Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2008. / Includes bibliographical references (leaves 127-143). Also available in electronic version.

Gamma-interferon stimulates human macrophages to produce of 7,8-dihydroneopterin (7,8-NP). 7,8-NP and its oxidation product neopterin are excellent inflammatory markers for a variety of chronic conditions, including atherosclerosis. The biological significance of 7,8-NP in atherosclerosis is not fully understood, but 7,8-NP has been shown to protect macrophage cells from oxidised low density lipoprotein (oxLDL). Cellular accumulation of oxLDL-derived lipids and oxLDL-induced cytotoxicity are major drivers of atherosclerotic plaque progression. This thesis investigated the mechanisms of 7,8-NP-mediated protection against oxLDL-induced damage to macrophage cells. The research assessed the relative contribution of the previously identifyed antioxidant capacity of 7,8-NP and its ability to down-regulate oxLDL uptake. OxLDL cytotoxicity was characterised by high intracellular oxidative stress within the first 12 hours of exposure, which was critical to oxLDL toxicity. Exogenously added 7,8-NP effectively scavenged the intracellular oxidants generated in response to oxLDL, shown by the oxidation of 7,8-NP to neopterin. The ability of 7,8-NP to alleviate oxidative stress during the critical time-frame of acute toxicity was the primary mechanism of protection. 7,8-NP was also found to down-regulate the levels of intracellular oxysterol esters in oxLDL-treated macrophages. This decrease was associated with the reduction of CD36 scavenger receptor protein and mRNA expression. The late onset of these processes in the second half of the 24 hour incubation highlighted their potential role in foam cell formation. Research indicated that 7,8-NP may play a role in the reverse cholesterol transport in these cholesterol ester-loaded cells. The CD36 down-regulation by 7,8-NP did not protect macrophages from acute oxLDL cytotoxicity. This research reveals novel detail about the mechanism of 7,8-NP protection of macrophages from cytotoxic effects of oxLDL. It is suggested that 7,8-NP may protect macrophage cells in the atherosclerotic plaques by scavenging ROS produced during acute cytotoxicity and alleviate oxysterol ester accumulation, thus stabilising macrophage cells during chronic oxLDL exposure.

Plasma neopterin is an excellent marker of inflammation and is found in elevated levels in plasma of patients with cardiovascular disease. Neopterin originates as the oxidation product of 7,8-dihydroneopterin (7,8-NP), which is secreted by human macrophages when stimulated with interferon-y during inflammation. 7,8-NP has been shown to be a very efficient free radical scavenger and a potent antioxidant which can protect macrophages from a range of oxidative stresses. The uptake of oxidised low density lipoprotein (oxLDL) by macrophages which lead to the formation of foam cells is a hallmark of early atherosclerotic lesions. OxLDL-induced cell death is also considered to be an important process in the formation of necrotic lipid rich plaques and in atherosclerotic plaque destabilisation. This thesis examined the extent of oxLDL-induced damaged to HMDMs and whether 7,8-NP can inhibit oxLDL-mediated cell death in HMDMs. Foam cells had previously been defined as cholesteryl ester (CE) macrophages that stained positive with oil red-O. This thesis shows that the foamy appearance and presence of lipid droplets stained with oil red-O was not dependent on accumulation of CE which raises the suitability of using oil-red-O staining to identify the foam cells. In addition, HPLC but not GC analysis showed an increased in CE levels of the macrophages when the macrophages were incubated with oxLDL. The HPLC approach spared the samples of lengthy manipulations that might cause ex vivo oxidation. It also avoided subjecting the samples to high temperature treatment that could alter the lipid composition and therefore quantification of the lipid contents. Previous studies showed that 7,8-NP is a potent antioxidant and cytoprotective agent. Exposure of HMDMs to 1 mg/ml oxLDL caused 50% loss of cell viability as measured by the MTT reduction and trypan blue exclusion assays. The development of apoptotic features including caspase-3 activity, cytochrome c release from mitochondria and phophatidyserine (PS) exposure was examined. OxLDL did not cause caspase-3 activation as shown by Western Blot analysis and did not cause DEVD-AMC cleavage in HMDMs. However, cytochrome c release and phosphatidylserine exposure were observed when HMDMs were incubated with oxLDL as shown by Western Blot analysis and Annexin V-FITC staining respectively. Dihydroethidium (DHE) staining showed that oxLDL treatment caused mitochondrial superoxide generation in HMDMs. OxLDL-induced oxidative stress appeared to cause a rapid loss of HMDMs' intracellular glutathione (GSH) as analysed by HPLC technique. Incubation of HMDMs' with buthionine sulfoximine (BSO) and diethyl maleate (DEM) caused similar loss in GSH as incubation with oxLDL but did not result in HMDMs' death. This showed that oxLDL-induced decrease in GSH alone was not sufficient to cause cell death. The loss of cell viability by oxLDL was inhibited by 7,8-NP in the concentration range of 50 to 200 lM. HMDMs' GSH loss caused by oxLDL was similarly inhibited by 7,8-NP supporting the idea that preventing the cellular GSH loss will protect the HMDMs from death. Incubation of HMDMs with 7,8-NP showed reduction in DHE fluorescence intensity staining suggesting that 7,8-NP inhibited or scavenged oxLDL-dependent generation of superoxide. 7,8-NP also effectively inhibited oxLDL-induced PS externalisation to the outer membrane but failed to inhibit the oxLDL-induced release of cytochrome c from mitochondria to the cytosol. The labelling of oxLDL with DiI showed that 7,8-NP significantly inhibited the uptake of oxLDL. However, the inhibitory effect was only measured at non-toxic concentration of oxLDL. The ability of 7,8-NP to inhibit oxLDL uptake raised the possibility that 7,8-NP protective effect against oxLDL involved modulation of the scavenger receptors'expression in particular SRA and CD36. The Western Blot analysis showed that incubation of HMDMs with 7,8-NP did not affect HMDMs' SRA protein expression. In 50% of the experiments, it was demonstrated that certain isoforms of CD36 protein were significantly down regulated by 7,8-NP suggesting that various factors might interact with 7,8-NP or CD36. The ability of 7,8-NP to protect HMDMs from oxLDL-induced death provides further evidence that this antioxidant is secreted by HMDMs to protect them against the oxidative damage in the highly oxidative environment of atherosclerotic plaque.

