published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

Primary Epstein-Barr virus (EBV) infection in childhood is typically asymptomatic, but infection later in life results in a mononucleosis with the illness severity ranging from asymptomatic to requiring hospitalisation. The illness is generally short-lived (four to six weeks following onset of symptoms), but persistent disabling symptoms (lasting up to 6 months or longer) are well described in around ten percent of individuals. The aim of this work was to characterise immunological and virological parameters in the peripheral blood, which correlate with persistence of symptoms following EBV-induced mononucleosis. Subjects were recruited prospectively following confirmed primary EBV infection, to allow blood samples and clinical data to be collected at multiple timepoints (baseline, 2 weeks, 4 weeks, 3 months and at least 12 months later). Subjects with 6 months or more of disabling symptoms were defined as ???cases??? with control subjects being those whose illness resolved within 6 weeks of enrolment. Cases were compared with control subjects in terms of: cellular EBV viral load in the peripheral blood by PCR; development of antibodies against EBV VCA (IgG and IgM) and EBNA-1 (IgG) by ELISA; proportions of peripheral blood leucocyte subsets and their activation status by flow cytometry; the magnitude, kinetics of development, and breadth of the CD8+ cytotoxic cell response by interferon-?? Elispot; cytokine levels in serum, and production by peripheral blood mononuclear cells ex vivo; and gene expression patterns in peripheral blood mononuclear cells by microarray. With the exception of gene expression, none of these parameters correlated with early resolution of symptoms or predicted clinical outcome following primary EBV infection. Antibody patterns suggest a tendency to Th2 type immune response may be associated with persistent illness. Preliminary analysis of the gene expression studies indicates that there are many genes involved in this complex disease requiring further investigation. Persistent illness following EBV infection is not associated with uncontrolled viral replication, or chronic immune activation due to an aberrant primary immune response.

Primary Epstein-Barr virus (EBV) infection in childhood is typically asymptomatic, but infection later in life results in a mononucleosis with the illness severity ranging from asymptomatic to requiring hospitalisation. The illness is generally short-lived (four to six weeks following onset of symptoms), but persistent disabling symptoms (lasting up to 6 months or longer) are well described in around ten percent of individuals. The aim of this work was to characterise immunological and virological parameters in the peripheral blood, which correlate with persistence of symptoms following EBV-induced mononucleosis. Subjects were recruited prospectively following confirmed primary EBV infection, to allow blood samples and clinical data to be collected at multiple timepoints (baseline, 2 weeks, 4 weeks, 3 months and at least 12 months later). Subjects with 6 months or more of disabling symptoms were defined as ???cases??? with control subjects being those whose illness resolved within 6 weeks of enrolment. Cases were compared with control subjects in terms of: cellular EBV viral load in the peripheral blood by PCR; development of antibodies against EBV VCA (IgG and IgM) and EBNA-1 (IgG) by ELISA; proportions of peripheral blood leucocyte subsets and their activation status by flow cytometry; the magnitude, kinetics of development, and breadth of the CD8+ cytotoxic cell response by interferon-?? Elispot; cytokine levels in serum, and production by peripheral blood mononuclear cells ex vivo; and gene expression patterns in peripheral blood mononuclear cells by microarray. With the exception of gene expression, none of these parameters correlated with early resolution of symptoms or predicted clinical outcome following primary EBV infection. Antibody patterns suggest a tendency to Th2 type immune response may be associated with persistent illness. Preliminary analysis of the gene expression studies indicates that there are many genes involved in this complex disease requiring further investigation. Persistent illness following EBV infection is not associated with uncontrolled viral replication, or chronic immune activation due to an aberrant primary immune response.

Thesis (M. Phil.)--University of Hong Kong, 2008. / Includes bibliographical references (leaf 102-115) Also available in print.

Twenty-two subjects with chronic/recurrent Epstein-Barr Virus (EBV) infection were compared with 22 controls to assess cognitive dysfunction. Subjects were compared on 15 measures of cognitive functioning from the Boston Diagnostic Aphasia Examination, Perceptual Speed, Wechsler Adult Intelligence Scale-Revised, Finger Tapping Test, Stroop Test, Trail-Making Test, Wisconsin Card Sorting Test and Revised Wechsler Memory Scale. They also were compared on measures of depression including the Beck Depression Inventory, Minnesota Multiphasic Personality Inventory (MMPI) depression subscale and SCL-90-Revised depression subscale. Group differences were assessed using discriminant analysis. Only some measures were included in this analysis, i.e. percent retention on Visual Reproduction and Logical Memory subtests of the Revised Wechsler Memory Scale, differential between time scores and between error scores on conditions three and two of the Stroop Test and total number of errors and perseverative errors on the Wisconsin Card Sorting Test. The Beck Depression Inventory was included to statistically remove depression effects from cognitive performances. Remaining measures were administered for exploratory and/or comparative purposes only. Results from discriminant analysis revealed significant group differences on the Beck, but not on any cognitive measure either before or after removal of depression effects. However, direction of group differences on cognitive measures occurred as expected. Also, a post-hoc multivariate analysis of variance revealed significant group differences on MMPI scales 1, 2 and 3 with EBV subjects showing higher elevations. Significant group differences also occurred on MMPI scale 7. Two categories of explanation for results are offered. The first suggests that cognitive deficits were missed due to shortcomings in study design. Remaining hypotheses address the possibility that no cognitive deficits occur with chronic/recurrent EBV infection. Suggestion for why EBV patients complain of cognitive deficits include discussion of hysteroid tendencies and intensification of sensations by a focus on somatic processes. The usefulness of assuming a multifactorial basis for symptoms associated with chronic/recurrent EBV infection, and the importance of abandoning the either/or approach of earlier investigators to hypothesizing about etiology, are discussed.

