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

人類免疫缺陷病毒 (HIV) 是人獲得性免疫缺陷綜合征 (愛滋病，AIDS) 的致病原，2010年全球有180萬人死於愛滋病，HIV/AIDS已成為全球健康的嚴重挑戰。人類免疫缺陷病毒與乙型肝炎病毒 (HBV) ，丙型肝炎病毒 (HCV) 的合併感染非常普遍，已演變成具有嚴重臨床後果的新健康問題。儘管對於人類免疫缺陷病毒的研究已有很大的進展，但由於受研究模型的限制，人體免疫系統對人類免疫缺陷病毒感染的應答，特別是對乙型肝炎病毒，丙型肝炎病毒與人類免疫缺陷病毒合併感染的免疫應答，仍值得進一步的闡明。 / 在本研究中，我們首先對深圳人類免疫缺陷病毒，乙型肝炎病毒，丙型肝炎病毒合併感染的流行情況進行研究。共選取914份人類免疫缺陷病毒感染者的血漿，經過對乙型肝炎病毒表面抗原 (HBsAg) 和抗丙型肝炎病毒抗體 (anti-HCV) 的檢測，發現10.9% (100/914) 的被檢測者是人類免疫缺陷病毒/乙型肝炎病毒合併感染，14.6% (133/914) 為人類免疫缺陷病毒/丙型肝炎病毒合併感染，3.7% (34/914) 為人類免疫缺陷病毒/乙型肝炎病毒/丙型肝炎病毒三重感染。多元邏輯回歸分析證明人類免疫缺陷病毒傳染的危險行為與合併感染顯著相關聯。大多數的人類免疫缺陷病毒/乙型肝炎病毒合併感染者都是通過性接觸感染人類免疫缺陷病毒，包括異性傳播與同性傳播 (95/100, 95%); 大多數的人類免疫缺陷病毒/丙型肝炎病毒合併感染者是靜脈注射吸毒者 (89/133, 66.9%); 人類免疫缺陷病毒/乙型肝炎病毒/丙型肝炎病毒三重感染者中，大多數是靜脈注射吸毒者 (28/34, 82.4%)。靜脈注射吸毒人群中，大部分是男性 (108/122, 88.5%)，約半數人的年齡介乎27至32歲 (56/122, 45.9%) 。有接近一半的經過血液和血液製品傳播人類免疫缺陷病毒的人是人類免疫缺陷病毒/丙型肝炎病毒合併感染者 (10/23, 43.5%) 。性別與人類免疫缺陷病毒感染的危險行為有顯著關係，大部份的靜脈注射吸毒者是男性。 / 進一步，我們利用酶聯免疫吸附測定法 (ELISA) 檢測深圳愛滋病陽性樣本血漿中白細胞介素27 (IL-27) 的濃度。結果顯示，對比健康參照者，人類免疫缺陷病毒單獨感染者，人類免疫缺陷病毒/乙型肝炎病毒合併感染者，人類免疫缺陷病毒/丙型肝炎病毒合併感染者的血漿IL-27濃度顯著升高。隨後我們進一步發現，人類免疫缺陷病毒單獨感染組，人類免疫缺陷病毒/乙型肝炎病毒，人類免疫缺陷病毒/乙型肝炎病毒/丙型肝炎病毒合併感染組之間的血漿IL-27濃度沒有顯著差異，而人類免疫缺陷病毒/丙型肝炎病毒合併感染組與人類免疫缺陷病毒/乙型肝炎病毒/丙型肝炎病毒三重感染組的血漿IL-27濃度差異顯著。我們還發現人類免疫缺陷病毒單獨感染組中，血漿IL-27濃度與CD4⁺ T 淋巴細胞數量顯著正相關 (r = 0.177, P = 0.034)。 / 我們進一步分析了人類免疫缺陷病毒和丙型肝炎病毒的病毒載量對血漿IL-27濃度的影響，發現HIV單獨感染組中人類免疫缺陷病毒載量與血漿IL-27濃度沒有顯著相關 (r = - 0.063, P = 0.679)，而人類免疫缺陷病毒/丙型肝炎病毒合併感染組中，人類免疫缺陷病毒載量與血漿IL-27濃度顯著正相關 (r = 0.362, P = 0.049)。人類免疫缺陷病毒/丙型肝炎病毒合併感染組中，人類免疫缺陷病毒載量與丙型肝炎病毒載量缺少顯著線性關聯 (r = - 0.072, P = 0.704)，而人類免疫缺陷病毒/丙型肝炎病毒合併感染組可根據人類免疫缺陷病毒與丙型肝炎病毒的病毒載量再細分成血漿IL-27濃度差異顯著的三組 (P = 0.014) ， 丙型肝炎病毒載量與血漿IL-27濃度缺少顯著關聯 (r = - 0.119, P = 0.530) 。 / 我們利用TaqMan®等位基因分型技術測定深圳男同性戀人群中IL-27 p28基因的單核苷酸多態性 (SNP)。結果顯示，人類免疫缺陷病毒感染組IL-27 p28 -964A/G 和4603G/A的基因型與健康男同性戀參照組的基因型沒有顯著差異， IL-27 p28 -964A/G 和4603G/A的等位基因比率也沒有顯著差異。結果也顯示，IL-27 p28 2905T/G的TG基因型可減少2.77倍的人類免疫缺陷病毒感染風險，等位基因G可減少2.72倍的人類免疫缺陷病毒感染風險。連鎖不平衡在IL-27 p28 -964A/G 和2905T/G 中存在 ( / 綜上所述， 在本研究中，我們首次調查了深圳人類免疫缺陷病毒，乙型肝炎病毒，丙型肝炎病毒合併感染的流行情況，並分析了合併感染的風險因素。 發現人類免疫缺陷病毒單獨感染者，人類免疫缺陷病毒/乙型肝炎病毒合併感染者， 及人類免疫缺陷病毒/丙型肝炎病毒合併感染者的血漿IL-27濃度比健康參照組顯著地升高；人類免疫缺陷病毒單獨感染組中，血漿IL-27濃度與CD4⁺ T淋巴細胞數量顯著正相關。人類免疫缺陷病毒/丙型肝炎病毒合併感染組中，人類免疫缺陷病毒載量與血漿IL-27濃度顯著正相關 (r = 0.362, P = 0.049)。分析深圳男同性戀人群IL-27 p28基因的單核苷酸多態性，發現IL-27 p28 2905T/G 與人類免疫缺陷病毒感染相關，GGG單型可降低男同性戀人群人類免疫缺陷病毒感染的風險。 / Human Immunodeficiency Virus (HIV) is the causative agent of Acquired Immunodeficiency Syndrome (AIDS); HIV/AIDS caused 1.8 million deaths world-widely in 2010 and became a major global health challenge. HIV co-infections with Hepatitis B virus (HBV), Hepatitis C virus (HCV) are common and have emerged into new health problems with severe clinical consequences. Since the discovery of HIV, massive progress in understanding of the pathogen has been achieved. Due to the restriction of research model, how human immune system responds to HIV infection, particularly, to HBV or HCV co-infections is still worthy further elucidation. / A cohort study was first conducted in Shenzhen regarding the seroprevalence of HBV, HCV infections among HIV-infected population. Totally 914 HIV positive individuals were recruited in the study and tested