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Diese Arbeit handelt von den multifunktionalen und immunmodulatorischen Biomolekülen Lactoferrin und dem Polysialinsäuren (PolySia), die beide Einfluss auf die Bildung und Aktivität von neutrophilen extrazellulären Fallen (engl. Abk. NET) haben. In vorangegangenen Arbeiten konnte gezeigt werden, dass nach Affinitätspräzipitation gegen PolySia auch Lactoferrin aus Körperflüssigkeiten wie Seminalplasma, Blut und Milch isoliert wird, obwohl Lactoferrin selbst keine PolySia-Ketten trägt. Dies ließ vermuten, dass eine Interaktion zwischen der linearen Zuckerkette PolySia und Lactoferrin stattfindet. Aus diesem Grund standen sowohl die Charakterisierung einer möglichen Interaktion zwischen Lactoferrin und PolySia sowie der Einfluss solch einer Interaktion auf deren biologische Aktivität im Fokus. Die Analysen zeigten, dass PolySia in Abhängigkeit der Kettenlänge über die N-terminale Region von Lactoferrin gebunden wird. Innerhalb der N-terminalen Region von Lactoferrin ist eine kleine basische Domäne namens Lactoferricin lokalisiert, die proteolytisch freigesetzt werden kann und antimikrobielle Eigenschaften besitzt. Unsere Analysen zeigten, dass freies Lactoferricin ebenfalls mit PolySia in Abhängigkeit der Kettenlänge interagiert und so wurde die Wirkung des antimikrobiellen Peptides Lactoferricin auf E. coli untersucht, während es gleichzeitig in Interaktion mit PolySia treten konnte. Es stellte sich heraus, dass PolySia egal mit welchem Polymerisationsgrad, keinen negativen Einfluss auf die antimikrobielle Wirkung von Lactoferricin hatte. Dadurch könnte sich diese Interaktion als ein interessantes, therapeutisches, antimikrobielles Pärchen darstellen. Darüber hinaus wurde der Einfluss von PolySia auf biologische Funktionen von Lactoferrin analysiert, wobei zwei Wirkungsweisen eines Zusammenspiels der beiden Biomoleküle aufgezeigt wurden. Einerseits verstärkt die Zugabe von PolySia die Fähigkeit von Lactoferrin, die Freisetzung von NET bei neutrophilen Granulozyten zu hindern. Dabei bildet es eine Art Schild um die Membranen der neutrophilen Granulozyten und hindert somit die Ruptur dieser und somit die Ausschüttung der NET-Fasern. Andererseits ist PolySia vermutlich in der Lage, den Austausch von Lactoferrin in bereits freigesetzte und mit Lactoferrin beladenen NET-Fasern durch externes Lactoferrin zu modulieren. Da die Ausschüttung von NET nicht nur positive Effekte wie die Bekämpfung von Pathogenen hat, sondern auch im Zusammenhang mit vielen Krankheiten steht, ist es von besonderer Bedeutung, die Regulations- und Modulationsmechanismen von NET aufzuschlüsseln. Diese könnten dann für zukünftige Therapieentwicklungen zur Verfügung stehen. Mit der Interaktion der natürlichen Biomoleküle PolySia und Lactoferrin bzw. Lactoferricin haben wir wahrscheinlich ein solches Paar gefunden.

Die vorliegende Arbeit beschäftigt sich mit der Untersuchung und Beschreibung der Eigenschaften der synthetischen Dekapeptide Cetrorelix und Ozarelix durch analytische Methoden und computergestützte Modellierung. Diese Moleküle sind hydrophobe, aggregierende Antagonisten des Gonadotropin-Releasing-Hormons (GnRH). Zusätzlich wurden amyloidbildende Peptidstrukturen als Modelle für die Assoziationsprozesse in hydrophoben Peptiden untersucht und visualisiert. Die intrinsische Fluoreszenz der GnRH-Antagonisten und zusätzlich der Peptide Teverelix und D-Phe6-GnRH sowie von verkürzten Fragmenten des Cetrorelix wurde untersucht. Ein Strukturmodell für die Beschreibung der Aggregation der Dekapeptide wurde erarbeitet. Der Aufbau eines Rechenclusters durch das Einbinden der Computer am Lehrstuhl in ein Linux-System zur Verteilung von Rechenprozessen über das Netzwerk ermöglichte die Bereitstellung der notwendigen Leistung zur Realisierung der Berechnungen. Es wurden Werkzeuge zur Modellierung der solvatisierten Aggregate von Peptiden ohne eindeutige Vorzugsstruktur programmiert und in ein Docking-System für beliebige Moleküle eingebunden. Verwendet wurde das Kraftfeld MMFF94 mit einer Erweiterung durch ein Verfahren zur dynamischen Berechnung von Partialladungen in Molekülstrukturen. Solvatisierte Aggregate der Dekapeptide und von bekannten amyloidbildenden Strukturen wurden modelliert (Docking). Berechnet wurden als aggregierend beschriebene Sequenzen und entsprechende Vergleichsstrukturen des Calcitonins, des Insel-Amyloid-Polypeptides, des beta2-Mikroglobulins, des Amyloid-beta-Proteins, des Lactoferrins und weitere Modellpeptide. Die wesentlichen Wechselwirkungen während der Aggregation konnten schließlich anhand von Dynamik-Simulationen der faltblattartigen Dimere des Cetrorelix und Ozarelix beschrieben werden. So wurden die Prozesse der hydrophoben Assoziation und Stabilisierung durch Wasserstoffbrücken von Peptiden veranschaulicht und auf molekularer Ebene erfolgreich analysiert. Die Visualisierung der erhaltenen Modellierungsergebnisse erfolgt durch die Darstellung der Strukturen und Dynamik-Simulationen als interaktive 3D-Modelle in einem für diese Arbeit aufgebauten Internetauftritt. This work discusses the analysis of the aggregation properties of the gonadotropin releasing hormone antagonists Cetrorelix, Teverelix, Ozarelix and of small amyloid forming model peptides by analytical fluorescence spectroscopy and molecular modelling. A high performance linux compute cluster was developed for calculation of molecular structures. Solvated aggregate clusters of peptides without defined secondary structure were modelled by molecular mechanics methods (forcefield mmff94) in combination with an advanced charge equilibration and docking technique. Molecular dynamics of solvated peptide dimers were implemented and the role of hydrophic association and hydrogen bond formation in hydrophobic peptide aggregates was explained. Finally, an aggregation model for the directed association of hydrophobic peptides is presented. The modelling results, 3d structures and dynamic simulations are visualized in an interactive web material.

