Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2005. / Includes bibliographical references (leaves 112-133). Also available in electronic version.

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

No description available.

Introduction: In pediatrics, ninety-eight percent of all chest pain complaints are non-cardiac in origin, with the leading cause being muscular skeletal pain. Of the two percent that are cardiac in origin, troponin levels are helpful in detecting cardiac muscle ischemia, most commonly present in myocardial infarction, pulmonary embolism, pericarditis, myocarditis, aortic dissection, heart failure, and cardiac contusion. There are multiple different troponin assays that detect troponin, usually using a two-site immunoezematic assay. Although values obtained may differ slightly between assays, there is not a statistically significant difference between assays. Several endogenous substances, such as rheumatoid factor, excess fibrin, alkaline phosphatase, and heterophile antibodies can cause statistically significant, though falsely increased troponin levels that do not rise and fall in the typical pattern seen in cardiac ischemia due to cross reactivity with the assays. We present the case of a fifteen-year-old male with chest pain and elevated troponins, but otherwise benign workup. It highlights the falsely elevated troponin levels seen with the cross-reactivity of heterophile antibodies and various troponin assays. Case Presentation: A 15-year-old male presents with a two-day history of intermittent, stabbing, 5/10 left sided chest pain, as well as substernal chest pressure that radiates to his left upper chest and shoulder. He is unable to identify any triggers of the pain or alleviating factors. Review of systems is negative for nausea, vomiting, diaphoresis, and shortness of breath. His past medical history is significant for pectus excavatum that has been corrected with Nuss bar procedure. He also reports being treated for strep pharyngitis two weeks prior. The patient presents to emergency department after having an irregular heart rate noted by the school nurse’s office. His vital signs are stable, and his physical exam is notable for a regular heart rate and rhythm. Pectus excavatum with well healed midline scar is present. A basic metabolic panel is normal. In the emergency department, serum troponins are elevated between 0.24-0.25 ng/ml (normal range 0.00-0.02), and a D-dimer is elevated at 500 ng/ml (normal range 0-230). Cardiology is consulted, and following an algorithm for chest pain in pediatric patients,1 a series of additional tests are performed. His electrocardiogram shows a normal sinus rhythm with minimal left axis deviation. Chest x-ray shows intact Nuss bars and is, otherwise, normal. Ventilation-Perfusion (VQ) scan is normal. An echocardiogram shows normal ventricular output and valvular function with an incidental finding of slight angulation of aortic arch with no significant gradient. Cardiology recommends trending troponins every 6 hours. A repeat EKG the following day shows normal sinus rhythm without evidence of ischemia or arrhythmia. Serial troponins remain elevated for the following 48 hours ranging 0.22-0.24 ng/ml. The patient denies any further cardiac symptoms despite the persistently elevated troponins, and he is discharged home with close follow-up with pediatric cardiology as well as a thirty-day looping event monitor. One week after discharge, in the cardiology clinic, troponin levels are still 0.23ng/ml. Creatine kinase MB level is obtained and is within normal limits. Due to persistently elevated troponins within a narrow range, a literature search is performed to determine if there are other causes for elevated troponins that neither rise nor fall. Several articles are found confirming cross reactivity between various antibodies and troponin assays that lead to falsely elevated troponin levels.2,3 Rheumatoid factor, antinucleic antibodies, and Epstein-Barr virus antibody titers are obtained. RF and ANA levels return within normal limits. However, EBV IgM levels are elevated , while EBV IgG levels are within normal limits, supporting the theory that the patient has a current infection of mononucleosis. The thirty day looping event monitor is uneventful, showing no dysrhythmia or ectopy. Serial echocardiograms continue to remain unchanged from the initial echo during the patient’s hospitalization. Serum samples are analyzed on four different troponin assays, and a vast variation in troponin levels is found, ranging from < 0.015 ng/ml (normal value) on the Siemens Vista/ cTnl to 0.23 ng/ml (significantly elevated) on Beckman Access/ AccuTnI+3. Serial dilution studies are performed on a Beckman Dxi800 instrument and reveal decreasing troponin values with increasingly diluted serum samples. Discussion: In pediatrics, ninety-eight percent of all chest pain complaints are non-cardiac in origin, with the leading cause being muscular skeletal pain.4 Of the two percent that are cardiac in origin, troponin levels are helpful in detecting cardiac muscle ischemia, most commonly present in myocardial infarct, pulmonary embolism, pericarditis, myocarditis, aortic dissection, heart failure, and cardiac contusion, over exertion, and rhabdomyolysis. Most troponin assays use a two-site immunoenzymatic assay. One study in 2016 found that various assays reported different troponin levels; however the 99th percentile cutoff values were in agreeance with each other.5 Heterophilie antibodies in the serum have been found to falsely elevate troponin levels due to cross-reactivity between the antibodies and troponin assays. Rheumatoid factor, fibrin, and alkaline phosphatase can also cause false positive results.2 Conclusions: Elevated troponins are commonly interpreted as a sign of myocardial injury that prompts an extensive cardiac evaluation. This case illustrates that a common pediatric illness, infection with Epstein-Barr virus, can cause a false elevation in serum troponins. Infectious mononucleosis, as well as autoimmune diseases, should be considered in the differential of patients who present with elevated troponins without an apparent cardiac source.

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Biochemistry / Doctoral / Doctor of Philosophy

No description available.

