Medicine (RMH) - Theses
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ItemThe HIT epitope( 2001)Heparin-induced thrombocytopenia (HIT) is a disorder found in some patients who developed thrombocytopenia after given heparin and similar glycosaminoglycans, with an incidence of 3%. There is little experimental study on the predisposition to HIT, while the pathogenesis of HIT has been investigated more widely. The currently favoured model is based on the activation of platelets FeγRII by HIT IgG, which only occur when the HIT IgG is in complex with PF4 and glycosaminoglycan. However, no study has looked into platelets activation by the HIT antibody beyond the binding to FeγRII. As the signal transduction pathways of platelets activation become increasingly understood, the activation of platelets by the HIT antibody await further in-depth studies. The involvement of endothelial cells in HIT is also an area that demands further detailed studies. Almost nothing is known about the immunogenesis of HIT, apart from isolated animal study based on idiotype. The possibility of involvement of cellular immunity in HIT is only studied very recently. HIT may be diagnosed by a functional assay based on activation of platelets or immunoasaay based on the detection of anti-PF4-hcparin antibody. A map of the HIT epitope is essential in elucidating the various aspects of HIT. It is now known that the majority, if not all HIT antibodies bind to a complex of hPF4 and heparin. Although the role of heparin or similar glycosaminoglycan is not fully understood, it is plausible that the HIT epitope is presented entirely on the PF4 tetramer. Heparin presumably serves to modify the presentation of the HIT epitope on the PF4 tetramers. One model contends that heparin alters the conformation of each PF4 tetramer, such that a neo-epitope is exposed on binding to heparin. On the other hand, when heparin is absent, the HIT epitope is hidden within the PF4 tetramer. An alternative model is based on the alignment of many PF4 tetramers by chains of heparin polysaccharides. Knowledge of the HIT epitope is crucial in unravelling the interaction between the HIT antibody and its antigen, and the subsequent platelets activation. This study uses site directed mutagenesis to create a comprehensive set of hPF4 mutant proteins. By measuring the binding avidity of these mutant PF4s with a batch of HIT sera, the contribution of the surface residues of PF4 tetramer is estimated. Subsequently, a single HIT population epitope is mapped on hPF4. Using graphical molecular modelling, several possible ways in which the HIT antibody, PF4 and heparin may interact with each other are examined. Viewing PF4 as a six faceted cuboid, the HIT epitope is found to be presented predominantly on one pair of opposite faces. The heparin binding helical pairs are located on a different pair of opposite faces. When a curve is drawn around the PF4 tetramer along the longitudinal axes of the two pairs of α-helices, the HIT epitope bearing faces are the pair that are crossed by this curve. This finding is significant because it is consistent with a model of interaction between PF4 and heparin, in which heparin chains bind to four pairs of lysine residues on each pair of α-helices in a orthogonal orientation between the longitudinal axes of the heparin chain and the helical pair. The finding is also consistent with a modified model in which heparin binds to the pair of opposite faces whose normal axes are directed perpendicular to the longitudinal axes of the paired α-helices. It is of significance to note that the pair of faces containing the HIT epitope are the only pairs which have a paucity of positively charged amino acid residues. If the HIT epitope resides on any of the other two pairs of faces, the HIT antibody will be forced to compete with heparin for binding to regions containing positively charged residues. The localisation of the HIT epitope on the pair of faces poor in positively charged residues is also significant, as PF4 tetramers are free to be aligned into arrays via cross-linking by heparin chains. These arrays of heparin cross-linked PF4 arrays have their HIT epitope bearing facets exposed. HIT IgG are facilitated to bind onto arrays of HIT epitopes aligned in optimal positions for coordinated binding of HIT IgG. The net effect of such coordinated binding is the oligomerisation of HIT IgG. When the oligomerised HIT IgG bind to Feγ receptors, the latter were forced to undergo lateral movement in the cell membrane in order to reach the binding targets. The resultant clustering of Feγ receptors on the cell membrane is expected to serve as trigger for downstream signal transduction which eventually leads to the activation of platelets. It may be some time yet before the finding of this study can be confirmed by 3D structural mapping of the HIT epitope using X-ray crystallography or NMR study. The tetrameric nature of PF4 and the tri-molecular interaction pose enormous barrier to this approach. In the meantime, the functionally determined HIT epitope will serve as a model for further experimental investigation and understanding of HIT.
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ItemThe ErbB family of receptor tyrosine kinases in vestibular schwannoma and meningioma( 2004)Neoplastic disease whether benign or malignant, is the aberrant uncontrolled proliferation of cells within any given compartment of the human body, leading to the formation of a tumour. Such proliferation of cells, known as transformation, is due to deregulation of cellular mechanisms that control normal growth and development, so that transformed cells become independent of factors that usually provide constraints for cell growth. Intracranial tumourigenesis essentially results in tumours of the brain, the meninges, the pituitary gland and/or the cranial nerves. (From Chapter 1)
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ItemDevelopments in the evaluation of oxygen uptake with emphasis on cardiopulmonary exercise testing, and its application to the preoperative assessment of surgical patients( 2009)The introduction of the Swan-Ganz catheter into clinical practice in 1985 facilitated a paradigm change in the management of patients undergoing major surgery. This development made it possible to measure cardiac output and calculate oxygen delivery and uptake in real time at the bedside. These advances together with the development of the portable programmable computer, meant that the attending clinician had access to more data than had ever been possible before. The modern Intensive Care Unit (ICU) evolved alongside these developments, enabling the use of a constellation of high technology equipment which was becoming available. At the same time there was considerable controversy around what was the most appropriate use of the ICU. This controversy was not resolved by the introduction of APACHE II in 1985 which allowed intensivists to correlate severity of illness with survival more reliably than previously. Our group published a paper in 1988, which showed that it was possible to identify high-risk surgical patients pre-operatively with the use of the Swan-Ganz catheter and schedule them for ICU admission post-operatively. In the late 1980s the metabolic cart became available clinically. Using this machine it was possible to measure oxygen uptake and calculate cardiac output non-invasively during exercise. The physiological data that became available with the use of this technology made possible reliable risk assessment of patients scheduled for major surgery. In 1989 a unique opportunity became available to the author to compare data from exercising patients obtained with a metabolic cart with data obtained synchronously with a Swan-Ganz catheter. Comparing this data enabled the investigation of the physiology of exercise in patients with cardiac failure in a way that would not have been possible by use of either a Swan-Ganz catheter or a metabolic cart alone. In 1993 our group published a paper from our laboratory about risk evaluation in surgical patients using Cardiopulmonary Exercise Testing (CPET). The data in this paper was pivotal to the belief that post-operative mortality was a function of cardiac failure and not myocardial ischaemia. In 1999 we published a prospective study involving 548 patients scheduled for major surgery. The results in this paper showed that mortality was inexorably linked to ventricular function. The data suggested that myocardial ischaemia per se was not the cause of post-operatively mortality, unless it was associated with cardiac failure. Other methods of pre-operative evaluation are discussed and it is argued that some of the common methods in use are flawed e.g. the use of metabolic equivalents and the use of stair climbing. The American College of Cardiology and American Heart Association (ACC/AHA) guidelines on pre-operative evaluation of non-cardiac surgical patients, place considerable emphasis on both these methods. This thesis contains a critique of some of the key aspects of these guidelines. An increasing numbers of patients presenting for surgery are taking beta-adrenergic blocking drugs. The use of these drugs has received close examination both from the ACC/AHA guidelines and a contemporary major multicentre trial. It is argued that the use of CPET in evaluating the use of these drugs is very important as management protocols for these patients can be generated from CPET data.