Die Gentherapie stellt einen neuartigen und viel versprechenden Ansatz für die zukünftige Behandlung angeborener oder erworbener Krankheiten dar, bei denen herkömmliche Therapieformen bislang nur geringe Effizienz zeigen. Hierbei ist die Lunge im Hinblick auf viele akute und chronische Erkrankungen wie beispielsweise Zystischer Fibrose, Asthma oder Tumoren ein wichtiges Zielorgan. Eine therapeutische Anwendung der Gentherapie setzt den erfolgreichen Transfer der applizierten Nukleinsäure in die Zielzelle voraus. Transkription eines Gens kann nur dann erfolgen, wenn die DNA den Zellkern erreicht. Die momentan größte Herausforderung besteht somit in der technologischen Entwicklung geeigneter Gentransfervektoren. Derzeitige Gentransfersysteme lassen sich in virale und nichtvirale Methoden einteilen. Obwohl Viren bislang deutlich höhere Transfektionseffizienzen zeigen konnten, bergen sie aufgrund immunogener und mutagener Eigenschaften unkalkulierbare Risiken. Dies führte zur Entwicklung nichtviraler Systeme, welche eine weit sicherere Alternative darstellen. Innerhalb der kationischen Polymere stellt Polyethylenimin (PEI) ein effizientes nichtvirales Gentransferagens dar, welches bereits Gegenstand vieler Untersuchungen war. Nichtsdestotrotz konnte im Vergleich zu viralen Systemen noch keine ausreichend hohe Transfektionseffizienz, insbesondere bei in vivo Applikationen, erreicht werden. Zusätzlich besitzt PEI eine hohe Zytotoxizität. Die Modifikation kationischer Polymere mit zielgerichteten Liganden scheint vor diesem Hintergrund eine viel versprechende Methode zur Schaffung intelligenterer Systeme darzustellen. Dies ist insbesondere im Hinblick auf eine höhere Genexpression und Selektivität erstrebenswert. Verschiedenste Liganden wie zum Beispiel Transferrin, Folsäure oder EGF wurden bereits erfolgreich für den Rezeptor-vermittelten nichtviralen Gentransfer beschrieben. Ziel dieser Arbeit war die Herstellung und Charakterisierung neuartiger zielgerichteter Gentransfersysteme auf der Basis von verzweigtem PEI. Verwendete Liganden waren dabei das Glykoprotein Lactoferrin, der Arzneistoff Clenbuterol und das Peptidhormon Insulin. Im Hinblick auf die spätere pulmonale in vivo Anwendung erfolgten die in vitro Untersuchungen bevorzugt mit Lungenzellen. Dabei wurden insbesondere Alveolarepithel- und Bronchialepithelzellen verwendet. Um das Potential eines Liganden abschätzen zu können, wurden zu Beginn jeder Studie in vitro Rezeptoruntersuchungen durchgeführt. Diese erfolgten durch Inkubation hergestellter Fluoreszenzfarbstoff-Ligand-Konjugate oder mittels Antikörper- basierter Methoden. Hierbei konnte beobachtet werden, dass Lactoferrin- Rezeptoren bevorzugt auf Bronchialepithelzellen exprimiert werden. Im Gegensatz dazu wurde eine hohe Anzahl von Rezeptoren für Clenbuterol und Insulin insbesondere auf Alveolarepithelzellen gefunden. Die Einbringung der Liganden in die Gentransferkomplexe erfolgte auf unterschiedlichen Wegen. Lactoferrin und Clenbuterol wurden jeweils durch biochemische Reaktionen kovalent an das PEI-Grundgerüst gekoppelt. Somit waren diese schon während der Komplexbildung mit Plasmid DNA anwesend. Im Gegensatz dazu wurde Insulin durch nichtkonvalente Adsorption aufgrund elektrostatischer Wechselwirkungen an die Oberfläche bereits komplexierter PEI/pDNA-Partikel gebunden. Für alle Komplexe wurden verschiedene molare Verhältnisse zwischen Polymer und Ligand hergestellt und untersucht. Bei der biophysikalischen Charakterisierung der Partikel konnte beobachtet werden, dass die Anwesenheit hoher Anteile der Liganden zu Komplexen mit einem größeren hydrodynamischen Durchmesser führte. Dies zeigte sich unabhängig von der Tatsache, auf welche Weise die Einführung des Liganden stattfand. Die Größenzunahme dürfte jedoch in unterschiedlichen Ursachen begründet sein. Während hohe Anteile kovalent an PEI gebundener Liganden (Lactoferrin bzw. Clenbuterol) wahrscheinlich die Komplexierung mit Plasmid-DNA stören, basiert ein Anstieg der Komplexgröße bei elektrostatischer Adsorption auf der Partikeloberfläche vermutlich auf einer Zunahme der Schichtdicke gebundenen Insulins. Diese Vermutungen wurden durch Messungen der Oberflächenladung der Polyplexe bestätigt. Die Adsorption von Insulin an der Oberfläche von PEI/pDNA-Komplexen führte zu einem drastischen Abfall des Zeta- Potentials bis hin zu negativen Werten, bei denen Aggregation der Partikel beobachtet werden konnte. Auch die Clenbuterol-Konjugation führte zu einer Reduzierung des Zeta-Potentials, welche jedoch deutlich schwächer ausgeprägt war. Im Gegensatz dazu resultierte die Kopplung von Lactoferrin diesbezüglich zu keiner relevanten Änderung. Gleichzeitig konnte mittels gelelektrophoretischer Untersuchungen jedoch gezeigt werden, dass in allen Fällen die Plasmid-DNA vollständig durch die Polymere komplexiert werden konnte. Bei in vitro Transfektionsstudien konnte für Lactoferrin-modifizierte Konjugate auf Bronchialepithelzellen ein Maximum der Genexpression bei einem Konjugationsverhältnis von 1 zu 20 zwischen Lactoferrin und PEI ermittelt werden. Dieses zeigte bei einem N/P-Verhältnis von 4 im Vergleich zu unmodifizierten PEI/pDNA-Partikeln eine signifikante 5-fache Steigerung der Transfektionseffizienz. Die Spezifität der Zunahme konnte durch Inhibierungsuntersuchungen mit einem Überschuss an Lactoferrin gezeigt werden. Auf Alveolarepithelzellen hingegen führte die Modifikation mit Lactoferrin zu keiner signifikanten Steigerung. In vivo Versuche zur Lunge wurden mittels Instillation und Vernebelung als Applikationsarten durchgeführt. Es konnte jedoch kein Vorteil der Genexpression durch die Lactoferrin-Modifizierung von PEI/pDNA-Komplexen beobachtet werden. Hier bleibt zu klären, inwiefern Lactoferrin in vivo strukturell erhalten bleibt und ob die verwendete Lactoferrin-Spezies eine Rolle spielt. Verschiedene Anteile an Clenbuterol wurden in die Transfektionskomplexe durch Herstellung von Mischungen aus PEI-g-Clen mit unmodifiziertem PEI eingeführt. Die höchste in vitro Gentransfereffizienz zeigte eine molare Zusammensetzung von 1,3 Clenbuterol- Liganden pro Molekül Polyethylenimin, welche bei einem N/P-Verhältnis von 8 im Vergleich zu PEI/pDNA-Polyplexen in einem signifikanten 14-fachen Anstieg der Luziferaseexpression resultierte. Dieser Vorteil war über einen weiten N/P-Bereich von 6-20 erkennbar. Der Abfall der Genexpression durch Zugabe eines Überschusses an freiem Clenbuterol ließ auf einen Rezeptor-vermittelten Mechanismus schließen. Die Clenbuterol-Modifikation der Gentransferpartikel führte auf zwei weiteren Zelllinien (murines Alveolarepithel, humanes Zervixkarzinom) ebenfalls zu einer signifikanten Steigerung der Transfektionseffizienz, wohingegen auf Bronchialepithelzellen kein Effekt festgestellt werden konnte. Diese Ergebnisse korrelierten mit der beta2-Rezeptorendichte auf den jeweiligen Zelllinien. In vivo Untersuchungen mit Clenbuterol als Ligand wurden auf drei verschiedenen Applikationswegen durchgeführt. Während bei der Instillation kein Unterschied zwischen den Gruppen erkennbar war, führte die Modifikation von PEI/pDNA-Partikeln mit Clenbuterol nach Vernebelung zu einem 1,6-fachen und nach intravenöser Applikation zu einem signifikanten 2,7-fachen Anstieg der Genexpression innerhalb der Lunge. Quantitative Bestimmungen im Lungengewebe mittels Real Time PCR zeigten, dass in diesen Fällen Clenbuterol-modifizierte Konjugate in der Lage waren, mehr Plasmid-DNA in das Zielgewebe zu transportieren. Bei der Auswertung des Verhältnisses von Genexpression und Plasmid-Menge innerhalb des Lungenhomogenisates konnte festgestellt werden, dass die Vernebelung die mit Abstand höchste Effektivität besitzt. Diese zeigte, bezogen auf die deponierte Menge an Plasmid-DNA, gegenüber der intravenösen Applikation eine etwa 110-fache und gegenüber der Instillation eine etwa 350-fache Steigerung der Luziferaseexpression. Unabhängig davon konnte mittels eines in vitro Versuches gezeigt werden, dass die Vernebelungsprozedur zu keiner Zerstörung komplexierter Plasmid-DNA führte, wohingegen unkomplexierte Plasmid-DNA den Scherkräften der Vernebelungskammer ausgesetzt war und dabei Schaden nahm. Die Oberflächenmodikation mit Insulin führte bei PEI/pDNA-Komplexen zu einer Steigerung der in vitro Transfektionseffizienz auf Alveolarepithel-, jedoch nicht auf Bronchialepithelzellen. Ein Verhältnis von 5 µg Insulin pro µg Plasmid-DNA resultierte in einem Maximum mit einer im Vergleich zu unmodifizierten PEI/pDNA-Partikeln 16-fach höheren Luziferaseexpression. Untersuchungen mittels Koinkubation freien Insulins zeigten, dass die Zunahme nicht auf proliferative Effekte des Liganden zurückzuführen war. Des Weiteren konnte die Notwendigkeit der Anwesenheit von verzweigtem Polyethylenimin gezeigt werden, nachdem die Addition von Insulin bei Verwendung von linearem PEI zu keiner Erhöhung der Genexpression führte. Insgesamt spielt auch die Toxizität der Gentransferkomplexe eine wichtige Rolle im Hinblick auf die spätere klinische Applikation. PEI ist als toxisches Gentransferagens bekannt. Hohe Toxizität des Polymers wurde bereits mehrfach in vitro, wie auch in vivo berichtet. Es konnte für alle drei Liganden demonstriert