Natural Killer (NK) cell, a crucial player of the human innate immune defense system, detects and kills virus-infected cells and cancer cells. Although the relevant molecular machineries involved in NK cell activation and NK-target cell interactions are largely known, how their collected dynamics regulate fast yet highly selective target cell killing in the complex environment of tissues is poorly understood. In traditional bulk killing assays, heterogeneity and kinetic details of individual NK-target cell interactions are masked, seriously limiting analysis of the underlying dynamic mechanisms. Therefore, the aim of my PhD study is to develop quantitative microscopy assays to elucidate, at the single cell level, real-time killing dynamics of epithelial cancer cells by primary NK cells purified from human blood. Results from my study not only identified the rate-limiting kinetics in NK-cancer cell interaction and mechanistically relevant heterogeneity in the process, but also characterized key molecular events and regulatory components of the NK cell machinery that were associated with the observed cytotoxic dynamics and heterogeneity. NK cells are considered promising candidate for cancer treatment, especially for eliminating residual cancer cells after conventional therapy. The fundamental knowledge acquired from my PhD study, in particular regarding how killing by primary NK cell varies between different target cancer cell types, provides new mechanistic insight that may help to develop this treatment strategy. And the quantitative microscopy assays that I developed are readily extendable to analysis of other cell-cell interaction dynamics, e.g., involved in cytotoxic T cell function.

Thesis (Ph. D.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 163-179).

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

No description available.

No description available.

Thesis (M.D.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 130-155).

Thesis (Ph. D.)--University of Queensland, 2002. / Includes bibliographical references.

Enterovirus 71 (EV71) belongs to the Enterovirus genus of the family Picornaviridae and is the major causative agent of hand, foot and mouth disease (HFMD). Although clinical manifestations of HFMD are usually mild and self-limiting, severe HFMD patients suffer from a diverse array of neurological diseases and sometimes these diseases are fatal. HFMD usually occurs in young children and gradually becomes a new threaten in Asia. Unfortunately, effective EV71 vaccine is not available to date and alternative treatments are still in debate. This is partially due to the lack of understanding of EV71 pathogenesis and host immune responses against EV71. Natural killer (NK) cells are key effector cells in host antiviral activities by directly killing viral-infected cells and producing cytokines and chemokines, especially in early phase of viral infection. After enteroviruses infection, NK cells were one of the most abundant cell types in the inflammatory infiltrate, and appeared to limit both enteroviruses replication and virus-induced disease in experimental mice model. However, role of human NK cells during EV71 infection, especially the direct interaction between EV71 and human NK cells, was not studied extensively. Clinical observation manifested that patients with severe EV71 infection have marked diminished NK cells in peripheral blood. Therefore we hypothesized that EV71 might directly target human NK cells as one of its immunoevasion strategies. Here, we demonstrated for the first time that fresh primary human NK cells were susceptible to EV71 infection. By flow cytometry and florescence microscope, EV71 capsid protein VP1 was able to be detected in viral-infected NK cells as soon as 6 hours after infection and peaked at 24 hour after infection. In the same time, EV71 viral RNA was detected by quantitative RT-PCR and the viral copies increased from 6 hour onwards to peak at 12 hours after infection. We further demonstrated the infectious entry of EV71 in human NK cells was depended on clathrin-mediated endocytosis. Next, we illustrated that EV71 infection could trigger NK cells apoptosis as evidenced by increased Annexin V+, PI+, and activated caspase 3+ cells in EV71-treated NK cells. We further proved that the cytotoxicity of NK cells was inhibited by EV71 infection and this inhibition might not be related with down-regulation of NKp46, but may be related to the increased apoptosis. In conclusion, our data suggested that EV71 might directly target and kill NK cells as a strategy to evade human innate immunity, which might facilitate virus replication, transmission and then contribute to viral-related pathogenesis. / published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

published_or_final_version / Medicine / Master / Doctor of Medicine

No description available.