- Molecular Basis of HCC --- p.3 / Chapter 1.1.6 --- Situation in China and Hong Kong --- p.4 / Chapter 1.2 --- The Hepatitis B Virus --- p.5 / Chapter 1.2.1 --- Morphology --- p.5 / Chapter 1.2.2 --- Structure of the HBV Genome --- p.6 / Chapter 1.2.3 --- Functional Domains of the HBV Genome --- p.9 / Chapter 1.2.4 --- Pathogenesis of HBV Infection --- p.11 / Chapter 1.3 --- HBx --- p.12 / Chapter 1.3.1 --- The HBV x Gene --- p.12 / Chapter 1.3.2 --- The HBX Protein --- p.13 / Chapter 1.3.3 --- "Preferential HBX Expression in Sera, Hepatitis, Cirrhosis and HCC" --- p.13 / Chapter 1.3.4 --- Cellular Localization of HBX --- p.14 / Chapter 1.3.5 --- Animal Studies --- p.15 / Chapter 1.3.6 --- Functional Studies on HBX --- p.15 / Chapter 1.3.7 --- Variations in the HBx Gene --- p.21 / Chapter 1.4 --- Objectives of this Study --- p.24 / Chapter Chapter 2 --- Methods and Materials Methods / Chapter 2.1 --- Paraffin Embedding of Patient Tissue Samples --- p.26 / Chapter 2.1.1 --- Tissue Processing --- p.26 / Chapter 2.1.2 --- Paraffin Embedding of Tissue Samples --- p.26 / Chapter 2.2 --- Sectioning of Paraffin Embedded Tissue Sections --- p.26 / Chapter 2.3 --- Immunohistochemical Staining of Paraffin Embedded Tissue Sections --- p.26 / Chapter 2.3.1 --- Dewaxing of Paraffin-Embedded Tissue Sections --- p.26 / Chapter 2.3.2 --- Rehydration of Tissue Sections --- p.27 / Chapter 2.3.3 --- Antigen Retrieval --- p.27 / Chapter 2.3.4 --- Quenching of Endogenous Hydrogen Peroxidase --- p.27 / Chapter 2.3.5 --- Blocking of Endogenous Biotin and Non-Specific Protein Binding --- p.27 / Chapter 2.3.6 --- Antibody Incubation and Color Development --- p.27 / Chapter 2.3.7 --- Counterstaining and Coverslip Mounting --- p.28 / Chapter 2.3.8 --- Interpretation of Immunostaining Results --- p.28 / Chapter 2.4 --- DNA Extraction from HCC Tissues --- p.28 / Chapter 2.4.1 --- Sectioning of Frozen HCC Specimens --- p.28 / Chapter 2.4.2 --- Proteinase K Digestion and Phenol Chloroform Extraction --- p.29 / Chapter 2.4.3 --- Ethanol Precipitation and Re-suspension in Tris-EDTA (TE) Buffer --- p.29 / Chapter 2.5 --- Quantitation and Purity Check of Extracted DNA --- p.29 / Chapter 2.6 --- Quality Check for Extracted Genomic DNA --- p.30 / Chapter 2.6.1 --- Agarose Gel Electrophoresis --- p.30 / Chapter 2.6.2 --- Polymerase Chain Reaction (PCR) of the β-globin Gene --- p.30 / Chapter 2.6.3 --- Analysis of PCR Fragments by Agarose Gel Electrophoresis --- p.30 / Chapter 2.7 --- Polymerase Chain Reaction Amplification of HBs and HBx Genes of the Hepatitis B Virus --- p.31 / Chapter 2.8 --- Southern Blot of HBx PCR Fragments --- p.31 / Chapter 2.8.1 --- Immobilization of DNA onto a Positively Charged Nylon Membrane and Pre-hybridization --- p.31 / Chapter 2.8.2 --- Radio-labeling of an HBV Probe --- p.32 / Chapter 2.8.3 --- Hybridization of a 32P-labeled HBV Probe and Film Exposure --- p.32 / Chapter 2.9 --- Cloning of PCR Fragments into pGEM®-T Vector for Sequencing --- p.33 / Chapter 2.9.1 --- Gel Extraction and Purification --- p.33 / Chapter 2.9.2 --- Ligation --- p.33 / Chapter 2.10 --- Transformation of Competent DH5a cells --- p.34 / Chapter 2.10.1 --- Preparation of Competent DH5α Using Calcium Chloride --- p.34 / Chapter 2.10.2 --- Heat Shock of Competent DH5α Cells --- p.34 / Chapter 2.10.3 --- Plating of Transformed Cells onto LB Agar Plates --- p.34 / Chapter 2.10.4 --- Screening of Transformants for Inserts --- p.35 / Chapter 2.11 --- Miniprep of Plasmid DNA --- p.35 / Chapter 2.11.1 --- Inoculation of Bacterial Clones --- p.35 / Chapter 2.11.2 --- DNA Extraction