werden, dass durch deren Einführung in PEI/pDNA-Komplexe die in vitro Toxizität, evaluiert durch die metabolische Zellaktivität, signifikant gesenkt werden konnte. Zusammenfassend konnte mit dieser Arbeit gezeigt werden, dass Lactoferrin, Clenbuterol und Insulin als zielgerichtete Liganden geeignet sind, in Verbindung mit verzweigtem Polyethylenimin intelligentere Systeme für den Gentransfer in die Lunge zu bilden. Diese zeichnen sich insbesondere durch ihre höhere Genexpression, durch selektive Unterscheidung zwischen Alveolar- und Bronchialepithel und durch geringere Toxizität aus., Gene therapy represents a novel and attractive approach for the future treatment of inherited or acquired diseases, where conventional clinical procedures have poor efficacy. The lungs are an important target organ for gene therapeutic intervention with respect to many acute and chronic diseases like cystic fibrosis, asthma and cancer. Therapeutic implementation of gene therapy requires the successful transfer of the applied nucleic acid into the target cell. Transgene expression results only when the DNA is transported into the cell nucleus. Presently, the greatest obstacle revolves around the engineering of appropriate gene delivery systems. Current gene delivery systems can be divided into viral and nonviral methods. Although viruses show much higher transfection efficiencies so far, their usage is limited due to safety concerns, such as immunogenicity and integration into the host genome. This has stimulated efforts into the development of nonviral systems, which represent a more safe alternative. Among cationic polymers, polyethylenimine (PEI) is an effective nonviral gene transfer agent and has already been subject of many investigations. Nevertheless, it shows relatively low transfection efficiency compared to viral systems, in particular for in vivo applications. In addition, PEI exhibits a high cytotoxicity. The modification of cationic polymers with targeting ligands seems to be a promising method to create more efficient systems, in particular with respect to both higher gene expression and selectivity. Numerous ligands have already been evaluated for nonviral receptor-mediated gene delivery, like transferrin, folic acid or EGF (see CHAPTER 1.5). The current work was aimed at the development and characterization of novel targeted gene transfer systems. Therefore, the glycoprotein lactoferrin, the chemical drug clenbuterol and the peptide hormone insulin were used to modify branched polyethylenimine. With regard to the pulmonary in vivo application, all in vitro investigations were preferentially performed on lung cells, in particular alveolar epithelial and bronchial epithelial cells. To evaluate the potential of a targeting ligand, initial in vitro receptor investigations were performed. For this purpose, cells were incubated with fluorescently labelled specific ligands or antibodies. It could be seen that lactoferrin receptors were predominantly expressed on bronchial epithelial cells, while in contrast, a high number of receptors for clenbuterol and insulin were found particularly on alveolar epithelial cells. Introduction of the ligands to the gene transfer complexes was performed in different ways. Lactoferrin as well as clenbuterol were conjugated through biochemical reactions to the PEI backbone. As a result of this, they were already present during complexation process with plasmid DNA. In contrast to that, insulin was bound to the surface of already complexed PEI/pDNA particles due to electrostatic adsorption. For all complexes, numerous molar ratios between polymer and ligand were evaluated. Biophysical characterization of the particles showed that the presence of high ratios of ligand to polymer resulted in complexes with larger hydrodynamic diameters, independently of the method used for introducing the targeting ligand. This increase of size could be due to different reasons. Whereas high ratios of ligands covalently attached to PEI (lactoferrin or clenbuterol) may interfere with the complexation to plasmid DNA, the increase of complex size for electrostatic adsorption on the particle surface may probably be due to greater coating thickness of the bound insulin. These assumptions were further confirmed by surface charge measurements of the polyplexes. Adsorption of insulin to the surface of PEI/pDNA complexes led to a strong decrease of zeta potential to negative values, concomitant with particle aggregation. Conjugation of clenbuterol also resulted in reduction of zeta potential, but only with lower impact. In contrast, coupling of lactoferrin did not result in any relevant changes. At the same time, gel retardation assays showed that plasmid DNA was successfully complexed by all polymers, irrespective of the formulation. In vitro transfection studies with lactoferrin-modified conjugates on bronchial epithelial cells showed a maximum of gene expression for a lactoferrin to PEI ratio of 1:20. Using a N/P ratio of 4, a significant increase in transfection efficiency compared to unmodified PEI/pDNA particles was observed. Specificity could be demonstrated in inhibition experiments with an excess of free lactoferrin. In contrast, modification with lactoferrin had no effect on alveolar epithelial cells. In vivo experiments were performed in mice via nasal instillation and aerosol application. However, with respect to gene expression, no benefit could be demonstrated by the modification of PEI/pDNA complexes with lactoferrin. Further studies will be necessary to address the questions, if the structure of lactoferrin stays intact in vivo and if the used lactoferrin species also plays an important role. Different amounts of clenbuterol were introduced into transfection complexes by mixing PEI g Clen with unmodified PEI. Highest in vitro gene expression efficiency was obtained with a molar composition of 1.3 clenbuterol ligands per molecule of PEI, which showed a significant 14-fold increase in luciferase expression compared to PEI/pDNA polyplexes at a N/P-ratio of 8. This could be demonstrated over a wide range of N/P-ratios from 6 to 20. After addition of an excess of free clenbuterol gene expression decreased, which indicated a receptor-mediated mechanism. Significant benefit of clenbuterol-modification could also be shown on two additional cell lines (murine alveolar epithelial, human cervix carcinoma), whereas no effect was seen on bronchial epithelial cells. These results correlated well with the density of beta2-receptors on respective cell lines. In vivo experiments in mice with clenbuterol as a targeting ligand were performed via three different application routes. Whereas no difference in gene expression within the lungs of mice was seen after nasal instillation, modification of PEI/pDNA particles with clenbuterol resulted in a 1.6-fold increase after aerosol delivery and a significant 2.7-fold increase following intravenous injection of complexes. Quantitative Real Time PCR measurements of lung tissue showed that clenbuterol-modified conjugates were able to transport a higher amount of plasmid-DNA into the target tissue. Calculating the ratio of gene expression versus the amount of quantified plasmid-DNA, aerosol delivery was identified to have the highest efficiency. Related to the deposited amount of plasmid-DNA, aerosol delivery showed a 110-fold increase of luciferase expression compared to intravenous application and a 350-fold increase compared to instillation. In addition, it could be demonstrated that aerosolisation procedure did not cause any damage to the complexed plasmid-DNA, whereas uncomplexed plasmid DNA was exposed to the shearing forces of the aerosolisation chamber and thus destroyed. Surface modification of PEI/pDNA complexes with insulin resulted in an increase of in vitro transfection efficiency on alveolar, but not on bronchial epithelial cells. A ratio of 5 µg insulin per µg of plasmid-DNA resulted in a maximum, showing a 16-fold higher luciferase expression compared to unmodified PEI/pDNA particles. Coincubation experiments with free insulin revealed that the increased expression was not caused by proliferative influences of the ligand. Furthermore, it could be demonstrated that this effect was polymer-specific. Addition of insulin in combination with linear PEI did not result in increased gene expression. Toxicity of gene transfer complexes also plays a major role with regard to their later clinical applications. PEI is known as a toxic gene transfer agent. High toxicity has been reported in several studies both in vitro and in vivo. It could be demonstrated for all three ligands, that after their introduction to PEI/pDNA complexes, in vitro toxicity, evaluated by measurements of the metabolic cell activity, was significantly reduced. In summary, this work demonstrates that lactoferrin, clenbuterol and insulin represent suitable targeting ligands, which form with branched polyethylenimine efficient nanoparticulate systems for successful gene transfer to the lung. Additionally, these complexes have the desired attributes of higher gene expression, lower toxicity and targeted delivery to alveolar or bronchial epithelial cells.