for HBsAg and anti-HCV antibodies. The results showed a 10.9% (100/914) HIV/HBV co-infection rate, 14.6% (133/914) HIV/HCV co-infection prevalence and 3.7% (34/914) HIV/HBV/HCV triple-infection prevalence. Multivariate logistic regression revealed that HIV transmission risk behavior was significantly associated with HIV, HBV, HCV co-infections. Most HIV/HBV co-infection cases got HIV through sexual contact including heterosexual and homosexual behaviors (95/100, 95%); while most HIV/HCV co-infection subjects were injection drug users (IDUs) (89/133, 66.9%). In the case of HIV/HBV/HCV triple-infection, IDUs accounted for a large ratio (28/34, 82.4%). Among IDUs, most of them were male (108/122, 88.5%) and nearly half were aged 27 to 32 years old (56/122, 45.9%). Near half people who got HIV through blood and blood products were HIV/HCV co-infected (10/23, 43.5%). Gender has a significant correlation with HIV risk behavior and most IDUs were male. / Next, we applied ELISA to test HIV positive clinical samples and proved that plasma interleukin-27 (IL-27) level was significantly elevated in HIV mono-infected, HIV/HBV co-infected and HIV/HCV co-infected subjects when compared with healthy controls. Later, we further revealed that plasma IL-27 titer was not significantly varied among HIV, HBV and HCV co-infections except between HIV/HCV co-infections and HIV/HBV/HCV triple-infections. We also observed a significant positive correlation between CD4⁺ T cell counts and plasma IL-27 titer within HIV mono-infected group (r = 0.177, P = 0.034). / We further analyzed the impact HIV and HCV viral loads on plasma IL-27 titer. We found there was no significant correlation between HIV viral load and IL-27 titer among HIV mono-infected individuals (r = - 0.063, P = 0.679); while a significant positive correlation was observed between HIV viral load and IL-27 titer in HIV/HCV co-infected individuals (r = 0.362, P = 0.049). In the case of HIV/HCV co-infection, there was no significant linear correlation between HIV and HCV viral loads (r = - 0.072, P = 0.704) but exist obvious subdivision of samples in terms of HIV and HCV viral loads with significant IL-27 titer variance (P = 0.014). No correlation was observed between HCV viral load and IL-27 titer (r = - 0.119, P = 0.530). / IL-27 p28 polymorphisms were genotyped with TaqMan® Allelic Discrimination Assay in Chinese men who have sex with men (MSM) population in Shenzhen and the results revealed that proportions of IL-27 p28 -964A/G and 4603G/A genotypes were not significantly different from the healthy controls; IL-27 p28 -964A/G and 4603G/A allele frequencies were similar between HIV positive MSM group and healthy control MSM group. Results also showed that for IL-27 p28 2905T/G polymorphism, TG genotype has a 2.77-fold decreased risk of HIV susceptibility and subjects with G allele has a 2.72-fold decreased risk of HIV susceptibility. Linkage disequilibrium (LD) coefficients were observed between IL-27 p28 -964A/G and 2905T/G ( / In conclusion, the seroprevalences of HBV and HCV infection among HIV positive population in Shenzhen were surveyed and risk factors associated with