Two types of (polymer-imaging agent-drug) complexes were prepared and characterized. These included block and graft copolymer complexes with magnetite nanoparticles and manganese ions. Magnetite block ionomer complexes (MBICs) were formed through binding of a portion of the anionic segment of poly(ethylene oxide)-b-poly(acrylic acid) (PEO-b-PAA) block copolymers with the magnetite nanoparticle surfaces. The remainder of the carboxylic acids were utilized to bind with high concentrations of the cationic antibiotic gentamicin (31 wt%). A near zero-order release of gentamicin (pH 7.4 in PBS) that reached ~35 wt% of the initial gentamicin within 10 hours was observed, and this was followed by slower release of another 7 % by 18 hours. These nanoparticles were efficiently taken up by macrophages and appeared to enhance intracellular antimicrobial activities of gentamicin. To increase the complex sizes and NMR T2 relaxivities, amine functional MBICs (MBICs-NH2) were first assembled by adsorbing the polyacrylate block of an aminofunctional poly(ethylene oxide)-b-poly(acrylic acid)) (H2N-PEO-b-PAA) copolymer onto magnetite nanoparticles. Amines at the tips of the H2N-PEO corona were then linked through reaction with a PEO diacrylate oligomer to yield MBIClusters where the metal oxides in the precursor nanoparticles were distinctly separated by the hydrophilic polymer. These MBIClusters with hydrophilic intra-cluster space had transverse relaxivities (r2's) that increased from 190 to 604 s-1 mM Fe-1 measured at 1.4 T and 37°C as their average sizes increased. The clusters were loaded with up to ~38 wt% of the multi-cationic drug gentamicin. MRI scans focused on the livers of mice demonstrated that these MBIClusters are very sensitive contrast agents. These results indicate that these complexes could be potential theranostic agents for dual imaging and drug delivery. Manganese graft ionomer complexes (MaGICs) comprised of Mn ions and a novel polyaminobisphosphonate-g-PEO copolymer were developed for use as T1 weighted MRI positive contrast agents. The graft copolymers were prepared by free radical copolymerization of ammonium bisphosphonate methacrylate monomers with PEO-acrylate macromonomers. The complexes exhibited good colloidal stability without release of free manganese and did not show any in vitro toxicity against mouse hepatocytes. The T1 relaxivities of the MaGICs were 2-10 times higher than that of a commercial manganese based contrast agent MnDPDP. These MaGICs with encapsulated anticancer drugs including doxorubicin, cisplatin and carboplatin have encapsulation efficiencies of 80-100 %. Drug release was sustained and depended on environmental pH, drug structure and drug concentration in the MaGICs. Moreover, these drug-loaded complexes exhibited high anticancer efficacy against MCF-7 breast cancer cells. The prominent MRI relaxivities and high anticancer efficacy suggest that these MaGICs have potential as effective dual imaging and chemotherapeutic agents. / Ph. D.

