published_or_final_version / abstract / Microbiology / Master / Master of Philosophy

Atherosclerosis is a chronic inflammatory disease leading to plaque buildup in the major arteries. The plaques consist of cholesterol, calcium, inflammatory cells, extracellular matrix and fibrous material. Under inflammatory conditions IFN-• stimulation of human monocytes and macrophages generates reduced pteridine, 7,8-dihydroneopterin (78NP) which has been shown to be an effective cytoprotective agent to some cell types against oxidative damage by reactive oxygen species (ROS). 7,8-dihydroneopterin is oxidized to fluorescent neopterin in the presence of hypochlorite (HOCl). Although a considerable amount of work has been published on the composition of neopterin in atherosclerotic plaques, very little is known about the variation of 78NP and other oxidative biomarkers across the length of the carotid and femoral and their contribution to plaque progression, which was researched in this work. Atherosclerotic plaques excised from patients with carotid and femoral plaques were sliced into 3-5 mm sections, and each section was analyzed for concentrations of neopterin, 7,8- dihydroneopterin, •-tocopherol, TBARS, DOPA, cholesterol, dityrosine, protein carbonyls •- aminoadipic semialdehyde (AAS) and •-glutamic semialdehyde (GGS), free and esterified 7- ketocholesterol (7-KC). Cultured live plaque as a source of 7,8-dihydroneopterin and neopterin was also investigated in this study. It was shown that carotid plaques significantly vary from femoral plaques, in the levels and range of most oxidative biomarkers. Carotid plaques showed a high variation in the biomarker concentrations between plaques but also between sections of an individual plaque. Femoral plaques on the other hand showed lower amounts of biomarkers with very little variation in biomarker concentrations. High variation with pterin concentrations and other biomarkers suggests dynamic and active changes in inflammation within the plaque. Collectively, it was observed that every plaque was unique with respect to its composition and correlations between the biomarkers. Though shown to be a well-known antioxidant and a radical scavenger, there is no published literature on 7,8-dihydroneopterin’s mode of entry into and out of the cell. To understand how it enters the cells could explain the difference in its protective ability of different cell types Abstract xxviii against oxidative stress-mediated cell death. Knowledge of transport of 7,8-dihydroneopterin will provide insights about its protection of monocyte/macrophage cell death which could potentially reduce atherosclerotic plaque growth and progression. As 7,8-dihydroneopterin is produced from guanosine, a nucleoside that is transported using specialized nucleoside transporters (equilibrative nucleoside transporters (ENT's) and concentrative nucleoside transporters (CNT's), their role was examined and characterized for 7,8-dihydroneopterin transport. It was found that 7,8-dihydroneopterin and neopterin are transported via nucleoside transporters in U937 cells, THP-1 cells and human monocytes. ENT 2 was the major transporter in U937 cells while ENT 1 transported bulk of 7,8-dihydroneopterin in THP-1 cells. Both ENT's and CNT's are involved in 7,8-dihydroneopterin uptake in human monocytes. In all the cell lines tested, 7,8-dihydroneopterin protection against AAPH mediated oxidative cell death was inhibited by nucleoside transport inhibitors, suggesting that nucleoside transporters are indispensible for 7,8-dihydroneopterin mediated intracellular protection against oxidative stress. Accurate measurement of neopterin, as a biomarker of inflammation in plaques and cells is critical aspect to assess disease progression. The current C18 HPLC method used in our laboratory for neopterin measurement lacks sensitivity due to interference of acetonitrile (ACN) over time. Acidic tri-iodide conversion of 7,8-dihydroneopterin to neopterin was also variable at times giving inconsistent measurement of neopterin so the manganese oxide (MnO2) method was looked at as an alternative. Electrochemical detector (ECD) was another option studied as it did not require any precolumn oxidation of 7,8-dihydroneopterin to neopterin. A new method using strong cation exchange (SCX) column was developed for a precise, sensitive neopterin assay which got rid of the ACN interference completely. The MnO2 method of 7,8-dihydroneopterin oxidation did not work with biological samples such as serum or plaque homogenates. Electrochemical detection was also found to be very unreliable and inconsistent.