co-infections were analyzed. Plasma IL-27 titer was significantly elevated in HIV mono-infected, HIV/HBV co-infected and HIV/HCV co-infected individuals. IL-27 level was correlated with CD4⁺ T cell counts within HIV mono-infected people. A significant positive correlation was found between HIV viral load and IL-27 titer in HIV/HCV co-infected individuals (r = 0.362, P = 0.049). IL-27 p28 2905T/G was associated with individual susceptibility to HIV infection and haplotype GGG showed a protective role in restricting HIV infection in MSM population. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / He, Lai. / "October 2012." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 135-155). / 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 (English) --- p.iii / Abstract (Chinese) --- p.vi / Acknowledgements --- p.ix / Contents --- p.x / List of Tables --- p.xv / List of Figures --- p.xvi / Abbreviations --- p.xvii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Human Immunodeficiency Virus --- p.1 / Chapter 1.1.1 --- HIV virology --- p.1 / Chapter 1.1.1.1 --- HIV structure and genome organization --- p.1 / Chapter 1.1.1.2 --- HIV life cycle --- p.3 / Chapter 1.1.1.3 --- HIV genotypes --- p.5 / Chapter 1.1.2 --- HIV epidemiology --- p.6 / Chapter 1.1.2.1 --- Global HIV epidemiology --- p.6 / Chapter 1.1.2.2 --- HIV epidemiology in China --- p.9 / Chapter 1.1.3 --- HIV pathogenesis --- p.13 / Chapter 1.1.3.1 --- Natural history of HIV infection --- p.13 / Chapter 1.1.3.2 --- HIV transmission --- p.15 / Chapter 1.1.3.3 --- HIV tropism --- p.17 / Chapter 1.1.4 --- Immune responses to HIV infection --- p.19 / Chapter 1.1.4.1 --- Innate immune response --- p.19 / Chapter 1.1.4.2 --- Adaptive immune response --- p.21 / Chapter 1.1.5 --- Diagnosis --- p.24 / Chapter 1.1.6 --- HIV prevention --- p.25 / Chapter 1.1.7 --- Anti-HIV therapy --- p.25 / Chapter 1.1.8 --- Hepatitis B virus, Hepatitis C virus infection --- p.26 / Chapter 1.1.8.1 --- HBV infection natural history, diagnosis, disease progression and epidemiology --- p.26 / Chapter 1.1.8.2 --- HCV infection natural history, diagnosis, disease progression and epidemiology --- p.30 / Chapter 1.1.9 --- HIV, HBV, HCV co-infections --- p.32 / Chapter 1.2 --- Interleukin-27 --- p.36 / Chapter 1.2.1 --- Biology of IL-27 --- p.36 / Chapter 1.2.2 --- IL-27 on immune system --- p.37 / Chapter 1.2.3 --- IL-27 anti-tumor properties --- p.38 / Chapter 1.2.4 --- IL-27 antiviral features --- p.40 / Chapter 1.2.5 --- IL-27 with hepatitis --- p.41 / Chapter 1.3 --- Single-nucleotide polymorphisms (SNPs) --- p.42 / Chapter 1.3.1 --- Types of SNPs --- p.43 / Chapter 1.3.2 --- Functions of SNPs --- p.43 / Chapter 1.4 --- Objectives of the study --- p.45 / Chapter Chapter 2 --- Seroprevalence of HBV, HCV infection among HIV positive individuals in Shenzhen --- p.52 / Chapter 2.1 --- Introduction --- p.52 / Chapter 2.2 --- Materials and methods --- p.54 / Chapter 2.2.1 --- Study participants --- p.54 / Chapter 2.2.2 --- Measure of HBV, HCV seroprevalence --- p.55 / Chapter 2.2.3 --- Statistical analysis --- p.60 / Chapter 2.3 --- Results --- p.61 / Chapter 2.3.1 --- HIV infection in