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Neutrophil activation plays a crucial role in the pathogenesis of the meconium aspiration syndrome. Therefore antiinflammatory strategies may offer therapeutic options. The methylxanthinderivative pentoxyphylline (PTX) is known to inhibit the tumor necrosis factor alpha-synthesis and neutrophil degranulation and thus may have beneficial effects on meconium-induced pulmonary inflammation. Effects of PTX on PMN-degranulation in neonatal whole blood have not yet been studied.Heparin-anticoagulated (3 IE/ml) whole blood of healthy neonates (n = 6) and adult volunteers (n = 6) was incubated for 45 min. Spontaneous PMN-degranulation was compared with meconium-induced (3 mg/ml) and PTX-inhibited (0,025 - 0,4 mg/ml) degranulation by means of elastase (EL) and lactoferrin (LF) release from azurophilic and specific granules. EL- and LF plasma concentration was measured by immunoluminometric methods.Spontaneous degranulation of neonatal PMN was found to be significantly increased after 15 minutes compared with cells from adults (EL and LF concentration: 674 and 660 ng/10 6 PMN vs. 284 and 261 ng/10 6 PMN). At 45 minutes adult PMN showed an acceleration of degranulation in contrast to neonatal cells (EL and LF: 1827 and 1232 ng/10 6 PMN vs. 1400 and 860 ng/10 6 PMN). In presence of PTX (0,4 mg/ml) spontaneous release of EL and LF from neonatal PMN was inhibited by nearly 70 % at 45 min. while degranulation from adult PMN was found to be completely inhibited at 15 min. and reduced by 82 % and 78 % at 45 min. In presence of meconium (3 mg/ml) an increased degranulation of EL from PMN of both neonates and adults (317 % and 170 %) could be observed while LF release was found to be increased from neonatal cells only (267 % and 113 % respectively). PTX inhibited meconium-induced EL release in blood of bath neonates and adults by 63 % and 66 %, while LF release was inhibited by 72 % and 57 % respectively.Neonatal PMN exhibit an increased degranulation from azurophilic and specific granules compared with cells from adults. PTX was found to be an effective inhibitor of spontaneous and meconium induced PMN degranulation and may offer new therapeutic options.