Kynurenine (KYN) is the first stable metabolite of the kynurenine pathway, which accounts for over 95% of tryptophan metabolism. Two previous studies by this research group reported elevated plasma KYN in Tourette syndrome (TS) patients when compared with age and sex matched controls and another study showed that KYN potentiated 5-HT2A-mediated head-shakes (HS) in rodents. These movements have been suggested to model tics in TS. This raised the questions how KYN acts in eliciting this response and whether it is an action of its own or of a further metabolite along the kynurenine pathway. In the liver, where most of the kynurenine pathway metabolism takes place under physiological conditions, the first and the rate limiting enzyme is tryptophan-dioxygenase (TDO) which can be induced by cortisol. In extrahepatic tissues the same step of the pathway is catalyzed by indoleamine-dioxygenase (IDO), which is induced by cytokines, predominantly interferon-y (INF-y). Plasma neopterin, which shows parallel increase with KYN following immune stimulation, was also found elevated in one of these studies positively correlating with KYN. In the present work animal studies suggested that KYN potentiates and quinolinic acid (QUINA) dose dependently inhibits the 5-HT2A-mediated HS response in mice. The potentiating effect seen with KYN was suggested to be an effect of KYN itself. Radioligand binding and phosphoinositide (PI) hydrolysis studies were done to explore the mechanisms by which kynurenine pathway metabolites could alter a 5-HT2A-receptor mediated response. None of the kynurenine pathway metabolites tested showed direct binding to 5-HT2A-receptors. PI hydrolysis studies with KYN and QUINA showed that KYN did not have any effect while QUINA inhibited 5-HT2A-mediated PI hydrolysis. Plasma cortisol determination in TS patients with elevated plasma KYN did not show elevated plasma cortisol levels, suggesting that the increase of plasma KYN in these TS patients is unlikely to be due to an increased TDO activity induced by increased cortisol. Attention deficit hyperactivity disorder (ADHD) is commonly associated with TS. Salivary cortisol detected in a group of children primarily affected with ADHD showed significantly lower salivary cortisol levels when compared with age and sex matched controls. Plasma tryptophan, KYN, neopterin, INF-y and KYN/tryptophan ratio and night-time urinary 6-sulphatoxymelatonin (aMT6s) excretion measured in a group of TS patients did not show any difference in their levels when compared with age and sex matched controls, but TS patients failed to show the expected positive correlation seen between plasma INF-y, neopterin and KYN and the negative correlation seen between plasma KYN and night-time urinary aMT6s excretion seen in healthy controls. The relevance of the kynurenine pathway, melatonin secretion and cortisol to Tourette Syndrome and associated conditions and the mechanism by which KYN and QUINA alter the 5-HT2A-receptor mediated HS response are discussed.