It is widely accepted that sleep disturbances occur in patients recovering from coronary artery bypass graft (CABG) surgery. This sleep disturbance, at least in theory, might retard or limit the return of functionality and exacerbate psychological states known to increase use of health care services and adversely affect prognosis. This study explored possible relations between sleep, self-rated aerobic physical capacity and physical activity in a sample of patients who underwent CABG surgery. Secondary analysis investigated the possible concurrent influences of post-CABG health complaints and depression on sleep function. Measures included the Pittsburgh Sleep Quality Questionnaire; the Veterans Specific Activity Questionnaire; the Paffenbarger Physical Activity Questionnaire; the Health Complaint Scale and the Beck Depression Inventory, Version II. The physical measure of body composition was also used. Fifty-five subjects completed baseline questionnaires for all measures just prior to surgery, excluding the Paffenbarger Physical Activity Questionnaire. Follow-up evaluations were repeated at 3 mo post-CABG for the same measures and the patients were also asked to report their patterns of post-surgical physical activity involvement. Correlation coefficients were calculated to determine whether a correlation existed between the measures. Significant correlations were found between pre-surgical and post-surgical sleep score, sleep subscales, functional capacity, depression and health complaints (p < 0.05). Post-CABG sleep and physical activity did not exhibit a significant correlation. A number of secondary analyses were performed in an effort to isolate possible influences of confounding factors, such as depression, body mass index > 27, and a ventricular ejection fraction (EF) < 30. In the low EF subgroup, overall sleep score and self-rated functional capacity were strongly correlated before surgery was performed (r = -0.85; p < 0.01). Stepwise regression equations were constructed to predict sleep outcome before and after surgery. Somatic health complaints, depression, and skinfold measures were found to be predictors for pre-surgical sleep (R2 = 0.52), as well as post-surgical sleep (R2 = 0.78). Fitness measures of functional capacity and physical activity were not a significant predictor of sleep. The findings of this study suggest that a modest correlation exists between functional capacity and sleep in CABG patients; however, predictors including health complaints, depression and skinfold measures serve as better indicators for sleep outcome before and after CABG surgery. / Master of Science

No description available.

The aims of the present investigation are fourfold: (i) to review the range of non-invasive methods that may be used to diagnose acute cardiac allograft rejection; (ii) to review the use of the bioptome in sampling the donor heart endomyocardium; (iii) to review the light microscopic and histological grading of acute cardiac rejection; (iv) to characterise the mononuclear populations in endomyocardial biopsy samples and correlate the findings with the light microscopic appearances of the same biopsy specimens.

No description available.

This study deals with the effects of branching in some high polymers on viscosity behaviour and on the rela­tionship between intrinsic viscosity and molecular weight. First, the preparation of branched polymers by means of graft polymerization is described. Evidence for the occurrence of grafting, which would result in the growth of polystyrene branches on a min, or backbone, chain com­posed of polystyrene or a copolymer of styrene and li-vinyl-cyclohexene-1, is reviewed, Next, the design, construction, and calibration of a flexible light scattering photometer is described. The performance of this instrument is shown to bs adequate for the determination of the molecular weight and size, as well as the second virial coefficient, of a high polymer in solu­tion. Finally, properties of the graft polymers in solu­tion are compared, with the corresponding properties of linear polystyrene. The relationships found between intrin­sic viscosity and molecular weight, as well as the values obtained for the second virial coefficient, confirm the ex­istence of branching in the graft polymers. On the other hand, it is shown that the viscosity slope constant, Huggins' k', the of which, is often used as an indication of branching, is not affected significantly by the branching present in the graft polymers. / Thesis / Doctor of Philosophy (PhD)