Approximately 3% of the patients with refractive errors wear contact lenses. Recent studies revealed contradictory results regarding the changes of the tear film in contact lens wearers. The aim of this study was to analyze the influence of wearing contact lenses on the tear protein patterns. The study was performed using a new method to analyze quantitatively the tear proteins based on digital image analysis of electrophoretical separations followed by multivariate statistical calculations.Two groups were examined: 121 healthy volunteers (KO) and 66 wearers of soft contact lenses (KL). The tear proteins were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). For each electrophoretic lane, a densitograph was built by digital image analysis. Subsequently a multivariate analysis of discriminance was performed.The concentration of the tear proteins lactoferrin, sIgA, lysozyme and albumin was slightly reduced. In contrast, the concentration of lipocalin was increased in the tears of contact lens wearers (p0.07). However, including all peaks found in each electrophoretic lane, the analysis of discriminance found a statistical significant difference between KO and KL (Wilks' Lambda = 0.88; p0.0001).Wearing contact lenses leads to significant changes in the composition of tear-film proteins. However, the pathomechanism of this alteration of the tear film is still unclear. Because of the large number of contact lens wearers, these alterations should be examined further.

Titelblatt, Inhaltsverzeichnis, Lebenslauf 1\. Einleitung 2\. Literatur 3\. Material und Methoden 4\. Ergebnisse 5\. Diskussion 6\. Zusammenfassung 7\. Summary Literaturverzeichnis, Die bedeutsamsten Erreger der Mastitis des Rindes stellen Bakterien dar. Unterstützend zu der körpereigenen Abwehr des Wirtstieres werden im allgemeinen Antibiotika zur Behandlung verwendet. Diese sollten nach Isolierung des Erregers und der Anfertigung eines Antibiogramms gezielt eingesetzt werden. Leider ist dieses nicht in allen Fällen möglich, da in etwa 10-40 % der zu untersuchenden Milchproben aus entzündeten Eutervierteln keine Erreger isoliert werden können. Ziel dieser Arbeit war es, neben der Wirksamkeit von Oxacillin in der Mastitistherapie (Untersuchungsteil 1) eine Erklärung für das Auftreten bakteriologisch negativer Mastitismilchproben zu finden (Untersuchungsteil 1 und 2). Der Untersuchungsteil 1 wurde auf einem Milcherzeugerbetrieb mit ca. 800 melkenden Kühen und 8000 kg gleitendem Herdendurchschnitt durchgeführt. Es wurden Tiere aus allen Laktationsstadien mit klinischen Mastitiden in die Untersuchung aufgenommen. Neben der Erhebung klinischer Befunde an den Tagen 0, 1, 2, 7, 14 und 21 wurde eine mikrobiologische Untersuchung an den Tagen 0, 7, 14 und 21 durchgeführt. In die Untersuchung aufgenommene Tiere wurden mindestens dreimal im Abstand von 24 Stunden mit 1000 mg Oxacillin intrazisternal behandelt. Die Behandlung wurde als klinischer Heilungserfolg eingestuft, wenn der Allgemeinzustand des Tieres ohne besonderen Befund und die Körperinnentemperatur, Bacterial infection is the most important cause of mastitis in cattle. Antimicrobial therapy is used for treatment. It should be used after isolation of the causative agent and the determination of its antimicrobial resistance pattern. Unfortunately, this is not possible in all cases, because 10 to 40 % of the milk samples from affected quarters yield no bacterial growth on culture. The objective of this study was to determine efficacy of Oxacillin in the antimicrobial therapy of mastitis (experiment 1) and to evaluate possible reasons for bacteriologically negative mastitis milk samples (experiment 2). Experiment 1 was conducted on a dairy farm with 800 cows in milk with a rolling herd average of 8000 kg per year. Animals from all stages of lactation with clinical mastitis were included in the study. Clinical findings were recorded on days 0, 1, 2, 7, 14 and 21. Milk samples were cultured for microbiological investigation on days 0, 7, 14 and 21. Animals included in the study were treated for at least three times in 24 hour intervals with 1000 mg Oxacillin intramammarily. Cases were classified as clinical cure if the attitude of the animal was back to normal and body temperature was below 39,0°C. The affected quarters had to be free of signs of acute inflammation like increased temperature, pain and/or swelling. The gross appearance of the milk had to be normal. The clinical cure rate was 60.8 % on day 14 and 48.1 % on the day 21. On average animals were treated 3.4 times with Oxacillin. The clinical cure rate on day 14 was 66.6 % (4 of 6 six animals) for mastitis due to Sc. agalactiae and 50 % (4 of 8 cases) for S. aureus mastitis. Clinical cure rates did not differ between cases with and without no bacteria isolated. The antimicrobial activity in milk has been described by many authors. In this study it was evaluated whether lysozyme, lactoferrin and the lactoperoxidase- thiocyanate-peroxide system (LPS) could be the reason for negative culture results in samples for cases of clinical mastitis. Experiment 2 was conducted on a dairy farm with approximately 2900 cows and a rolling herd average of approximataly 7100 kg. Animals from all stages of lactation with clinical mastitis were included in the study. Animals were examined clinically and milk samples were collected for microbiological investigation and to test for inhibitory substances. Lysozyme concentrations were determined using the " lysoplate-technique" which is based on the lysis of Micrococcus lysodeicticus suspended in the agar. Lactoferrin concentrations were measured in an enzyme linked immunosorbent assay (ELISA, Meisel, 1990). Activity of the LPS was determined spectrophotometrically (Shindler et al., 1976; Bjoerck and Mullan, 1993). The concentrations or activities of the three factors were significantly higher in diseased quarters than in quarters without clinical signs of mastitis. No correlations could be determined between the three factors and milk production, parity and stage of lactation. The concentration of lysozyme increased with severity of the clinical signs (local swelling and changes in secretion). The concentration of lactoferrin was significantly lower in quarters with only limited tissue alterations than in quarters with medium or severe alterations (p