Formation of functional collateral circulation, to repair blocked or damaged arterial blood flow, is an important process in amending adverse outcomes after acute coronary occlusion events. Inadequate capillary growth during pressure overloads impairs myocardial perfusion, often contributing to the progression of coronary heart disease and ischaemia. Considered to be the critical rate-limiting step in physiological angiogenesis, the binding of VEGF (vascular endothelial growth factor) to VEGFR (vascular endothelial growth factor receptors) is essential for the growth and repair of arteries. Conversely, VEGF mediated angiogenesis has also been shown to promote atherosclerosis through arterial wall thickening. However, an alternatively spliced soluble form of VEGFR-1 (sFlt-1) has been shown to inhibit VEGF activity. sFlt-1 binds and sequesters free extracellular VEGF and/or heterodimerizes with VEGFR preventing the angiogenic pathway occurring. As a result, the primary pathway of angiogenesis does not occur. In recent years this has led to debate over the nature of sFlt-1 in the VEGF system. However, the level of sFlt-1 found in cardiovascular disease (CVD) patients, as well as its stability in plasma, has allowed for current research into its involvement with ischemic disorders to take place. Enhanced T-cell activity that results in increased production of interferon-γ has been shown to have involvement in the pathogenesis of CVD. 7,8-dihydroneopterin (7,8 NP) production by monocytes and macrophages is primarily in response to stimulation by interferon-γ (IFN-γ) released by activated T-lymphocytes. When combined with neopterin, the oxidised product of 7,8 NP, the total neopterin is accounted for which is a measure of the total macrophage activation by interferon-γ. Therefore, the levels of total neopterin observed may reflect the level of cell-mediated immunity within individuals which could contribute to mortality post CVD event. Progression of coronary heart disease is often clinically silent, without signs or symptoms. For this reason, the ability of markers to monitor progression is a powerful tool for predicting cardiovascular risk and the level of preventative treatment required. This study shows, that in 514 stable post-ACS (MI or unstable angina) patients, above median baseline sFlt-1, total neopterin and 7,8 NP levels, were strong predictors of mortality over a median 5 year period. Furthermore, above median sFlt-1 levels were specifically predictive of CVD death (p=0.001). This suggests that sFlt-1, total neopterin and 7,8 NP may be useful markers for risk prediction in CVD patients, post-acute event, with potential to aid prognosis in previously diagnosed patients. In support of these findings, levels of sFlt-1 measured in plasma taken from patients, immediately prior to undergoing carotid endarterectomy procedures (n=27), were significantly raised in comparison to age and gender matched healthy controls (p<0.001). Furthermore, levels of sFlt-1 in patient and control groups were shown to be independent of both age and gender. Another aspect of the study, analysis of excised live plaque tissue from carotid endarterectomy patients, showed the presence of live inflammatory cell populations. Macrophages, in the plaque sections, could be stimulated in the presence of IFN-γ to produce significantly elevated (p<0.01) levels of the antioxidant 7,8 NP. Since bivariate analysis of 7,8 NP and sFlt-1, in plasma from the endarterectomy patients, yields a positive correlation (r=0.323, p<0.01), further analysis of live plaque may give insight into the association between inflammation and hypoxic up-regulation of sFlt-1. It is now generally accepted, in diseases with complex pathogenesis, that particular biomarkers are predominantly indicative of only a single variable in a wide range of contributing factors. The data generated in this study highlights the potential for sFlt-1, neopterin and 7,8 NP to be used as contributing biomarkers in the prognosis of patients suffering from CVD, which if confirmed, may have important clinical implications in the medical community.

Thesis (M. Sc.)--University of Canterbury, 2008. / Typescript (photocopy). Includes bibliographical references (leaves 74-83). Also available via the World Wide Web.