by Alkaline Lysis and Phenol/Chloroform --- p.35 / Chapter 2.11.3 --- Ethanol Precipitation and Re-suspension in TE Buffer --- p.35 / Chapter 2.11.4 --- Confirmation of Positive Clones --- p.36 / Chapter 2.12 --- Sequencing of pGEM®-T Cloned HBx PCR Fragments --- p.36 / Chapter 2.13 --- Construction of the HBx-GFP Plasmid --- p.36 / Chapter 2.13.1 --- PCR Amplification of HBx Gene Inserts --- p.36 / Chapter 2.13.2 --- Confirmation of HBx Insert Sequence by DNA Sequencing --- p.37 / Chapter 2.13.3 --- Restriction Digest of HBx-pGEM®-T Plasmids to Obtain HBx Inserts --- p.37 / Chapter 2.13.4 --- Restriction Digest of pEGFP-Nl Cloning Vector for Cloning --- p.37 / Chapter 2.13.5 --- Ligation of HBx Inserts into the pEGFP Cloning Vector --- p.37 / Chapter 2.14 --- Large Scale Plasmid DNA Preparation --- p.38 / Chapter 2.15 --- Cell Culture --- p.39 / Chapter 2.16 --- Transfection using LipofectAminéёØ --- p.39 / Chapter 2.16.1 --- Seeding of Cells for Coverslip Growth --- p.39 / Chapter 2.16.2 --- Transfection using LipofecAminéёØ --- p.39 / Chapter 2.17 --- Cell Fixation and DAPI Staining Materials --- p.40 / Chapter 2.18 --- Chemicals --- p.41 / Chapter 2.19 --- Antibodies --- p.41 / Chapter 2.20 --- "Formalin-fixed, Paraffin Embedded Tissues of HCC Tissues from Xiamen" --- p.41 / Chapter 2.21 --- Frozen Liver Tissues --- p.41 / Chapter 2.22 --- PCR Reagents --- p.43 / Chapter 2.23 --- Primers --- p.43 / Chapter 2.24 --- Plasmid --- p.43 / Chapter 2.25 --- Enzymes --- p.43 / Chapter 2.26 --- Ligation Reagents --- p.43 / Chapter 2.27 --- Cloning Vectors --- p.45 / Chapter 2.28 --- Competent Cell --- p.45 / Chapter 2.29 --- Hela and HepG2 Cell Line --- p.45 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Hepatitis B Virus Status of HCC Patients from Hong Kong and Xiamen --- p.46 / Chapter 3.2 --- Immunohistochemical Studies of the HBx Protein in Hong Kong and Xiamen HCC --- p.46 / Chapter 3.2.1 --- Cross Reaction of Anti-99 with Cytokeratin 18 (CK18) --- p.46 / Chapter 3.2.2 --- HBx Expression in HCC Patient Tissue Samples from Hong Kong --- p.50 / Chapter 3.2.3 --- HBxAg Staining in HCC Tissue Samples from Xiamen --- p.50 / Chapter 3.3 --- Agarose Gel Electrophoresis of DNA Extracted from Frozen Liver Tissues --- p.50 / Chapter 3.4 --- PCR Amplification of the β-globin Gene --- p.55 / Chapter 3.5 --- PCR Amplification of the HBs Gene from Liver Samples of HCC Patients from Hong Kong --- p.55 / Chapter 3.6 --- PCR Amplification of the HBx Gene from Liver Samples of HCC Patients from Hong Kong --- p.55 / Chapter 3.7 --- Amplification of the HBx Gene from Serum Samples of Chronic Hepatitis B Virus from Hong Kong Using Nested PCR --- p.61 / Chapter 3.8 --- Southern Blot of HBx PCR Fragments --- p.61 / Chapter 3.9 --- Cloning and Sequencing of the HBx Gene in HCC and Chronic Hepatitis B Patient Samples from Hong Kong --- p.61 / Chapter 3.10 --- Expression Pattern of Wild-type HBx-GFP Fusion Protein in Transiently Transfected HeLa and HepG2 Cells --- p.73 / Chapter 3.11 --- Expression Patterns of HBx-GFP with and without Mutations at Codons 130 and 131 in HeLa and HepG2 Cell Line --- p.78 / Chapter 3.12 --- Growth Kinetics of HeLa Cells Transfected with GFP and Wild-type HBx-GFP with and without Mutations in Codons 130 and131 --- p.81 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- HBxAg Expression in Tumorous and Surrounding Non-tumorous Tissues --- p.83 / Chapter 4.2 --- "Detection of the HBx Gene in Sera, Non-tumorous and Tumorous Tissues" --- p.84 / Chapter 4.3 --- HBx Gene Mutations in Chronic Hepatitis and HCC --- p.85 / Chapter 