The routine clinical diagnosis of sicca syndrome remains difficult; the results of the standard tests, such as the Schirmer test, tear film break-up time and the rose Bengal test, correlate neither with the course of the disease nor with one another.We introduce two procedures that can be used to differentiate patients with sicca syndrome from healthy individuals on the basis of tear fluid protein patterns. Electrophoretic separation of the tear proteins or measurement by high-performance liquid chromatography (HPLC) was followed by digital image processing and multivariate discriminant analysis.In addition to analysis of tear protein patterns, these methods permit diagnostic classification. Statistical evaluation reveals whether an unidentified sample is to be classified as sicca syndrome or "healthy". Both HPLC analysis and protein electrophoresis detect differences in protein patterns between the tear fluid of healthy individuals and patients with sicca syndrome and point to a diagnosis accordingly.Both of the techniques presented - electrophoresis and HPLC - could potentially be used for diagnostic purposes in the detection of sicca syndrome. The HPLC method of tear protein analysis is more reliable, lends itself more readily to automation and achieved greater success rates in the experiments we describe; for these reasons it is the preferred approach.

[no abstract]

Localized depositions of amyloid in the seminal vesicles may occur in elderly men. Earlier immunohistochemical studies have failed to identify immunoreactivity of known amyloid material. In this autopsy study, all seminal vesicles of males older than 50 years were histologically examined to determine incidence and phenotype of seminal vesicle amyloidosis. Seven out of 50 patients (14%) showed depositions of amyloid in the seminal vesicles. These amyloid depositions as well as one additional case were characterized histochemically, immunohistochemically and electronmicroscopically. All but two of these patients (75%) showed simultaneously amyloid depositions in the heart. Lactoferrin immunoreactivity was found in 6 patients (75%). Lactoferrin is an iron-binding, bacteriostatic glycoprotein, which is produced in the seminal vesicles. Four patients with lactoferrin positive amyloid in seminal vesicle showed different amyloid depositions in the heart (immunoglobulin light chain amyloid AL-lambda). Two cases (25%) showed the same amyloid type in heart and seminal vesicles (prealbumin-transthyretin type amyloid). Our study shows that most amyloidoses of the seminal vesicles are organ-limited depositions of lactoferrin. These forms of localized amyloidosis have to be separated from senile systemic amyloidosis with seminal vesicle involvement.

Neutrophilic granulocytes in the lower respiratory tract are of decisive importance for the elimination of pathogenic germs in bacterial pneumonia. On the other hand, the liberation of phagocyte products (e.g. elastase) can result in tissue damage in the parenchyma of the lungs. For this reason, we determined in patients suffering from acute pneumonia (n = 21), in patients with acute pneumonia associated with immunosuppression (n = 12), in patients who had overcome their pneumonia (n = 9) and in controls (n = 17) in bronchoalveolar lavage (BALF) and in plasma, the concentration of the locally produced granulocyte products myeloperoxidase (MPO), lactoferrin (LF) and elastase-alpha 1 proteinase complex (ELA) as well as of the alpha 1 proteinase inhibitor (alpha 1 Pi) and alpha 2 proteinase inhibitor (alpha 2 Pi) via chemoluminescence immunoassay, and compared the same with the differential cell count in the BALF. The protein concentrations were referred to the albumin concentration (Alb) for standardisation. This concentration did not differ significantly between the various patients and control groups. The BALF concentration of ELA in the group with pneumonia (median: 86.3 micrograms/l or 8.5 micrograms/mg Alb) was about eight times higher than in the group of patients suffering from pneumonia with immunosuppression (median: 16 micrograms/l or 1.0 micrograms/l Alb, p less than 0.001) or in whom the pneumonia was no longer present (17.6 micrograms/l or 0.5 micrograms/mg), and approximately 40 times higher than in the control group (3 micrograms/l or 0.2 micrograms/mg, respectively). Similar results were obtained for LF (61 micrograms/mg Alb vs. 11.3; 16.8 and 5.9 micrograms/mg; p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