Thesis (M. Phil.)--University of Hong Kong, 2005. / Title proper from title frame. Also available in printed format.

Five atherosclerotic carotid and femoral plaques were sliced longitudinally. Each section was analysed for the concentrations of neopterin, α-tocopherol, TBARS, DOPA, dityrosine, protein carbonyl, protein and cholesterol. The spatial concentrations of the oxidative and inflammatory markers were diverse across and between the individual plaques suggested by the lack of consistent correlations and trends. The only correlation that occurred twice within the individual plaques was a positive correlation between α-tocopherol and cholesterol levels. In the combined plaque analysis which included data from eight previously studied plaques, neopterin, protein carbonyl and protein concentrations all had significant positive correlations and α-tocopherol concentrations positively correlated to cholesterol and negatively to TBARS. Thus overall the level of protein may influence protein carbonyl concentration and α-tocopherol may provide an antioxidant effect towards lipid peroxidation. Furthermore, the plaques were divided into three zones, pre-bifurcation, bifurcation and post-bifurcation, associated with shear stress levels. The neopterin concentrations were significantly high within the pre- and post-bifurcation region and the opposite trend occurred with the to peroxyl radical driven TBARS levels. The protein and cholesterol content in the postbifurcation was high, possibly due to the low and/or oscillatory shear stress occurring at these sites. The overall composition of the plaque, either thrombosed, heavily calcified or neither, also identified significant trends in marker concentrations between the plaques. The calcified plaques had significantly low levels of protein, cholesterol, α-tocopherol, DOPA and dityrosine whereas the thrombosed plaques had significantly high protein, α-tocopherol and dityrosine concentrations. The medication and symptoms presented by the patient had no major influence of the overall concentration of the markers within the plaques. Therefore even though individually the plaques have varied biochemical compositions, common influences were dictate the spatial and overall concentration of the markers within and across the plaques. Further potential markers were investigated for detection within plaque. AAS and GGS for replacement of the protein carbonyl assay as a more specific marker for protein oxidation, as well as the oxysterol 7-ketocholesterol detected simultaneously during α-tocopherol analysis. The 7-ketocholesterol would increase the information on lipid oxidation occurring in the plaque without increasing the volume of the limited homogenate required for the analysis. Investigation was also carried out on the mechanism of protein oxidation in human plasma that may provide mechanisms and interactions to protein oxidation within plaques.

