Techn. Universiẗat, Diss., 2005--München.

In humans of all ages, the cell wall-less and genome-reduced species Mycoplasma pneumoniae can cause infections of the upper and lower respiratory tract. The well-documented occurrence of major peaks in the incidence of community-acquired pneumonia cases reported world-wide, the multifaceted clinical manifestations of infection and the increasing number of resistant strains provide reasons for ongoing interest in the pathogenesis of mycoplasmal disease. The results of recent studies have provided insights into the interaction of the limited virulence factors of the bacterium with its host. In addition, the availability of complete M. pneumoniae genomes from patient isolates and the development of proteomic methods for investigation of mycoplasmas have not only allowed characterization of sequence divergences between strains but have also shown the importance of proteins and protein parts for induction of the immune reaction after infection. This review focuses on selected aspects of the humoral host immune response as a factor that might influence the clinical course of infections, subsequent protection in cases of re-infections and changes of epidemiological pattern of infections. The characterization of antibodies directed to defined antigens and approaches to promote their induction in the respiratory mucosa are also preconditions for the development of a vaccine to protect risk populations from severe disease due to M. pneumoniae.

In unserer Studie sollte die Genauigkeit der PCR zur Bestimmung von Zytokinspiegeln ermittelt werden. Mittels Blutproben von mit A. Fumigatus infizierten Mäusen, sollte eine Aussage bezüglich der Immunantwort getroffen werden. Wir griffen TNFα, IL-12p40 und IL-10 heraus, um einschätzen zu können, ob die Immunantwort eher humoral oder zellvermittelt abläuft. Zur möglichen Bestimmung der Sensitivität und Genauigkeit, wurden die crossing points der Standardverdünnungsreihen jeweils einmal in einem Lauf dreifach, ausserdem jeweils in drei unabhängigen Läufen von einander einfach eingesetzt, und miteinander verglichen. Unsere Ergebnisse decken sich mit den Ergebnissen aktueller Literatur und Etablierungen anderer Zytokine. Die Etablierung des ELISAs sollte dem Vergleich zwischen mRNA-Ebene und Proteinebene dienen. Zur richtigen Einordnung unserer Arbeiten mit dem Immunoassay müssen die Limitierungen der Ergebnisse beachtet werden. Die Versuche zur Quantifizierung der mRNA murinen TNFαs aus den Versuchsserien misslang. Auch die erzielten Ergebnisse mit Protein-basierten Nachweisverfahren konnten letztendlich nicht suffizient beurteilt werden. Die großen Schwankungen in der Konzentration und die Widersprüchlichkeit im Vergleich der Ergebnisse aktueller Literatur, machen eine Verfälschung durch Kontamination mit Proteinen aus lysierten Zellen sehr wahrscheinlich. Die erzielten Ergebnisse der RT-PCR anhand der Inter- und Intra-Assay- Vergleiche jedoch können nachfolgenden Projekten dazu dienen, hauptsächlich das Instrument LightCycler in seiner Sensitivität und Genauigkeit einschätzen zu können, und so die ermittelten Daten besser verarbeiten zu können. / In this study we analysed the accuracy of PCR for identification of murine cytokine levels. Using blood samples of with A. Fumigatus infected mice, we wanted to reach a conclusion concerning the immune reaction. We picked TNFα, IL-12p40 and IL-10 to decide if the immune reaction is humoral or cell mediated. For determination of sensitivity and accuracy we compared the crossing points of the dilution standard series. We used the standard series once three times in one run and on the other hand in three independent runs. Our results correspond with the results in current literature. The establishment of the ELISA should have served for comparing the mRNA level with the protein level. But the tests failed. We couldn´t find mRNA of murine TNFα, and there was big variability in the concentration of protein. Probably the falsification happend because of the contamination with proteins from lysis of cells. But the conclusions from the RT-PCR inter- and intra-assay comparison could be helpful to assess the LightCycler instrument in its sensitivity and its accuracy and so facilitate the assessment of the results.

In humans of all ages, the cell wall-less and genome-reduced species Mycoplasma pneumoniae can cause infections of the upper and lower respiratory tract. The well-documented occurrence of major peaks in the incidence of community-acquired pneumonia cases reported world-wide, the multifaceted clinical manifestations of infection and the increasing number of resistant strains provide reasons for ongoing interest in the pathogenesis of mycoplasmal disease. The results of recent studies have provided insights into the interaction of the limited virulence factors of the bacterium with its host. In addition, the availability of complete M. pneumoniae genomes from patient isolates and the development of proteomic methods for investigation of mycoplasmas have not only allowed characterization of sequence divergences between strains but have also shown the importance of proteins and protein parts for induction of the immune reaction after infection. This review focuses on selected aspects of the humoral host immune response as a factor that might influence the clinical course of infections, subsequent protection in cases of re-infections and changes of epidemiological pattern of infections. The characterization of antibodies directed to defined antigens and approaches to promote their induction in the respiratory mucosa are also preconditions for the development of a vaccine to protect risk populations from severe disease due to M. pneumoniae.