Graft-versus-Host Disease (GvHD) stellt einen häufigen, den Gesamterfolg einer allogenen hämatopoetischen Stammzelltransplantation limitierenden Faktor dar. Bei dieser Komplikation attackieren vor allem alloreaktive T-Lymphozyten des Stammzellspenders gesunde Körperzellen des Patienten. Infolgedessen kommt es zu Gewebeschäden in den Zielorganen Haut, Leber und Darm. Die Behandlung der GvHD erfordert eine effektive Immunsuppression, was wiederum Graft-versusTumor-Effekte kompromittiert und den Rückfall der malignen Grunderkrankung bedingen kann. Viele Patienten sprechen aus bisher ungeklärten Gründen nicht auf die klassische immunsuppressive Therapie mit Steroiden oder second-line Therapien an. Neue zelluläre Therapien zur Behandlung der refraktären GvHD sind auf dem Vormarsch, bedürfen aber einer weiterführenden klinischen Testung, auch um die exakten Wirkungsmechanismen zu verstehen. Idealerweise könnten neue Testsysteme das GvHD-Potential von allogenen Stammzellpräparaten oder aber das immunsuppressive Potential von neuen GvHD-Therapien vorhersagen, bevor diese in klinischen Studien eingesetzt werden. Ziel der vorliegenden Arbeit war es, ein erstes, in multiplen Replikaten einsetzbares, humanes organotypisches Gewebemodell zur Simulation einer GvHD-Reaktion am Beispiel der Haut zu etablieren. Zu diesem Zweck wurden artifizielle humane Hautmodelle unter statischen (KollagenHautmodelle) und dynamischen Kulturbedingungen (vaskularisierte Hautmodelle) generiert. Die Injektion unstimulierter PBMCs (engl. peripheral blood mononuclear cells) führte zu keinen histomorphologischen Veränderungen in den KollagenHautmodellen. Im Gegensatz dazu hatte die Injektion vorstimulierter allogener PBMCs eine Zerstörung der epidermalen Strukturen der Kollagen-Hautmodelle zur Folge, welche vergleichbar waren mit Gewebeschäden bei einer akuten GvHD der Haut. Dieselben Schädigungen der Epidermis wurden durch die Injektion von Mediumüberständen vorstimulierter PBMCs in die Kollagen-Hautmodelle erreicht. Im Kulturmedium der Kollagen-Hautmodelle wurden hohe Konzentrationen von Interleukin 2 und 17, Interferon gamma sowie Tumornekrosefaktor alpha gemessen, wodurch auf die Beteiligung von Zytokinen an der inflammatorischen Reaktion geschlossen werden konnte. Auch im komplexeren vaskularisierten Hautmodell verursachte die Injektion vorstimulierter PBMCs histomorphologische Veränderungen entsprechend einer akuten Haut-GvHD sowie einen zeitabhängigen Anstieg proinflammatorischer Zytokine. Zusammenfassend zeigen die Resultate dieser Arbeit, dass die Induktion einer starken Inflammations- und Immunreaktion in artifiziellen humanen Hautmodellen, welche histomorphologisch eine GvHD imitiert, möglich ist. Dieses Modell könnte als Grundlage für die Entwicklung eines klinisch relevanten Testsystems zur Bestimmung des GvHD-Restpotentials oder zur Festlegung der immunsuppressiven Kapazität innovativer Zellpräparate dienen. Somit könnten humane artifizielle GvHDModelle in klinischen Studien eingesetzt werden und die Erfahrungen aus Tiermodellen ergänzen sowie erste in vitro Ergebnisse im humanen System liefern, welche dann mit dem tatsächlichen klinischen Resultat verglichen werden könnten. / Graft-versus-Host Disease (GvHD) remains the most important limiting factor for the success of allogeneic hematopoietic stem cell transplantation. This major complication is caused by alloreactive donor T-lymphocytes that attack healthy tissues of the recipient leading to severe tissue damage within the target organs skin, liver and gut. Treatment of GvHD requires effective immunosuppression, which in turn impairs Graft-versus-Tumor activity and enhances the risk for relapse of the malignant disease. However, for still unknown reasons many patients do not respond to standard immunosuppressive therapy with steroids or to second-line therapies. Development of novel cellular therapies that gain more and more clinical relevance due to their high anti-tumor potency lead to a strong demand for advanced test platforms to further investigate their underlying functional mechanisms and exclude off-tumor effects against healthy tissues. Ideally, new test systems could be used for the prediction of the GvHD potential of allogeneic stem cell products or for prediction of an immunosuppressive potential of novel GvHD therapies before entering clinical studies. The aim of this study was to establish a GvHD test system based on human organotypic skin models allowing the simulation of GvHD reactions in the skin in multiple replicates. To this end, artificial human skin models were generated under static (collagen skin model) and dynamic culture conditions (vascularized skin model). Injection of unstimulated peripheral blood mononuclear cells (PBMCs) did not cause histomorphological changes in collagen skin models. In contrast, injection of prestimulated PBMCs resulted in disruption of the epidermis of collagen skin models mimicking acute skin GvHD. The same disruption of the epidermal layer was observed using cell culture supernatants of prestimulated PBMCs, suggesting the involvement of proinflammatory cytokines. Indeed, measurement of cytokine levels in culture supernatants revealed an increase of interleukin 2 and 17, interferon gamma and tumor necrosis factor alpha. In addition, injection of prestimulated PBMCs into more complex vascularized skin models also caused disruption of the epidermal layer and an increase of proinflammatory cytokine levels in a time dependent manner. Taken together, these findings demonstrate that it is possible to induce a strong immune reaction and inflammatory tissue damage in artificial human skin models mimicking histomorphological patterns of acute skin GvHD. Therefore, this model could contribute to the development of a clinically relevant GvHD test platform for prediction of the GvHD potential or immunosuppressive capacity of innovative cell products. Thus, artificial human GvHD models may be employed in clinical studies in order to gain first in vitro results in a human system and to extend information from animal models, which can then be compared to the actual clinical outcome.

