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This dissertation, Mechanism of Bacillus Calmette Guerin-induced Immune Response by Ka-wa, Benny, Cheung, 張嘉華, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled Mechanism of Bacillus Calmette Guerin-induced Immune Response submitted by Cheung Ka Wa, Benny for the degree of Master of Philosophy at The University of Hong Kong in August 2003 Mycobacterium tuberculosis (MTb) is a major cause of death in the world and approximately one-third of the world's population is infected with this bacterium. Following infection, different branches of the immune system are mobilized to mount defense against the pathogen. Of the immune responses mounted by the host, induction of cytokines including tumor necrosis factor (TNF) and apoptosis in macrophages are the two major defense mechanisms to limit pathogen invasion. Cytokines are pleiotropic proteins released in response to pathogens. Apoptosis is a process in which a cell dies in a programmed manner, instead of uncontrolled cell lysis or necrosis. Cytokines interact with each other to form a complex network of pathways. For example, interferon (IFN) and TNF were shown to interact through common pathways including the induction of protein kinase PKR. PKR is an IFN-inducible, dsRNA-activated protein kinase. As a signal transducer, it regulates the induction of cytokine gene expression including IFN-α and IFN-β in response to virus infection through nuclear factor kappa B (NF-κB). It also mediates TNF- and virus-induced cellular cytotoxicity via activation of p53 and caspases. I hypothesize that PKR is a pattern recognition molecule and is involved in pathogen-induced immune responses against viruses and bacteria. In light of the critical role of TNF in immunity and its cytotoxic effects being mediated by PKR, the role of PKR in the regulation of immune response against MTb is examined using Bacillus Calmette Guerin (BCG) as a mycobacterial model in vitro. The signaling mechanism of BCG-induced cytokine gene expression was first investigated. Primary human blood monocytes were treated with BCG and were assayed for cytokine expression by RT-PCR. Western analysis was used to examine for PKR and mitogen-activated protein kinase (MAPK) phosphorylation, whereas electrophoretic mobility shift assay was used to study nuclear translocation of NF-κB. Results showed that BCG stimulates the induction of cytokine gene expression in human blood monocytes. This cytokine induction process is regulated by PKR through its activation and phosphorylation following exposure to BCG. Downstream of PKR phosphorylation is the activation of MAPK and translocation of NF-κB into the nucleus. NF-κB in turn mediates the transcription of specific cytokine genes. In light of the apoptogenic role of PKR in cell death, I investigated whether cell death process induced by BCG is dependent on PKR. Promonocytic U937 cells were treated with phorbol esters for cellular differentiation. This was followed by co-culture with BCG. Cells were assayed for cell survival, immunostaining for apoptotic markers, and RT-PCR for apoptotic gene expression. Results demonstrated that BCG induces cell death in U937 cells through apoptosis and necrosis. The cell death process is regulated by PKR following its activation by BCG. The activated PKR in turn mediates the activation of caspases and down-regulation of Bcl-2. The recruitment and activation of these cell death-related molecules finally leads to death of BCG-infected cells. Taken together, PKR pla

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