In the mammary gland of cattle there is a complex defense system of non-specific and specific reactions available preventing the invasion of pathogenic bacteria. Most infections occur via the teat canal, so teat canal keratin (SKK) is of particular importance in non-specific defense of the gland. The SKK serves as a physical barrier, and bacteriostatic and/or bactericidal effects of SKK lipids and proteins against certain mastitis bacteria could be demonstrated. By increasing the concentrations of lactoferrin and lysozyme in milk a reduction of mastitis frequencies could be observed. However, those high concentrations in the proteins occur only during the dry period of the cow. An improvement of the mastitis situation would also appear possible by increasing phagocytosis. The numerous trials intended to reduce mastitis by improving specific protection showed no significant success. Therefore, the most successful and cheapest means to achieve udder health remains the strict and consistent hygiene of housing, animals and mammary glands.

Typical alterations of the white blood cell count are often missed during the acute course of infectious diseases. Activiation and degranulation of granulocytes are followed by elevation of E alpha 1 PI and lactoferrin plasma concentrations under these conditions. The aim of our study was the evaluation of the diagnostic significance of these granulocyte parameters in relation with the absolute granulocyte count in infected pediatric patients. A total number of 106 patients at the age of 1 day to 16 years were studied. 25 children suffered from viral, 26 from localized and 23 from systemic bacterial infections, 32 children exhibiting no signs of infection served as controls. Results of the study are given as medians and ranges. Total granulocyte count was elevated above controls (4.8; 2.2-12.7/nl) only in patients with localized bacterial infections (13.3; 5.5-36.5/nl). E alpha 1 PI and lactoferrin plasma concentrations correlated well (r = 0.72) and were found to be significantly elevated in patients with localized bacterial infections (856; 363-4820 micrograms/l and 748; 206-2078 micrograms/l) and septicemia respectively (661; 256-2078 micrograms/l and 871; 160-9550 micrograms/l). A clearcut differentiation of septic and locally infected patients was given by the ratio of E alpha 1 PI and total granulocyte counts. Significantly elevated E alpha 1 PI concentrations of patients exhibiting viral infections (295; 86-690 micrograms/l) may suggest effective granulocyte activation under this condition. Finally we conclude that E alpha 1 PI and lactoferin plasma concentration related to total granulocyte counts in infected patients may serve as a helpful indicator of granulocyte activation during the acute course of the disease.

The concentrations of sodium, potassium, calcium, protein and lactoferrin and the enzymatic activity of salivary kallikrein, phosphohexoisomerase, alpha-amylase, lysozyme and IgA were measured in the parotid saliva of 12 patients suffering from Sjögren's syndrome. Compared with a control group (n = 31) remarkable differences were found in the composition of the parotid saliva in Sjögren patients. The possible reasons for these sialochemical alterations are discussed.

Having demonstrated transferrin (TF) and the TF receptor in periretinal membranes, we now present results of quantitative studies of TF and lactoferrin (LF), another protein with iron-binding properties. Normal human vitreous contains 74 +/- 7 mg/l TF, but less than 50 micrograms/l LF (ELISA). The TF levels determined in vitreous aspirates from patients with proliferative intraocular diseases [traumatic proliferative vitreoretinopathy (PVR), idiopathic PVR, and proliferative diabetic retinopathy (PDR)] were higher. A statistically significant difference between the levels in the vitreous in the three types of proliferative intraocular disease and in physiological vitreous was not observed. In contrary to TF, LF could not be labeled in surgically obtained membrane specimens using immunochemistry. Apparently LF does not have the same importance for cell proliferation in proliferative intraocular diseases as is suggested for TF.

One of the greatest advantages of feeding exclusively breast-milk is the continuous provision of immunoglobulin A, especially during the first days of life, and of leucocytes with macrophage function as well as unspecific, antiinfectious agents like lactoferrin, lysozyme and neuraminic acid. It seems, that the organism is protected against allergic reactions at the mucosa level of the small intestine caused by the penetration of "foreign" protein by feeding exclusively breast-milk especially during the first weeks and months of life. During the first months of the infant's life an increased supply of iron results from the higher content of iron in breast-milk as compared to cow's milk, and the better absorption of the iron from breast-milk. Just because of this (the better provision with iron from natural food) solid foods should not be added to the infant's diet before 6 months of age. One of the disadvantages of breast-feeding is the passage of unwanted substances from breast-milk to the infant. First of all the chlorinated hydrocarbons have to be mentioned within this context. However, a decreasing tendency can be assumed according to recent investigations. An increasing tendency in breast-milk, though not confirmed, seems possible only for the polychlorinated biphenyls. Nevertheless, for the pediatrician no reason to advise against breast-feeding results from the unwanted admixtures of chlorinated hydrocarbons in breast-milk. One should rather vigorously propagate to feed as many children as possible exclusively with breast-milk over a period of 4 to 6 months.

The group of tumour markers contain antigens and cell products which can be demonstrated in tumour cells by immunocytochemical methods (immunofluorescence, immunoperoxidase) and can, thus, be analysed for the classification of tumour. In human salivary gland tumours the distribution of cytoplasmatic antigens as components of the cytoskeleton, the occurrence of cell membrane antigens and of enzymatic cell products is demonstrated. Prekeratin, as an intermediate-sized filament protein, is a specific marker of epithelial tumours, whereas vimentin is a marker of mesenchymal cells. A special feature is the occurrence of prekeratin and vimentin in spindle-shaped cells of pleomorphic adenomas. The tumour-associated carcinoembryonic antigen (CEA) is found in glandular tumours and highly differentiated keratinized squamous cell carcinomas. With regard to enzymatic cell products, lactoferrin is present in glandular tumours and amylase in acinic cell tumours, but lysozyme is not detectable. The implementation of tumour markers contributes not only to an improvement in tumour diagnosis, but opens up new aspects in the cyto- and histogenesis of tumours.