4.3.1 --- Codon 127 (HBV nt 1752-1754) --- p.85 / Chapter 4.3.2 --- Codons 130 and 131 (HBV nt 1761-1766) --- p.86 / Chapter 4.3.3 --- Lack of Correlation between HBx Gene Mutations and Lack of HBxAg Expression --- p.87 / Chapter 4.4 --- Cellular Localization of HBxAg in Transiently Transfected Cells Lines --- p.88 / Chapter 4.5 --- Functional Difference Between Wild-type and Mutant HBX Protein --- p.89 / Chapter Chapter 5 --- Conclusions and Directions for Further Studies / Chapter 5.1 --- Conclusions --- p.91 / Chapter 5.2 --- Directions for Further Studies --- p.92 / References --- p.93 / Appendix / Chapter A1 --- Recipes of Reagents Used in this Study --- p.109 / Chapter A2 --- Schematic Setup of Downward Capillary Transfer of DNA --- p.112 / Chapter A3 --- Circle Map of the pGEM®-T Cloning Vector and Construct of the HBx-pGEM®-T Plasmid --- p.113 / Chapter A4 --- Circle Map of the pEGFP-Nl Cloning Vector and Construct of the HBx-GFP Plasmid --- p.114

Lam Siu Kuen. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 77-79). / Abstracts in English and Chinese. / Acknowledgments --- p.ii / English Abstract --- p.iv / Chinese Abstract --- p.vi / Table of Contents --- p.vii / List of Tables --- p.x / List of Figures --- p.xii / List of Appendices --- p.xiv / Chapter Chapter 1. --- Introduction --- p.1 / Chapter Chapter 2. --- Research Background --- p.7 / Chapter 2.1 --- Epidemiology of Hepatitis B Virus (HBV) --- p.7 / Chapter 2.2 --- The prevalence of HBV around the world --- p.12 / Chapter 2.3 --- The prevalence of HBV in Hong Kong --- p.16 / Chapter 2.4 --- Standard medical treatment --- p.17 / Chapter Chapter 3. --- Literature Review --- p.20 / Chapter Chapter 4. --- Data compilation --- p.31 / Chapter 4.1 --- Prince of Wales Hospital's Dataset --- p.31 / Chapter 4.2 --- Expert Opinion and other published data --- p.35 / Chapter 4.3 --- Definition of health states under study --- p.36 / Chapter Chapter 5. --- Empirical Findings I --- p.39 / Chapter 5.1 --- Estimation of disease costs from Department of Hepatology --- p.39 / Chapter 5.1.1 --- Methodology and sample size --- p.39 / Chapter 5.1.2 --- Summary of costs included in the analysis --- p.42 / Chapter 5.1.3 --- Descriptive analysis --- p.43 / Chapter 5.1.4 --- Calculation method --- p.44 / Chapter 5.1.5 --- Empirical results --- p.47 / Chapter 5.2 --- Estimation of direct medical cost from the Department of Oncology --- p.51 / Chapter 5.2.1 --- Methodology and sample size --- p.51 / Chapter 5.2.2 --- Summary of Costs included in the analysis --- p.52 / Chapter 5.2.3 --- Descriptive analysis --- p.52 / Chapter 5.2.4 --- Calculation method --- p.54 / Chapter 5.2.4 --- Empirical results --- p.58 / Chapter 5.3 --- Kernel estimators --- p.61 / Chapter 5.4 --- Sensitivity to cost variations in medical procedures --- p.63 / Chapter Chapter 6. --- Empirical Findings II --- p.65 / Chapter 6.1 --- Estimation of indirect medical costs --- p.65 / Chapter 6.1.1 --- Methodology --- p.65 / Chapter 6.1.2 --- Calculation method --- p.67 / Chapter 6.1.3 --- Empirical results --- p.68 / Chapter 6.2 --- Estimation of indirect cost (HCC-deceased) --- p.70 / Chapter 6.3 --- Premature death --- p.71 / Chapter 6.4 --- Limitation --- p.72 / Chapter Chapter 7. --- Conclusion --- p.75 / Bibliography --- p.77 / Tables --- p.80 / Figures --- p.120 / Appendices --- p.128