Shenzhen --- p.61 / Chapter 2.3.2 --- Seroprevalence of HBV, HCV infection among HIV positive individuals in Shenzhen --- p.61 / Chapter 2.4 --- Discussion --- p.65 / Chapter 2.4.1 --- HIV infection in Shenzhen --- p.65 / Chapter 2.4.2 --- HIV, HBV, HCV co-infections in Shenzhen --- p.68 / Chapter 2.4.3 --- Limitations of the study --- p.71 / Chapter Chapter 3 --- Upregulation of Interleukin-27 titer in HIV infected persons --- p.78 / Chapter 3.1 --- Introduction --- p.78 / Chapter 3.2 --- Materials and methods --- p.80 / Chapter 3.2.1 --- Study participants --- p.80 / Chapter 3.2.2 --- Measure of HIV, HBV, HCV infection --- p.80 / Chapter 3.2.3 --- Detection of IL-27 in plasma --- p.81 / Chapter 3.2.4 --- CD4 counting --- p.84 / Chapter 3.2.5 --- Statistical analysis --- p.84 / Chapter 3.3 --- Results --- p.84 / Chapter 3.3.1 --- Demographics of study participants --- p.84 / Chapter 3.3.2 --- Upregulation of IL-27 levels in HIV infected persons --- p.85 / Chapter 3.3.3 --- Correlation of plasma IL-27 titer with CD4⁺ T cell count --- p.86 / Chapter 3.4 --- Discussion --- p.86 / Chapter Chapter 4 --- Impact of HIV, HCV viral loads on Interleukin-27 titer among Antiretroviral Therapy- Naïve HIV positive Chinese --- p.95 / Chapter 4.1 --- Introduction --- p.95 / Chapter 4.2 --- Materials and methods --- p.96 / Chapter 4.2.1 --- Study participants --- p.97 / Chapter 4.2.2 --- HIV, HBV and HCV Serological assays --- p.97 / Chapter 4.2.3 --- CD4 counting --- p.97 / Chapter 4.2.4 --- Detection of plasma IL-27 --- p.98 / Chapter 4.2.5 --- Quantification of HIV, HCV viral loads --- p.98 / Chapter 4.2.6 --- Statistical analysis --- p.102 / Chapter 4.3 --- Results --- p.102 / Chapter 4.3.1 --- Demographics of study participants --- p.102 / Chapter 4.3.2 --- Plasma IL-27 was elevated in HIV-positive persons --- p.103 / Chapter 4.3.3 --- Correlation of IL-27 titer and CD4⁺ T cell count --- p.103 / Chapter 4.3.4 --- Correlation of HIV viral load and IL-27 titer --- p.104 / Chapter 4.3.5 --- Correlation of HCV viral load and IL-27 titer --- p.104 / Chapter 4.4 --- Discussion --- p.105 / Chapter Chapter 5 --- Association of Interleukin-27 polymorphisms with the susceptibility to HIV infection in a Chinese men who have sex with men population --- p.116 / Chapter 5.1 --- Introduction --- p.116 / Chapter 5.2 --- Materials and methods --- p.118 / Chapter 5.2.1 --- Study participants --- p.118 / Chapter 5.2.2 --- HIV screening --- p.118 / Chapter 5.2.3 --- Genomic DNA extraction --- p.119 / Chapter 5.2.4 --- IL-27 p28 -964A/G, 2905T/G and 4603G/A genotyping --- p.120 / Chapter 5.2.5 --- Statistical analysis --- p.121 / Chapter 5.3 --- Results --- p.122 / Chapter 5.3.1 --- Demographics of study participants --- p.122 / Chapter 5.3.2 --- IL-27 genotypes and allele frequencies in HIV MSM and healthy MSM controls --- p.122 / Chapter 5.3.3 --- LD analysis and haplotype analysis --- p.123 / Chapter 5.4 --- Discussion --- p.124 / Chapter Chapter 6 --- Summary and perspectives --- p.130 / Chapter 6.1 --- Summary --- p.130 / Chapter 6.2 --- Perspectives --- p.132 / Bibliography --- p.135

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.