T cell infiltration into the intestine occurs after priming and activation in the mesenteric lymph nodes and Peyer’s patches and subsequent trafficking via the blood circulation. We hypothesized that additionally to the vascular trafficking route, a fraction of T cells in the Peyer’s patches directly migrate into the adjacent lamina propria of the small intestine. To test this hypothesis, we employed a mouse model of acute Graft-versus-Host Disease to study the direct T cell migration from the Peyer’s patches to the adjacent lamina propria. First, we analyzed the border of Peyer’s patches on histological sections and found that the Peyer’s patch is not enclosed by a capsule or basement membrane. Thus, the tissue architecture allows for direct access to the surrounding tissue. With whole-mount light sheet fluorescence microscopy we quantified a three-dimensional gradient of T cells around Peyer’s patches on day 2.5 and day 3 after transplantation. This gradient evened out at day 4 and day 6 when high numbers of T cells started to evenly infiltrate the intestine from the blood circulation. We confirmed that gradient-forming T cells around Peyer’s patches resided within the tissue parenchyma of the lamina propria and not inside lymphatic vessels. To positively prove that the recently activated donor T cells around Peyer’s patches have egressed directly from that patch, we established a protocol for intravital photoconversion of T cells inside Peyer’s patches. 12 h after photoconversion inside a single Peyer’s patch, photoconverted T cells resided only around this particular Peyer’s patch and not elsewhere in the small intestine. This indicated that the T cells did not infiltrate via the blood but migrated to the adjacent lamina propria of the small intestine. Dynamic intravital two-photon microscopy revealed that these T cells next to the Peyer’s patch migrated in a random pattern. This suggested that these cells did not follow a positive chemoattractive gradient once they had reached the lamina propria. Laser-capture microdissection combined with RNA sequencing of the mucosa near the Peyer’s patch identified a wide range of migration-promoting factors. These included chemokines, co-stimulatory receptors and migration-associated intracellular molecules, which are candidates to promote this direct migration from Peyer’s patches. Altogether, we demonstrate for the first time that additionally to the vascular trafficking route, a fraction of T cells migrates directly from the Peyer’s patch to the surrounding mucosa. This mechanism implies so far unrecognized regional specification of Peyer’s-patch-primed T cells. Our findings may impact treatment strategies to avoid intestinal inflammation or foster immunity after oral vaccination. / T-Zell Infiltration in den Darm erfolgt nach Primen und Aktivierung in den mesenterialen Lymphknoten und Peyerschen Plaques durch Rezirkulation über die Blutbahn. Wir stellten die Hypothese auf, dass zusätzlich zur vaskulären Route ein Teil der T-Zellen im Peyerschen Plaque direkt in die angrenzende Lamina propria des Dünndarms wandert. Um diese Hypothese zu testen, setzten wir ein Mausmodell für eine akute Graft-versus-Host Reaktion ein, um die direkte Migration von T Zellen aus den Peyerschen Plaques in die angrenzende Lamina propria zu untersuchen. Zuerst