Chronic infection with the hepatitis C virus infection (HCV) affects over 170 million individuals worldwide. In this thesis the natural history and management of hepatitis C in North- East London was investigated. The prevalence of cirrhosis in patients with chronic hepatitis C rises with increasing duration of infection. In Asian patients infected at birth, infection over 60 years causes cirrhosis in 71 % of infected individuals. Since the rate of fibrosis progression in Asian patients is the same as that seen in Caucasian patients, it is likely that similar rates of cirrhosis will be seen in all patients who are infected with HCV for over 60 years. Factors found to be associated with fibrosis progression were:- age and alcohol excess. Insulin resistance was associated with fibrosis progression. However, elevated serum ferritin or hepatiC iron were not. Knowledge of hepatitis C in the East of London was examined and found to be poor despite the Department of Health information campaign. Educational meetings and postal surveys improved the level of knowledge of HCV. However as our group only assessed knowledge immediately after completion of the sessions, such a testing regime does not address long-term knowledge retention. We examined current and novel management strategies for patients with chronic HCV. Current therapy involves pegylated interferon and ribavirin. We found that insulin resistance was a poor predictor of sustained virological response. Chinese herbal treatments for hepatitis C are widely used but poorly studied. Our group designed a randomised controlled double blind study to assess whether Chinese herbal treatment is effective and results from this study show that recruitment and retention in trials of alternative therapies are problematic and that the herbal remedy had little effect on viraemia and quality of life, although liver function tests did improve a little.

Hepatitis C virus (HCV) infection has become a global public health concern with over 130 million people chronically infected and over 350,000 deaths every year from HCV-related liver diseases. GB virus-B (GBV-B) infection in tamarins is a surrogate model for acute HCV infection. Whilst HCV infection commonly leads to chronicity, GBV-B is naturally cleared. To better understand this natural clearance, this project aimed to study the associated humoral immune response to GBV-B. Additionally, GBV-B-specific antibodies were produced with the aim of characterising the pathology of the virus. Previously, there was no available GBV-B neutralisation assay to identify antibodies in this animal model. Therefore, a GBV-B neutralisation assay, based on a method that is known to be successful for the closely-related HCV, was developed. This method involved producing pseudotyped retroviral particles (PV) expressing the GBV-B envelope that could infect a human hepatocarcinoma cell line. GBV-B PV production was confirmed by western blotting. Future studies can now test archived tamarin sera in this assay for the presence of neutralising antibodies. Neutralising antibodies found through this model could be epitope mapped, and incorporated into HCV vaccine design strategies. To study the pathology of GBV-B infection, GBV-B-specific antibodies were also produced using two techniques in parallel- classical hybridoma technology and ribosome display. Antibodies targeting the nucleocapsid core protein of GBV-B have been previously detected in tamarins and served as the target for production of GBV-B antibodies using both aforementioned technologies. GBV-B core-specific antibodies were successfully isolated using both technologies and can now be used in downstream techniques, such as immunohistochemistry, to characterise the pathology of GBV-B infection thereby further validating the use of the animal model.