Um estudo de caso-controle para investigar a associação entre a hanseníase e infecção pelo vírus da hepatite B (VHB) foi conduzido no período de 1992/93, na cidade de Goiânia e municípios contíguos - Estado de Goiás. Avaliou-se, também, a distribuição espacial da hanseníase neste aglomerado urbano. Inicialmente, os indivíduos com suspeita clínica de hanseníase foram submetidos a exames baciloscópicos e histopatológicos, independentemente da rotina do Programa de Controle de Hanseníase. Do total de 855 pacientes recémdiagnosticados de hanseníase, 600 eram residentes em área urbana, e foram categorizados em casos multibacilares (31,3 por cento ), paucibacilares (51,8 por cento ) e prováveis (16,8 por cento ). Foi realizada análise descritiva desta casuística, havendo nítida predominância do sexo masculino na forma multibacilar de hanseníase. A distribuição espacial dos pacientes possibilitou, através da análise exploratória das taxas de detecção, discriminar estratos de risco intra-urbano. Para o estudo de caso-controle, 552 pacientes de hanseníase de 1 O a 70 anos foram incluídos. Os controles (N =552) foram selecionados de indivíduos com ausência de sinais e sintomas sugestivos de hanseníase oriundos da demanda espontânea de ambulatórios de 7 unidades de saúde, localizadas na região de procedência dos casos. Os participantes - casos e controles - foram entrevistados para avaliar fatores de risco para hanseníase e infecção pelo vírus da hepatite B. Foram coletadas amostras de sangue para detecção de marcadores ao vírus da hepatite B pela técnica de ELISA. Comparou-se a prevalência de marcadores de exposição (anti-HBc), de imunidade (anti-HBs) e de portador (AgHBs) entre casos e controles. Foram avaliados como potenciais fatores de confusão: sexo, idade, condições sócio-econômicas, estado nutricional, cicatriz vacinal de BCG e utilização dos serviços de saúde. Casos e controles foram similares quanto às características sócio-econômicas e nutricionais indicando que o princípio de selecionar controles da mesma base populacional que os casos parece ter sido adequado. Cicatriz vacinal de BCG esteve estatisticamente associada aos diferentes tipos de hanseníase. Houve maior proporção de indivíduos hospitalizados nos útimos 5 anos entre casos que em controles indicando que o emparelhamento por local de residência não eliminou completamente as diferenças entre os grupos em relação ao uso dos serviços de saúde. Entre os participantes do estudo, 18,1 por cento dos casos e 19,6 por cento dos controles foram soropositivos ao anti-HBc. Em análise multivariada, utilizando-se o modelo de regressão logística politômica, a associação da hanseníase e anti-HBc entre casos e controles apresentou odds ratio de 0,9 (IC95 por cento O, 7-1 ,3) para a categoria de multibacilar; 1,0 (IC 95 por cento 0,7-1,3) para a de paucibacilar e 1,1 (IC95 por cento 0,8-1,5) para a de provável. Estes resultados mostraram que subgrupos de casos e os controles estiveram igualmente expostos ao vírus da hepatite B. As proporções de indivíduos imunes foram semelhantes nos grupos de casos (9,2 por cento ) e controles (10,2 por cento ). Casos multibacilares responderam à exposição viral com formação de anticorpos protetores, qualitativa e quantitativamente de maneira semelhante aos pacientes paucibacilares e grupo controle. Os resultados dos índices de persistência de infecção (PPI) indicaram não haver diferença quanto ao clearance do antígeno viral nos subgrupos de casos e controles. Os resultados obtidos nesta investigação mostraram nos subgrupos de casos e controles: (i) prevalências semelhantes dos marcadores de exposição, de imunidade e de estado de portador; (ii) capacidade similar para produção de anticorpos protetores, avaliada através dos percentuais do marcador anti-HBs e, quantitativamente, através do Índice de Elisa e (iii) baixa