Medicine (St Vincent's) - Theses
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Item“A tale of two cities”: Studies on host control of chronic hepatitis B infection & Comparative evaluation of methods for quantifying cytokine production( 2016)Globally, an estimated 240 million people are living with chronic hepatitis B (CHB) [1], and the number is approximately 218000 in Australia [2]. Inactive hepatitis B e antigen (HBeAg)-negative patients are the most prevalent group, who are defined by with sustained virological response (SVR). This state is associated with favorable prognosis and low risk of liver complications. Through monitoring the serological markers and immune markers by flow cytometry, as well as advanced techniques such as transcriptome mapping, progress has been made on unveiling the mechanism of host immunological control of the CHB infection. Furthermore, it is of great clinical significance to accurately identify relapse of patients (Chapter 1). Few studies have focused on effectively identifying the treatment-naïve inactive carriers (IC), and we are particularly interested in predicting sustained inactivation through monitoring the serum HBV DNA level and HBsAg titer. Based on a retrospective/longitudinal study, we report that HBV DNA is a better predictor over HBsAg, and it is not beneficial to add HBsAg into the prediction model. The cutoff value of HBV DNA>2.5log IU/mL is effective for identifying IC with the NPV 93.6% for 12 months and 92.3% for 24 months. In another words, IC with baseline HBV DNA load<2.5 log IU/mL is likely to maintain the sustained inactivation for another 1 and 2 years, with a probability over 90% (Chapter 2). We also developed a protocol for effectively selecting peripheral blood mononuclear cells (PBMCs) subpopulations- including monocytes, Natural Killer (NK) cells, and rest of the PBMCs from inactive HBeAg negative patients undergoing clinical relapse after NA therapy cessation. Corresponding selection kits (from Miltenyi Biotec or Stemcell Technologies) were compared, and sample quality and the purification of the sample was analysed. Extracted mRNA samples will undergo expression profiling in the future, expected to pinpoint the biomarkers and unveil the mechanism of the carrier relapse (Chapter 3). For host immunity studies, accurate assessment of cytokine profile is an important perspective. We performed the comparative evaluation of two most commonly used methods for quantifying cytokines, flow cytometry and ELISA, based on the model of TLR stimulated human monocytes. Fundamentally different biological patterns were ascertained regarding cytokines from the two assays, and were mainly evidenced by the more comprehensive information that was available by flow cytometry (Chapter 4). For applying this optimized methodology, which is presenting results from flow cytometry and ELISA in parallel, to CHB studies, we adopted a human hepatocyte cell line, HepG2 (Chapter 5).