The vitamin D receptor (VDR) is a transcription factor mediating genomic responses to the biologically active form of vitamin D, 1,25(OH)2D3, a key modulator of the immune system. Knowledge on how these polymorphisms modulate the vitamin D endocrine system and confer risk of disease is hindered by the fact that several of the associated allelic variants are located in introns or are synonymous and likely serve as markers within an extended haplotype covering disease-causing alleles. The functional relevance of VDR polymorphisms need to be studied in the context of the haplotype, comparing haplotypes with the process of DNA transcription, protein levels and biological function. These functional studies should be performed using techniques reflecting the in vivo, naturally occurring milieu as close as possible. VDR has several known allelic variants including a FokI restriction fragment length polymorphism in exon II, BsmI and ApaI polymorphisms in the intron VIII, and a synonymous TaqI variant in exon IX. The aim of the current study was the identification of sequence variants in VDR, to define haplotype patterns in the Caucasian population and to understand the functional consequences of single nucleotide polymorphisms (SNPs) and haplotypes. Methods: EBV transformed B-lymphocyte cell lines, from twenty-three individuals, within the Caucasian population were established. Polymorphisms and haplotypes in VDR were identified by genotyping and sequencing. Quantification of the VDR protein level measured with flow cytometry was studied together with the genotype and haplotype data to determine possible influence of genotypes or haplotype and VDR protein levels. Biological function was analysed by the percentage inhibition that each individual experienced in the presence of 1,25(OH)2D3, measured by the Alamar Blue assay and the Trypan blue dye exclusion method. Results: The results showed thatVDR genotypes and haplotypes may not influence VDR protein level although certain genotypes and haplotypes significantly influenced biological function. It was proposed that VDR variants may account for significant influences on cellular responsiveness to 1,25(OH)2D3 as mediated by VDR. Conclusions: The findings of the current study suggest that individual SNPs and haplotypes of VDR influences quality of the repose in the presence of 1,25(OH)2D3, rather than quantity of the VDR levels. This knowledge may permit a rational choice of polymorphisms to use in epidemiology studies or improve our understanding of the significance of VDR genetic polymorphisms on biological function. Keywords: functionality, polymorphism, haplotype, vitamin D receptor, VDR, 1,25(OH)2D3, structure-function analysis, biological responsiveness. / Prof. L. Bornman

Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

published_or_final_version / Microbiology / Master / Master of Medical Sciences

Nasopharyngeal carcinoma (NPC) is a common cancer in Southeast Asia. While external radiotherapy is the mainstay of treatment, adjuvant chemotherapy is required in advanced disease. Current chemotherapy heavily relies on cisplatin and docetaxel. The disease relapse rate is relatively high with poor survival chance for recurrent or metastatic disease. Development of novel therapeutic strategies against the disease is clearly needed. Bortezomib and suberoylanilide hydroxamic acid are respectively classified as proteasome inhibitor and histone deacetylase inhibitor. Bortezomib and SAHA induce apoptosis in various cancers including renal cell carcinoma, hepatoma and mantle cell lymphoma. However, the effect of bortezomib and SAHA on NPC cells was not mentioned. We sought to study the molecular and cellular effects of the bortezomib and SAHA on NPC cells hoping to look for drug alternatives in NPC treatment. Since SAHA reactivates EBV in NPC cells, the combined effect of bortezomib and SAHA on EBV lytic cycle was also evaluated. NPC proliferation was assessed by MTT assay. 5 EBV-positive NPC cell lines authenticated by Short Tandem Repeats (STR) profiling were used as most NPC in Chinese contains EBV. Isobologram and combination index analysis confirmed that the anti-proliferative effect on NPC mediated by the drug combination was synergistic. 30 nM bortezomib and 5μM SAHA were chosen for further studies on apoptosis because the synergism of the drugs was maximal at these concentrations. NA and C666-1 were chosen for further studies because C666-1 was the only NPC cell line that consistently harboured native NPC and the combination index was lowest in NA among the rest of the NPC cell lines. Bortezomib led to apoptosis in NPC cells. The effect was more pronounced after the addition of SAHA as evidenced by greater TUNEL positive population and earlier cleavage of poly ADP ribose polymerase (PARP). In previous cancer studies, ROS induction was commonly suggested pathways of bortezomib and SAHA’s antiproliferative effects. Staining with dichlorofluorescein diacetate (DCFH-DA) revealed enhanced reactive oxygen species (ROS) level in cells treated with both drugs. At the same time, addition of N-acetyl cysteine, a ROS scavenger, markedly reduced their effect on cytotoxicity. SAHA is known for its effect on EBV lytic cycle induction. Yet, the addition of bortezomib diminished SAHA-induced viral load, lytic protein expression and EBV infectivity. The expression of Latent Membrane Protein 1 (LMP1) was much lower in NPC treated with both drugs than in NPC treated with SAHA alone, which would reduce NF-κB activation. This, together with reduced EBNA1 expression upon treatment with both drugs, would theoretically reduce oncogenic activity. In conclusion, bortezomib and SAHA induced ROS-driven apoptosis of NPC in a synergistic manner and bortezomib inhibited SAHA-induced EBV lytic cycle. It suggests that bortezomib and SAHA are potential drug candidates for the treatment of nasopharyngeal carcinoma. / published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Research in Medicine

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 198-213) Also available in print.