The protective effect of breast feeding against infections is well proved by the experiences in underdeveloped countries and in industrial countries as well as shown by numerous investigations in several populations of different social structure. Various factors are meant to be responsible for this special property of human milk, the importance of which is to be discussed. The lysozyme and the lactoferrin are two different antibacterial factors well known since a long time, the former of which is mainly directed against grampositive organisms and against gramnegative ones only under special conditions. Lactoferrin is effective against E. coli and Staphylococcus as well. The neuraminic acid of which the human milk contains a larger quantity than cow's milk also inhibits the growth of E. coli and Staphylococcus. A further factor consisting of isomers of linoleic acid protects mice against lethal Staphylococcus - infections. The most important antibacterial principles of human milk are meant to be specific immunoglobulins, specially secretory IgA. The immunoglobulins are mainly important for the young baby in the early stage of life. The fecal bifidusflora specific for the breast-fed infant is also meant to be protective against several infections. The factors of human milk provoking this special kind of intestinal flora are to be discussed. In the artificial-fed infant bifidus flora like that of breast-fed one can arise by application of lactulose. Nowadays, bifidus-flora can probably be obtained by application of beta-lactose. It is suggested, that infants with bifidus-flora provoked by one of this means are protected against intestinal infections. The results of all investigations on mother's milk lead to the conclusion, that breast-feeding is the optimal kind of alimentation in the first time of life, not only concerning the composition of the food, but concerning protection against infections.

A short historical review of the antimicrobial effects of siderophilin, the iron chelator in human plasma and serum, is followed by a presentation of the rational basis for assigning it a role in the overall defence mechanisms of the host against infectious disease. Details are given of the qualitative and quantitative activities of normal and pathological sera on the growth and nutrition of several pathogenic bacteria and the fungus Candida albicans as governed by the percentage iron saturation of their contained siderophilin. Staphylococcus aureus is differentiated from Staphylococcus albus by its ability to grow in normal serum whose percentage iron saturation affects not only the metabolism of Staphylococcus aureus but also its production of diffusible factors and sensitivity to antibiotics. The protein iron chelator of bodily secretions, ekkrinosiderophilin, found in relatively high concentrations in human milk is likewise evaluated for its antimicrobial properties and their relevance to the health of the newborn.

As compared to 20 controls, 14 patients with chronic pancreatitis showed a significantly higher lactoferrin level in pure pancreatic secretion. The determination of lactoferrin levels in pure pancreatic secretion is an important parameter in the diagnosis of chronic pancreatitis.

The diagnostic relevance of measuring calcium and lactoferrin in duodenal juice collected after stimulation is unclear. Concentration and output of these compounds were therefore analyzed after maximal stimulation of the pancreas according to Ribet, the duodenal juice being collected during an endoscopic procedure as described earlier. Kinetics of secretion showed a maximum within the first 10 minutes. Values of calcium and lactoferrin were not statistically different in normal persons (n = 32) and patients with chronic pancreatitis (n = 11). There was a good correlation to concentration of bilirubin (p less than 0.001), however no correlation to concentrations of immunoreactive trypsin and lipase. It must be assumed, that calcium and lactoferrin in duodenal juice are not only of pancreatic origin. These measurements are therefore useless in the diagnosis of pancreatitis.

With the isolation of secretory IgA and secretory component from human colostrum following the route communicated by Kobayashi some differences revealed with respect to the preparative results. Mainly problems arising from lactoferrin and IgM encouraged us to offer some varied laboratory instructions.

Bronchopulmonary defense system protects the organism effectively against various inhaled material. This system, including mechanical, biochemical and immunological mechanisms, is reviewed.

A short review on the origin of the human spermaplasma proteins and their different methods of examination is given. It follows a description of the results of 44 men over 50 years of age concerning albumin and immunoglobulin IgA and IgG. These results were obtained with an immunodiffusion technique.

The importance of the mammary gland as a potential immunological organ is characterized in this literature survey by the following aspects: immunoglobulins in colostrum and mother's milk, immunological active cells in colostrum and mother's milk, nonspecific factors in the mother's milk. The importance of the early nursing is emphasized from the immunological point of view.

Iron, iron-binding capacity, lactoferrin and total protein were determined in the plasma and pleural fluid of 30 patients with cardiac failure (n = 10), infectious/inflammatory disease (n = 9) and metastatic carcinoma (n = 11). In 16 patients pleural transferrin and ferritin was also measured. Plasma iron and total iron-binding capacity were reduced in inflammatory and neoplastic disease, whereas hyposideremia with normal iron-binding capacity was seen in patients with heart failure. Plasma lactoferrin was reduced in metastatic carcinoma. Exudates (protein greater than or equal to 30 g/l; infectious/inflammatory: 9/9, carcinomatous: 10/11) had significantly higher iron, lactoferrin, transferrin and ferritin concentrations than transudates (protein less than 30 g/l; heart failure: 10/10, carcinomatous: 1/11). Statistically, infectious/inflammatory exudates could be distinguished from neoplastic exudates by a higher median iron concentration (non-parametric Wilcoxon-Mann-Whitney test). Overlap of the respective ranges, however, did not allow a clear-cut differential diagnosis in individual cases. Pleural lactoferrin concentrations, on the other hand, correlated with the pleural granulocyte count and nonspecifically reflect the degree of granulocytic inflammation. Positive pleural/plasma correlations of protein and of iron concentrations were found in exudates only. Within exudates and transudates, on the other hand, total protein correlated with transferrin but not with iron concentrations. Therefore, and because of the substantially higher pleural/plasma ratio for iron than for transferrin concentrations, a quantitatively important, non-transferrin bound iron pool in pleural fluids, most probably ferritin, must be assumed.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute overload with an iron salt in mice enhanced the virulence of a relatively avirulent type of Salmonella typhimurium in a dose-dependent fashion. Iron administration also partly abolished specific immunity. Ferritin iron did not display the virulence-enhancing effect.