analysierten wir die Randzonen um die Peyerschen Plaques mit histologischen Schnitten und konnten bestätigen, dass der Peyersche Plaque von keiner Kapsel oder Basalmembran umschlossen ist, sodass die Gewebearchitektur den direkten Zugang des umliegenden Gewebes zulässt. Mithilfe der Lichtblatt-Fluoreszenzmikroskopie von Dünndarm-Komplettpräparaten quantifizierten wir einen dreidimensionalen T-Zell Gradienten um Peyersche Plaques an den Tagen 2,5 und 3 nach allogener Stammzelltransplantation. Dieser Gradient verschwand zwischen an Tag 4 und Tag 6, als eine hohe Anzahl an T-Zellen begann, den Darm gleichmäßig über die Blutbahn zu infiltrieren. Wir bestätigten, dass die Gradienten-bildenden T-Zellen im Gewebe der Lamina propria und nicht in lymphatischen Gefäßen saßen, um zirkulierende Zellen von der Gradientenbildung auszuschließen. Um direkt zu beweisen, dass die T-Zellen um dem Peyerschen Plaque unmittelbar aus diesem Plaque ausgewandert sind, haben wir ein Protokoll für intravitale Photokonversion von T-Zellen im Peyerschen Plaque etabliert. 12 h nach der Photokonversion in einem einzelnen Peyerschen Plaque befanden sich die T-Zellen nur um diesen bestimmten Plaque herum. Dies zeigt, dass die T-Zellen das Gewebe nicht über die Blutbahn infiltrierten, sondern direkt in die angrenzende Lamina propria des Dünndarms gewandert waren. Dynamische intravitale Zweiphotonenmikrokopie offenbarte, dass diese T-Zellen um den Peyerschen Plaque nach zufälligem Schema wanderten. Dies legte nahe, dass diese T-Zellen keinem positiven Chemokingradienten folgten, sobald sie die Lamina propria erreicht hatten. Laser-Mikrodissektion kombiniert mit RNA-Sequenzierung der Mukosa nahe des Peyerschen Plaques identifizierte eine große Auswahl an migrationsfördernden Faktoren. Hierunter waren Chemokine, kostimulatorische Rezeptoren und intrazelluläre migrationsassoziierte Moleküle, welche Kandidaten sind, diese direkte Migration aus den Peyerschen Plaques zu fördern. In dieser Arbeit zeigen wir erstmalig, dass zusätzlich zur vaskulären Route ein Teil der T Zellen direkt vom Peyerschen Plaque in die umliegende Mukosa wandert. Dieser Mechanismus impliziert bislang unerkannte regionale Spezialisierung von T Zellen, welche in Peyerschen Plaques aktiviert wurden. Diese neuen Befunde können zukünftige Behandlungsstrategien gegen intestinale Entzündungserkrankungen oder für Immunreaktionen nach oraler Impfung beeinflussen.

Im Rahmen dieser Arbeit wurden 72 pädiatrische Patienten, die eine allogene Stammzelltransplantation erhielten, auf die Spender- und Empfänger-Zytokinpolymorphismen TNF-α, TGF-β1, IL-10, IL-6 und IFN-γ untersucht. Die Proben wurden mittels DNA-Extraktion, sequenzspezifischer PCR und Gelelektrophorese analysiert und auf deren Einfluss auf die Entstehung und den Verlauf maligner Erkrankungen sowie auf die Entstehung einer GvHD untersucht. / Within the scope of this work, 72 pediatric patients who received allogeneic stem cell transplantations were examined for the donor and recipient cytokine polymorphisms TNF-α, TGF-β1, IL-10, IL-6 and IFN-γ. We analyzed the samples by means of DNA extraction, sequence-specific PCR and gel electrophoresis and investigated their influence on the development and course of malignant diseases as well as on the development of a GvHD.

No description available.