Lipopolysaccharide or LPS is localized to the outer leaflet of the outer membrane and serves as the major surface component of the bacterial cell envelope. This remarkable glycolipid is essential for virtually all Gram-negative organisms and represents one of the conserved microbial structures responsible for activation of the innate immune system. For these reasons, the structure, function, and biosynthesis of LPS has been an area of intense research. The LPS of a number of bacteria is composed of three distinct regions - lipid A, a short core oligosaccharide, and the O-antigen polysaccharide. The lipid A domain, also known as endotoxin, anchors the molecule in the outer membrane and is the bioactive component recognized by TLR4 during human infection. Overall, the biochemical synthesis of lipid A is a highly conserved process; however, investigation of the lipid A structures of various organisms shows an impressive amount of diversity. These differences can be attributed to the action of latent enzymes that modify the canonical lipid A molecule. Variation of the lipid A domain of LPS serves as one strategy utilized by Gram-negative bacteria to promote survival by providing resistance to components of the innate immune system and helping to evade recognition by TLR4. This review summarizes the biochemical machinery required for the production of diverse lipid A structures of human pathogens and how structural modification of endotoxin impacts pathogenesis.

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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.

Mit vorliegender Arbeit wurde geprüft, ob an Dislocatio abomasi (DA) erkrankte Rinder vor dem Auftreten klinischer Symptome Veränderungen im Fettstoffwechsel aufweisen und ob Beziehungen zum Endotoxin-Metabolismus bestehen. Dazu wurde ein breites Untersuchungsspektrum zum Fett- und Leberstoffwechsel [b-Hydroxy-Butyrat (BHB), Freie Fettsäuren (FFS), Cholesterol, Tria-cylglycerol (TG), Phospholipide (PL), a-, b-, prä-b-Lipoproteine (LP), Bilirubin, Aspartat-Amino-Transferase (ASAT), Gamma-Glutamyl-Transferase (GGT), Glutamat-Dehydrogenase (GLDH), Lactat-Dehy-drogenase (LDH), Glucose], freies Endotoxin, Endotoxineffektoren [Anti-Lipid A-Antikörper (IgG), C-reaktives Protein (CRP), Leukozytenzahl, Gesamteiweiß, Albumin, Eisen (Fe)] sowie Nat-rium (Na), Kalium (K), Calcium (Ca), Chlorid (Cl), Magnesium (Mg), anorganisches Phosphat, Harnstoff, Creatinin und die Creatinkinase im Blut untersucht. / The displacement of abomasum (DA) frequently occurs in high yielding dairy cows. There is a lack of knowledge of its etiology. This paper examines whether dairy cows with DA show changes in the fat metabolism already in the initial stage, i.e. before clinical symptoms occur. The paper also analyses whether a relationship exists between endotoxin and the fat metabolism. Therefore a large variety of parameters were examined in blood: parameters of the fat and liver metabolism (betahy-droxybutyrat, free fatty acids, cholesterol, TG, phospholipids, alpha-, beta- and pre-beta-lipoproteins, bilirubin, ASAT, GGT, GLDH, LDH, glucose), free endotoxin, anti-lipid-A-antibodies (IgG), C-reactive protein, number of leukocytes, total protein, albumin, Fe as well as Na, K, Ca, Cl, Mg, anorganic phosphate, urine, creatinin and creatinkinase

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Modification of the lipid A domain of lipopolysaccharide (LPS) is important for the pathogenesis and virulence of various Gram-negative bacteria. The major lipid A species of Helicobacter pylori is significantly different from that of Escherichia coli. H. pylori lipid A contains fewer acyl chains and phosphate groups with only one Kdo sugar attached to the disaccharide backbone. However, H. pylori produces a minor lipid A species that resembles E. coli lipid A, suggesting that the major lipid A species results from the action of specific modifying enzymes. This work describes two enzymes, a lipid A phosphatase and a phosphoethanolamine (pEtN) transferase, involved in modifying the 1-position of H. pylori lipid A. H. pylori lipid A contains a pEtN unit directly linked to the 1-position of the disaccharide backbone. This is in contrast to the pEtN units found in other pathogens, which are attached to the lipid A phosphate group to form a pyrophosphate linkage. Using in-vitro assay systems, we demonstrate that the modification of the 1-position of H. pylori lipid A is a two-step process involving the removal of the 1-phosphate group by LpxEHP followed by the addition of a pEtN residue catalyzed by EptAHP. As compared to wild-type H. pylori, lpxEHP mutants are extremely sensitive to the cationic peptide polymyxin, thus, demonstrating the importance of modifying the 1-position of lipid A. Furthermore, this work describes another enzyme, YeiU (renamed LpxT), which specifically utilizes the carrier lipid undecaprenyl pyrophsphate (C55-PP) to modify the 1-position of E. coli lipid A. Typically, E. coli lipid A is a hexa-acylated disaccharide of glucosamine in which monophosphate groups are attached at positions 1 and 4'; however, a small fraction contains a diphosphate moiety at the 1-position (lipid A 1-diphosphate). 32P-labeled lipid A obtained from lpxT deficient mutants produces only lipid A, and complementation with a plasmid expressing LpxT restores lipid A 1-diphosphate formation. Inhibition of lipid A 1-diphosphate synthesis was demonstrated by sequestering C55-PP with the cyclic polypeptide antibiotic bacitracin. In conclusion, this work describes two novel pathways for lipid A modification at the 1-position in Gram-negative bacteria.

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Flavanols are polyphenols that are known to have many beneficial effects on the body. Cocoa is a major source of these flavanols. However, research on the potential anti-obesity and anti-diabetic health benefits of cocoa flavanols is lacking in the literature. Furthermore, the effect that the size of these flavanols has on the extent of its beneficial properties is also unclear. The objective of this study was to evaluate the dietary effects of cocoa flavanols on the onset of obesity, insulin resistance and impaired glucose tolerance and to determine the impact that the size of these compounds has on the magnitude of this effect. Cocoa extract was fractionated into a monomer-, an oligomer-, and a polymer-rich fraction. Six groups (n=9) of C57BL/6J mice were fed either a control low-fat diet, a control high-fat diet, or a high-fat diet supplemented with 25 mg/kg*BW of cocoa extract or one of the three cocoa fractions. After 12 weeks on these diets, the oligomer-rich fraction proved to be most effective in preventing weight gain, fat mass accumulation, elevated fasting blood glucose and impaired glucose tolerance in diet-induced obese mice. This is the first long-term feeding study to examine the relative activities of cocoa constituents on diet-induced obesity and insulin resistance. / Master of Science in Life Sciences

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