probabilidade de persistência da antigenemia mensurada pelo PPI. Em conclusão, não houve evidências epidemiológicas de uma associação entre hanseníase e infecção pelo vírus da hepatite B, avaliada através de estudo de caso-controle, conduzido em área de baixa endemicidade ao VHB e alta endemicidade de hanseníase. / A case-control study was conducted in Goiânia, Central Brazil, a highly endemic area for leprosy and Iow endemic region for hepatitis B virus (HBV) infection. The purpose was to investigate the association between leprosy types and hepatitis B infection. The spatial distribution of leprosy in urban area was assessed. Between 1992 and 1993, newly detected leprosy cases (N=855) were investigated and 600 cases lived in the urban area. They were classified in multibacillary (31.3 per cent ), paucibacillary (51.8 per cent ) and probable cases (16.8 per cent ) according to histopathological and baciloscopic exams, independently of the leprosy control routine. The majority of multibacillary cases was males. Detection rates of leprosy were calculated by mapping cases and several risk strata were identified by using exploratory data analysis. This methodology seems to be particularly useful for targeting control activities in urban areas. Cases were 552 leprosy patients from the urban area and adjacent counties, between the ages of 1O and 70 years who self-referred or were referred to the main outpatient clinic for treatment in the region. 552 controls were selected from among self-referred outpatients from 7 health centers geographically located in areas where the cases came from. The main criteria for eligibility for control subjects was that they must not have any signs or symptoms indicative of leprosy. Blood samples were collected for all participants to determine serological markers of HBV infection and tested by enzyme immunoabsorbent assay technique (ELISA). Cases and controls were interviewed in order to evaluate risk factors for leprosy and hepatitis B vírus (HBV) infection. Prevalence of HBV exposure (anti-HBc), immunity (anti-HBs) and carrier status (AgHBs) were compared among cases and controls. Cases and controls were also compared for age, sex, socio-economic conditions, nutritional status, BCG scars and previous hospitalization. The participants had similar socio-economic pattern and also nutrition status, suggesting that the source of control selection was adequate for controlling for the most common confounding variables. BCG vaccine appeared to provide protection against multibacillary and paucibacillary types of leprosy and percentage of hospitalization was higher among cases. Prevalence of anti-HBc was similar among leprosy cases (18.1 per cent ) compared to controls (19.6 per cent ). An analysis of association between anti-HBc infection and leprosy types in terms of odds ratio, calculated by polytomous logistic regression, showed no positive association: multibacillary (OR=0.9 CI95 per cent 0.7-1.3); paucibacillary (OR= 1.0 CI95 per cent 0.7-1.3) and probable (OR= 1.1 CI95 per cent 0.8-1.5). The main findings of the case-control study were: (i) cases and controls had similar leveis of viral exposure, immune and carrier status (íi) the persistence of antigen response (PPI) was low among cases and controls respectively; (iii) ELISA índices were similar among multibacillary, paucibacillary and control group indicating that all participants mount antibody response to viral infection. In conclusion, there was no association between multibacillary leprosy and HBV infection in this setting.

by Zhong Sheng. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (p. 186-203). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web.

by Winnie Yeo. / Thesis (M.D.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (p. 213-256). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web.