During the erythrocytic stage, the malaria parasite digests host cell haemoglobin into amino acids. Toxic haeme is released and is incorporated into an insoluble non-toxic crystal called haemozoin. Haemozoin is released into the blood stream along with the merozoites when the erythrocyte bursts and is phagocytosed by circulating monocytes and macrophages resident in tissues. Phagocytosed haemozoin impairs many functions of the monocytes, including antigen presentation and adhesion to T cells, differentiation and maturation to dendritic cells, erythropoiesis and thrombopoiesis, but stimulates the release of proinflammatory cytokines and activation of metalloproteinase 9 expression. In response to interferon-g secretion by T-helper cells subtype 1, monocytes secrete neopterin, which is used as a marker of a cell mediated immune response. Neopterin is an oxidation product of 7,8-dihydroneopterin, produced by the dephosphorylation of 7,8- dihydroneopterin triphosphate which results from the conversion of guanosine triphosphate that is catalysed by GTP-cyclohydrolase 1. Elevated plasma and urine neopterin levels have been detected in malaria infections and are associated with severe anaemia, respiratory distress, peak temperatures as well as fever- and parasite-clearance times. It has also been reported that monocytic U937 cells treated with P. falciparum-infected red blood cell lysate secrete elevated levels of neopterin. Antimalarial drugs are known to modulate the functions of monocytes, including inhibition of cytokine release, changes in phospholipid metabolism, decrease in expression of cytoadherance receptors as well as TNF receptors and MHC Class I and II molecules, changes in the production of reactive oxygen and nitrogen intermediates, and decreased phagocytosis. However, the effects of antimalarial drugs on haemozoin phagocytosis and GTP-cyclohydrolase 1 mRNA expression by monocytes are unknown. This study aimed to determine the effects of seven antimalarial drugs, amodiaquine, artemisinin, chloroquine, doxycycline, primaquine, pyrimethamine and quinine, on the phagocytosis of latex beads and b-haematin, a synthetic equivalent of haemozoin. Phagocytosis of b-haematin and latex beads by two monocytic cell lines, J774A.1 and U937, as well as peripheral blood mononuclear cells were monitored by enumeration and a novel spectrophotometric method. Patterns of inhibition and activation differed with each cell type investigated, due to the differing stages of cell differentiation. In general, artemisinin, primaquine, pyrimethamine and quinine activated the phagocytosis of b-haematin, whereas amodiaquine and chloroquine inhibited b-haematin phagocytosis. Doxycycline had different effects on each cell type investigated. Artemisinin, chloroquine, primaquine and quinine inhibited latex bead phagocytosis. The remaining drugs had minimal effects on latex bead phagocytosis. Thus, the effects of antimalarial drugs on monocyte phagocytosis appear to be dependent on the substance being phagocytosed. The effects of antimalarial drugs, b-haematin, latex beads, non-infected- and P. falciparuminfected cell lysates on interferon-g-induced neopterin secretion by U937 cells was monitored by GTP-cyclohydrolase 1 mRNA expression using quantitative PCR. Artemisinin, primaquine and quinine down-regulated the interferon-g-induced expression of GTPcyclohydrolase 1 mRNA, but by no greater than 1.7-fold. b-haematin up-regulated mRNA expression by 1.2-fold whereas P. falciparum-infected red blood cell lysate down-regulated the mRNA expression of GTP-cyclohydrolase 1 by 1.6-fold. Quinine and artemisinin, currently used to treat malaria, increased b-haematin phagocytosis suggesting that quinine and artemisinin might promote increased phagocytosis of infected red blood cells and enhance clearance of the parasite from circulation. Increased b- haematin phagocytosis also reduces ICAM-1 expression on the monocyte surface, thereby leading to reduced cytoadherance and sequestration, thus increasing the number of circulating monocytes to phagocytose infected red blood cells. Down regulation of GTPcyclohydrolase 1 mRNA expression by quinine and artemisinin suggested that the drugs reduce the responsiveness of the monocyte to interferon-g. Thus, quinine and artemisinin might also decrease the production of interferon-g-induced proinflammatory cytokines by monocytes, and potentially play a role in maintaining the balance between the pro- and antiinflammatory cytokines that determines the progression from acute to severe malaria. Therefore, in addition to the drug’s ability to kill the malaria parasite, the immunomodulatory effects of the antimalarial drugs may play a role in controlling the pathophysiology associated with the malaria infection. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.

Tez (Tıpta Uzmanlık) - Süleyman Demirel Üniversitesi, Tıp Fakültesi, Dermatoloji Anabilim Dalı, 2005. / Bibliyografya var.

Tez (Tıpta Uzmanlık) - Süleyman Demirel Üniversitesi, Tıp Fakültesi, Fiziksel Tıp ve Rehabilitasyon Anabilim Dalı, 2006. / Bibliyografya var.

Tez (Tıpta Uzmanlık) - Süleyman Demirel Üniversitesi, Tıp Fakültesi, Göğüs Hastalıkları Anabilim Dalı, 2006. / Bibliyografya var.

Tez (Tıpta Uzmanlık) - Süleyman Demirel Üniversitesi, Tıp Fakültesi, Çocuk Sağlığı ve Hastalıkları Anabilim Dalı, 2003. / Bibliyografya var.

Thesis (Ph. D.)--University of Canterbury, 2006. / Typescript (photocopy). Includes bibliographical references (leaves 233-271). Also available via the World Wide Web.

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.

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