Sofosbuvir is a uridine based nucleotide inhibitor of the hepatitis C viral (HCV) polymerase that is the backbone of many treatment regimens. In combination with drugs targeting other viral enzymes (including the poorly potent guanosine analogue ribavirin or highly potent inhibitors of viral NS5A or protease) most patients clear virus and resistance to sofosbuvir is rare, allowing effective retreatment with sofosbuvir. Patients with Genotype 3 HCV respond less well than other genotypes and response is reduced in those previously exposed to interferon. Here we show that patientderived virus from patients with Genotype 3 HCV who relapse to sofosbuvir-based therapies have a reduced sensitivity to SOF in an in-vitro phenotyping assay. Analysis of viral sequencing data revealed two distinct polymorphisms (A150V and K206E) in the HCV polymerase that are associated with treatment failure and in-vitro; they reduce sofosbuvir sensitivity against genotype 3 hepatitis C virions. However both polymorphisms modify the cellular response to type I interferon and in cells lacking response to interferon the impact on sofosbuvir sensitivity is minimal. The A150V polymorphism reduces the response to interferon 70 fold whereas the K206E substitution has minimal effects on interferon in isolation but in combination with A150V reduces the response 100 fold. Preliminary data indicates that the A150V polymorphism interferes with the late response to type I interferons enabling the virus to overcome the induction of interferon-stimulated genes. These data indicate a complex interaction between direct acting antiviral drugs and the innate antiviral response.

Chronic Hepatitis B (CHB) related cirrhosis and hepatocellular carcinoma (HCC) account for more than 750,000 deaths per year. Current therapies for CHB are limited in achieving HBsAg decline/loss and thus there remains a pressing need for curative treatment strategies. Although, Pegylated Interferon-α (Peg-IFNα) may be used, the majority of patients progress to nucleos(t)ide analogue (NUC) therapy due to treatment failure. Peg-IFNα and NUCs used in isolation act differentially on the immune response; Peg-IFNα induces NK cell activation and NUC therapy may partially restore T cell function. NK cells are important antiviral effectors, highly enriched in the liver, with the potential to regulate immunopathogenesis in persistent viral infections. Here we examined the NK cell pool in HBeAg-positive CHB patients treated with Peg-IFNα and whether changes in the NK cell repertoire are induced when patients are 'primed' with Peg-IFNα and importantly, whether these changes are sustained or further modulated long-term after switching to sequential NUC therapy. The cumulative expansion of CD56bright NK cells driven by 48-weeks of Peg-IFNα was maintained at higher than baseline levels throughout the subsequent 9 months of sequential NUCs. Peg- IFNα-expanded NK cells showed further augmentation in their expression of the activating NK cell receptors during sequential NUCs. The expansion in proliferating, functional NK cells and HBsAg reduction was greater and more pronounced following sequential NUCs than in patients treated with de novo NUCs. This highlights the potential benefit of Peg-IFNα- priming, providing mechanistic insights for the further optimisation of treatment strategies to achieve sustained responses. Sustained boosting of NK cells on sequential NUCs following Peg-IFNα-priming has not previously been described raising the potential of 'long-lived' NK cell populations in keeping with their emerging adaptive features. These findings provide a mechanistic and immunological rationale to explore combination/sequential treatment strategies for CHB, including on-treatment immune responses in the liver, whilst awaiting the emergence of new therapies in the field.