No description available.

Infection with Epstein-Barr virus (EBV) during adolescence results in an immunopathological disease, Infectious Mononucleosis (IM), in around 25% of cases. A role for Regulatory T cells (Treg) in IM has yet to be established. These suppressive cells may affect the well-characterised cytotoxic T cell (CTL) response to EBV and thus the level of viral persistence and reactivation, potentially creating an environment conducive to the outgrowth of EBV-infected cells and tumour development. The work in this thesis examines the frequency and functional capacity of Treg in primary EBV infection. The results show that the frequency of Treg within the CD4+ T cell population of IM patients was reduced with borderline significance (p=0.05) compared with healthy controls as revealed by fluorescence activated cell sorting. Treg function was confirmed using suppression assays on peripheral blood mononuclear cells (PBMC) from healthy controls but could not be assessed in IM patients due to low cell numbers. EBV-specific Treg function was analysed using Interferon (IFN)-γ ELISPOT assays in which PBMC from IM patients and healthy controls were stimulated with phytohaemagglutinin (PHA) and EBV peptides in the presence or absence of Treg. The IFN-gamma response of PBMC to PHA stimulation was significantly reduced in IM patients compared to healthy controls (p=0.009) but the IFN-gamma response to EBV peptides did not alter, irrespective of the presence or absence of Treg. Investigation of FOXP3 expression by immunohistochemistry provided evidence of Treg presence and preliminary data indicated an increased expression in IM tonsil sections compared with healthy tonsil sections. The proliferative responses and cytokine profiles of healthy controls, as measured by proliferation assays and ELISAs, in response to stimulation with the recall antigen PPD did not significantly alter upon the addition of latent membrane protein (LMP)-1 peptide. In IM patients, the same treatment resulted in a significant reduction in IFN-gamma (p=0.026) but no significant differences in IL-10 production or cell proliferation. The significantly reduced frequency of Treg in peripheral blood of IM patients and abundant FOXP3 expression in IM tonsils provides evidence for a Treg role in primary EBV infection. One plausible explanation is the recruitment of Treg to the site of primary infection by an as yet unidentified EBV-specific mechanism. Clarification of Treg activity in IM may expose opportunities for immunomanipulation during early stages of infection.

Epstein-Barr virus (EBV) is a gamma herpes virus persistently infecting over 90% of the adult population worldwide. It has been aetiologically linked to a number of human malignancies, including more than 90% of post transplant lymphoproliferative disease (PTLD), 50% of Hodgkin’s lymphoma (HL), virtually all undifferentiated nasopharyngeal carcinoma (NPC), and approximately 10% of gastric carcinoma (GC). As EBV infection in healthy individuals is mainly controlled by virus specific cytotoxic T lymphocytes (CTLs), we hypothesise that engineering T cells with chimeric T cell receptors (cTCRs) specific for EBV latent membrane proteins (LMPs) will confer on these cells the ability to target and kill the malignant cells of cancers associated with Epstein-Barr virus. Thus, the aim of this project was generate these engineered T cells and to set up a severe combined immunodeficient (SCID) mouse model in which to test their effectiveness. Three EBV-infected cell lines derived from HL, NPC and GC gave rise to tumours in 11 of 12 (92%), 12 of 12 (100%) and 10 of 10 (100%) SCID mice respectively, when 1x107 cells were injected subcutaneously. Immunohistochemical analysis showed that the HL SCID tumours were CD4-, CD15-, CD20+, CD30+, consistent with a HL Reed-Sternberg cell phenotype, and NPC and GC SCID tumours expressed the epithelial cell marker cytokeratin. Furthermore, all tumours expressed EBVencoded RNAs (EBERs) and LMP1. This was identical to parent cell line expression patterns, and hence growth in vivo did not affect cell phenotype. T cells were successfully transduced with a retroviral vector encoding a CD19-specific cTCR (CD19- cTCR) with a mean transduction rate of 13%±6%. Transduced cells were cytotoxic for HL-derived L591 cells in vitro, with specific lysis of 24%±11% at an effector to target ratio of 20:1. This was significantly higher than specific lysis seen in mock transduced cells (p>0.05). At a tumour inoculation dose of 5x106, in vivo sc transfer of 5x107 CD19-cTCR transduced cells was able to prevent HL tumour development in 6 of 6 (100%) test mice, whereas 17 of 22 (77%) control mice and 2 of 3 (66%) mice treated with unmodified EBV-specific CTLs developed tumours. Moreover, iv transfer of 5x107 CD19-cTCR transduced cells mediated complete regression of HL SCID tumours in 3 out of 6 (50%) mice. Phage display selection experiments to isolate a single chain antibody fragment (scFv) specific for viral LMPs for incorporation in a cTCR were performed. Linear, biotinylated and cyclised biotinylated peptides derived from the external reverse turn loops of LMP2 were used as target antigens. Despite extensive testing, no reactive clones specific for the peptides were identified. The ability of CD19-cTCR transduced cells to specifically lyse HL cells in vitro, and clear tumour burden in vivo, supports a future role for engineered T cells in the treatment of HL. Despite the lack of success in isolating a scFv for LMP2, the use of viral antigen specific, cTCR redirected T cells remains in principle a valuable therapeutic alternative for EBV-associated malignancies. The SCID models for HL, NPC and GC will provide a useful preclinical tool for investigation of their efficacy in vivo.