Verfasser: Gerald Stumpf Titel: Feldstudie zu fieberhaften Erkrankungen des Pferdes unter besonderer Berücksichtigung der Ehrlichiose und freien Endotoxins Institut: Institut für Bakteriologie und Mykologie der Veterinarmedizinischen Fakultät der Universität Leipzig Eingereicht im Dezember 2005 Bibliographische Daten: 92 Seiten, 13 Abbildungen, 15 Tabellen, 134 Quellenangaben Schlüsselworte: Pferd – Fieber – Ehrlichiose – Endotoxin – CRP – Neopterin – PCR Die vorliegende Arbeit beschäftigt sich mit den in der ambulanten Praxis vorkommenden fieberhaften Erkrankungen des Pferdes. Ein Ziel bestand in der Analyse und Verifizierung der klinischen Beobachtungen in der Praxis und der routinemäßigen Laborparameter. Durch die Verwendung zusätzlicher Parameter wurde deren diagnostische Bedeutung am erkrankten Pferd untersucht, um so den diagnostischen Rahmen bei der ätiopathogenetischen Aufklärung des Fiebers unbekannter Genese zu erweitern. Anhand eigener Untersuchungen von Erkrankungen mit Fieber unbekannter Genese im Vergleich zu anderen fieberhaften Krankheiten des Pferdes wurde die Bedeutung des Fiebers unbekannter Genese beim Pferd herausgestellt und das Krankheitsgeschehen charakterisiert. In die Laboruntersuchungen wurden die Entzündungsparameter Neopterin und CRP zusätzlich einbezogen. Mittels Antikörpernachweisen wurde die Beteiligung von Endotoxinen an der Entstehung von Fieber unbekannter Genese untersucht. Außerdem wurde die PCR zum Nachweis von Ehrlichiose bei spontan fieberhaft erkrankten Pferden eingesetzt. Bei 195 (50,9%) von insgesamt 383 Patienten wurde Fieber unbekannter Genese (Fiebertyp 1) registriert, im Jahresdurchschnitt waren es 65 (56 - 72) Pferde (Tab. 2). Dieser Fiebertyp hatte demnach die größte Bedeutung (p0,05). Es tritt in den Herbst und Wintermonaten am häufigsten auf. Rasse- und Altersprädispositionen ist nicht feststellbar. Es handelt sich um ein mit Fieber einhergehendes Krankheitsgeschehen mit geringer Kontagiosität. Der CRP-Wert liegt bei den Patienten mit Fieber unbekannter Genese im Normbereich. Das bedeutet, in Übereinstimmung mit OKIN et al. (2005), AGRAWAL (2005) und DE MAAT (2004), dass es nicht zu den entsprechenden mit Gewebszerfall einhergehenden Entzündungsreaktionen gekommen war. Das Neopterin wurde hier erstmals bei Pferden mit Fieber unbekannter Genese. Dieser Wert war erhöht (x=7,29). Es ist ein unspezifischer Parameter für den negative Korrelation (r=0,33, p0,05) mit Anti-Endotoxin-IgG festgestellt wurde. Die Erhöhung der Immunglobuline der Klasse IgG (Antiendotoxin-IgM: x=1,18 und Anti-Lipid-A-IgM: x=1,5) weist auf eine Beteiligung freier Endotoxine an der Entstehung von Fieber unbekannter Genese beim Pferd hin. Das Vorkommen von Anaplasma phagocytophilum (Ehrlichia equi) wurde erstmals in Deutschland beim Pferd molekulargenetisch nachgewiesen. Obwohl die ätiologische Diagnosestellung in dieser Studie nur begrenzt erweitert werden konnte, ergeben sich nach der hier vorgenommenen Charakterisierung des Fiebers unbekannter Genese beim Pferd für die Zukunft gute Ansatzpunkte für den gezielten Einsatz der PCR mit selektiv ausgewählten Primern, um den diagnostischen Rahmen zu erweitern. / Author: Gerald Stumpf Titel: Fieldstudy of febrile diseases in the horse in particular condition of ehrlichiosis and free endotxin Institute: Institute of Bacteriology and Mycology, Faculty of Veterinary Medicine, University of Leipzig Submitted in December 2005 Bibliographic data: 92 pages, 13 figures, 15 tables, 134 references Key words: Horse – Fever – Ehrlichiosis – Endotoxin – CRP – Neopterin - PCR The importance and prevalence of fever of unknown origin in horses was investigated, characterised and compared with other febrile conditions in horses. Inflammatory and stress parameters such as neopterin and CRP were included in the investigated laboratory profile. The level of IgG was investigated by an immunological approach for checking the involvement of endotoxins in the genesis of fever. PCR was used for the detection of ehrlichiosis in horses with spontaneous fever. In total, 383 horses with febrile conditions were examined within a period of 3 years. In 195 horses (50.9%) fever of unknown origin was diagnosed (fever type 1) indicating that this is the most important fever type in horses (p0.05) mainly seen in autumn and winter. In contrast, an age or race predispostion could not be detected. Typically, this fever type is characterised by a low contagiosity. The horses with fever of unknown oigin showed CRP values within the physiological range. This is indicative for the lack of tissue damage and respective inflammatory processes and supports the findings of OKIN et al. (2005), AGRAWAL (2005) und DE MAAT (2004). To the knowledge of the author, this is the first report on investigations of neopterin in horses with fever of unknown origin. Neopterin is an unspecific parameter and showed a negative correlation (r=0.33, p0.05) with anti-endotoxin IgG. The mean neopterin value in horses with fever of unknown oigin was increased (7.29). The observed increase of anti-endotoxin-IgM and anti-Lipid A-IgM indicates the involvement of endotoxins in the genesis of fever of unknown origin in horses. Moreover, this is the first report about the molecular genetic detection of Anaplasma phagocytophilum (Ehrlichia equi) in horses in Germany. In conclusion, the characterisation of fever of unknown origin in this thesis may stipulate further targeted investigations using PCR with selective primers to improve the etiological diagnosis of this disease.

Thesis (Ph. D.)--University of Canterbury, 2008. / Typescript (photocopy). Includes bibliographical references (p. 211-250). Also available via the World Wide Web.