Introdução: A Organização Mundial da Saúde estima que cerca de 71 milhões de pessoas estejam vivendo infectadas pelo vírus da hepatite C (HCV), o que corresponde a 1% da população mundial. A infecção crônica pelo HCV é uma das principais causas de cirrose hepática e carcinoma hepatocelular. O tratamento farmacológico visa a erradicação do vírus e melhora da atividade inflamatória. Em 2015, três fármacos de ação direta (DAA), simeprevir (SMV), sofosbuvir (SOF) e daclatasvir (DCV), foram incluídos no protocolo brasileiro de tratamento da hepatite C crônica. Estudos demonstram que o uso destes medicamentos está associado a elevadas taxas de resposta ao tratamento e a menor incidência de reações adversas em relação às terapias baseadas no uso do interferon e de inibidores de protease de primeira geração. Outros trabalhos indicam que pode haver associação entre a presença de determinadas mutações de resistência viral com a resposta ao tratamento farmacológico. Objetivos: Verificar a efetividade e a segurança dos esquemas de tratamento da hepatite C crônica que envolvem os DAA e analisar se a presença das mutações de resistência impacta negativamente na resposta ao tratamento. Casuística e Métodos: Trata-se de um estudo quase experimental de braço único realizado no Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto. Foram incluídos indivíduos maiores de 18 anos, infectados pelo genótipo 1 do HCV, que iniciaram tratamento com esquema que continha SOF em associação com DCV ou SMV. Foram coletados dados sociodemográficos, antropométricos, clínicos e realizou-se levantamento da ocorrência de reações adversas durante o tratamento, por meio do sistema informatizado do hospital. A coleta de sangue, para verificação da presença de mutações de resistência viral, foi realizada em até 15 dias antes do início do tratamento. O principal desfecho considerado foi a RVS12, definida como a ausência de carga viral detectável na 12ª semana após o fim do tratamento. Resultados: 262 indivíduos foram incluídos no estudo, dos quais 58,0% eram do sexo masculino e 79,4% foram classificados como brancos. A média de idade calculada para a amostra foi 55 anos, com desvio padrão de 10 anos. Quanto aos esquemas de tratamento propostos, 49,6% dos pacientes receberam tratamento com SOF e DCV e 50,4% fizeram uso de SOF e SMV. A taxa global de RVS12 foi de 92,7% (93,9% para SOF + DCV e 91,7% para SOF + SMV). Não foram identificados fatores associados a RVS, segundo a análise estatística. As reações adversas mais comuns foram anemia, náuseas, cefaleia e fadiga. Não foi encontrada evidência de associação entre a presença de mutações associadas a resistência ao tratamento e a falha no tratamento. Conclusão: O uso de esquemas de tratamento compostos por SOF e DCV ou SOF e SMV apresentou alta taxa de RVS e bom perfil de tolerabilidade em pacientes com genótipo 1 do HCV. / Background: The World Health Organization estimates that about 71 million people are living with hepatitis C virus (HCV), which accounts for 1% of the world population. Chronic HCV infection is one of the major causes of liver cirrhosis and hepatocellular carcinoma. The pharmacological treatment aims to eradicate the virus and improve inflammatory activity. In 2015, three direct-acting antivirals (DAA), simeprevir (SMV), sofosbuvir (SOF) and daclatasvir (DCV) were included in the brazilian protocol for the treatment of chronic hepatitis C. Studies have shown that the use of these drugs is associated with higher rates of response to treatment and to lower incidence of adverse reactions, compared to therapies based on the use of interferon and first-generation protease inhibitors. Other studies indicate that there may be an association between the presence of certain viral resistance mutations and the response to pharmacological treatment. Aims: To verify the effectiveness and safety of chronic hepatitis C treatment regimens involving DAA and to analyze whether the presence of resistance mutations negatively affects SVR rates. Methods: It is an almost experimental single-arm study performed at the Clinics Hospital of the Ribeirão Preto Medical School. We included individuals older than 18 years old, infected with HCV genotype 1, who started treatment with a regimen containing SOF in combination with DCV or SMV. Sociodemographic, anthropometric and clinical data were collected and the occurrence of adverse reactions during the treatment was investigated through the computerized system of the hospital. Blood collection for search for viral resistance mutations was performed within 15 days prior to initiation of treatment. The main outcome was SVR12, defined as the absence of detectable viral load at the 12th week after the end of treatment. Results: 262 subjects were included in the study, of which 58.0% were male and 79.4% were white. The mean age calculated for the sample was 55 years, with standard deviation of 10 years. Regarding the treatment regimens, 49.6% of the patients received SOF and DCV and 50.4% used SOF and SMV. The SVR12 rate was 92.7% (93.9% for SOF + DCV and 91.7% for SOF + SMV). No factors associated with SVR were identified according to the statistical analysis. The most common adverse reactions were anemia, nausea, headache and fatigue. No evidence of association was found between the presence of mutations associated with treatment resistance and treatment failure. Conclusion: The use of treatment regimens composed of SOF and DCV or SOF and SMV showed a high rate of SVR and a good tolerability profile in patients with HCV genotype 1.