This thesis investigates the epidemiology of hepatitis B virus infection (HBV) and estimates HBV-related liver disease burden in Vietnam and Australia using a cross-sectional study design and mathematical modelling. A population-based seroprevalence survey was undertaken in rural Northern Vietnam. In a sample of 870 study participants, prevalence of anti-HBV core antibody (anti-HBc) and hepatitis B virus surface antigen (HBsAg) was 68.2% and 19.0%, respectively, and hepatitis B e antigen (HBeAg) was detected in 16.4% of the HBsAg-positive group. Factors associated with HBV infection (anti-HBc and/or HBsAg-positive) were age 60 years or older (adjusted odds ratio (AOR), 3.82; 95% CI, 1.35??10.80; P = 0.01), residence in Vu Thu district (AOR, 3.00; 95% CI, 2.16??4.17; P <0.001), hospital admission (AOR, 2.34; 95% CI, 1.33??4.13; P = 0.003) and history of acupuncture (AOR, 2.01; 95% CI, 1.29??3.13; P = 0.002). Household contact with a person with liver disease (AOR, 2.13; 95% CI, 1.29??3.52; P = 0.003), reuse of syringes (AOR, 1.81; 95% CI, 1.25??2.62; P = 0.002) and sharing of razors (AOR, 1.69; 95% CI, 1.03??2.79; P = 0.04) were independent predictors of HBsAg positivity. Alanine aminotransferase (ALT) level was elevated (>40 IU/L) in 43% of the HBsAg-positive group; the proportion of elevated ALT was higher in HBeAg-positive (65%) compared with HBeAg-negative (39%) (P = 0.02). Based on data from the seroprevalence study, other prevalence estimates and HBV natural history parameters, a mathematical model was used to estimate HBV-related liver disease burden in Vietnam. Estimated chronic HBV prevalence increased from 6.4 million cases in 1990 to around 8.4 million cases in 2005 and was projected to decrease to 8.0 million by 2025. Estimated HBV-related liver cirrhosis and hepatocellular carcinoma (HCC) incidence increased linearly from 21 900 and 9400 in 1990 to 58 650 and 25 000 in 2025. Estimated HBV-related mortality increased from 12 600 in 1990 to 40 000 in 2025. To estimate HBV-related HCC incidence among Australians born in the Asia-Pacific region (APR), a mathematical modelling was developed utilising HBV natural history parameters, HBV prevalence estimates in APR countries and immigration data. Chronic HBV cases among the APR-born population increased rapidly from the late 1970s, reaching a peak of 4182 in 1990. Chronic HBV prevalence increased to more than 53 000 in 2005. Estimates of HBV-related HCC increased linearly from one in 1960 to 140 in 2005, with a projected increase to 250 in 2025. Universal HBV vaccination programs in countries of origin had limited impact on projected HBV-related HCC to 2025. HBV-related HCC survival was analysed in a population-based linkage study in New South Wales (NSW), Australia. Between 1994 and 2002, 278 HCC cases notified to the NSW Cancer Registry were linked to chronic HBV infection notifications to the NSW Health Department. The majority of cases were male (83.5%) and overseas born (93.6%); Asian-born cases accounted for 72.1%. Median survival following HCC diagnosis was 15 months. HCC survival was poorer among older age groups (P <0.001), and among cases with regional spread (HR 3.23; 95% CI, 1.83??5.69; P <0.001) and distant metastases (HR 3.85; 95% CI, 2.44??6.08; P <0.001). Sex, region of birth, and study period (1994??1997 versus 1998??2002) were not associated with HCC survival. The results of these studies show that HBV infection remains a major public health challenge in highly endemic countries such as Vietnam. HBV-related liver disease burden in Vietnam was estimated to increase for at least two decades despite the introduction of a universal infant HBV-vaccination program. Similarly, HBV-related HCC among Australians born in the APR was estimated to continue to increase over the next two decades. Survival for HBV-related HCC even in settings such as Australia continues to be extremely poor. Strategies are required to expand HBV treatment to individuals with chronic HBV infection who are at greatest risk of progression to advanced liver disease.

This thesis examines the results of a hepatitis B vaccination questionnaire study that was completed by medical officers, nurses, carers of the developmentally disabled, and correctional officers in the Greater Western Sydney area of New South Wales, Australia. The main aim of the study was to contrast these four high risk occupational groups for their acceptance of hepatitis B vaccination, seroconversion status, and behavioural, attitudinal, motivational and institutional determinants of their vaccination status. The results of the thesis revealed that medical officers and nurses were the most likely to be tested and vaccinated for seroconversion, while DD carers and correctional officers had the largest number of Not Vaccinated respondents. The findings of the questionnaire are discussed in some detail. It is also suggested that groups differ in the degree of hepatitis risk anxiety they experience as well as the degree of control felt over their health status. A number of indications for personal, institutional and governmental interventions to increase vaccination levels are discussed. / Doctor of Philosophy (PhD)

Master of Dental Surgery / This work was digitised and made available on open access by the University of Sydney, Faculty of Dentistry and Sydney eScholarship . It may only be used for the purposes of research and study. Where possible, the Faculty will try to notify the author of this work. If you have any inquiries or issues regarding this work being made available please contact the Sydney eScholarship Repository Coordinator - ses@library.usyd.edu.au