Myelodysplastic syndromes (MDS) belong to the most common hematological neoplasms in the elderly population, characterized by ineffective hematopoiesis, peripheral cytopenia and the risk of transformation into acute myeloid leukemia. A dysregulated innate immune response and pro-inflammatory bone marrow microenvironment play a crucial role in the MDS pathogenesis by providing chronic inflammation which makes those pathways the perfect candidate for future therapeutics. Specifically, it has been shown that the alarmin S100A9, an important ligand for dri-ving inflammation and promoting tumor progression, is elevated in MDS patients. Previous expe-riments performed in the Stem Cell Lab 2 provided evidence that mesenchymal stromal cells (MSCs), an important component of the BM niche with immunomodulatory capacity, can be tar-geted by the novel oral small molecular drug Tasquinimod (TASQ, Active Biotech) which has demonstrated S100A9 inhibitory activity. The inhibition of inflammation-related molecules such as IL-1b, IL-18, PD-L1, resulted in a significant improvement of the hematopoietic support by MSCs. However, almost nothing is known about potential effects of TASQ in the context of immunomo-dulation. Therefore, we aimed in this project to understand the mechanisms of S100A9+/- TASQ concerning the immunomodulatory capacity of MDS-MSCs in response to T cell-mediated in-flammation by analyzing adhesion (ICAM1, VCAM1), immune checkpoint (PDL1, PDL2), anti-inflammatory cytokine (COX2, IDO1), chemokines (CCL2, IL8) and extracellular matrix-related (COL4A2, COL1A1) gene expression with quantitative real-time PCR. We observed a general de-crease in the aforementioned genes except for COL4A2 and COL1A1 upon treatment with TASQ, though T cell-mediated inflammation and activity remained unaffected, suggesting that inhibition of S100A9 reduces the inflammation-mediated immunomodulatory potential of MDS-MSCs.:Motivation Aim Methods Result Conclusion

Optimale Konservierungsmethoden sind erforderlich, um die bedarfsgerechte Verfügbarkeit kardiovaskulärer Transplantate für den Ersatz geschädigter Gewebe (Herzklappen oder Gefäße) zu garantieren. Die konventionelle Kryokonservierung (engl.: Conventional Frozen Cryopreservation, CFC) ist derzeit der Standard zur Konservierung kardiovaskulärer Allografts. Jedoch limitieren Immunreaktionen deren Langzeitfunktionalität. Die Alternative der eisfreien Kryokonservierung (engl.: Ice-free Cryopreservation, IFC) wurde kürzlich entwickelt. In der Arbeit wurde die Reaktion des humanen Immunsystems auf allogene kardiovaskuläre Gewebe nach Anwendung unterschiedlicher Konservierungsmethoden umfänglich charakterisiert. Zusätzlich wurde Glutaraldehyd (GA)-fixiertes Gewebe untersucht, um die Ergebnisse in den Gesamtkontext der Gewebekonservierung einzuordnen. Die Analyse des konservierten humanen Aortengewebes, welches als Modellmaterial diente, ergab, dass die Gewebestruktur nach IFC erhalten blieb, jedoch die metabolische Aktivität sowie Apoptose und Nekrose des Gewebes durch IFC reduziert wurde. Dies spiegelte sich auch in der verminderten Freisetzung von Zytokinen aus IFC-Gewebe wider. Funktionelle In-vitro-Tests zeigten deutlich, dass Immunzellen verstärkt in Richtung der löslichen Faktoren aus CFC- und GA-fixiertem Gewebe, jedoch nicht aus IFC-Gewebe migrieren. In Kokulturen der Makrophagen auf dem Aortengewebe konnte ausschließlich bei Makrophagen, welche auf GA-fixiertem Gewebe kultiviert wurden, eine Polarisation zum M1-Phänotyp festgestellt werden. Weiterhin zeigte sich, dass lediglich Faktoren des CFC-Gewebes in der Lage waren, die Aktivierung und Proliferation von T-Zellen zu verstärken. Insgesamt belegen diese Daten detailliert, dass IFC die Eigenschaften des Gewebes selektiv moduliert und dadurch eine verringerte Aktivierung des Immunsystems stattfindet. Die Ergebnisse verdeutlichen, dass IFC eine aussichtsreiche Strategie zur verbesserten Konservierung darstellt. / Optimal preservation methods are needed, to ensure constant availability of biological matrices for the replacement of damaged cardiovascular structures (heart valves or vessels). Conventional frozen cryopreservation (CFC) is currently the gold standard for cardiovascular allograft preservation. However, inflammation and structural deterioration limit transplant durability. The recently developed method of Ice-free cryopreservation (IFC) might be a superior method. The aim of this study was to characterize the reaction of the human immune system to allogeneic cardiovascular tissues after different cryopreservation methods. Regarding some aspects, the cryopreservation was compared to glutaraldehyde (GA) fixation, which is another common tissue preservation method. Human aortic tissue served as a proof-of-principle material for heart valves and vascular allografts. First, the histological and metabolic features of the differently preserved aortic tissues were analyzed. Tissues preserved by IFC exhibited typical architecture but significantly lower metabolic activity and the absence of necrotic or apoptotic cells. The reduced release of cytokines from IFC-tissue reflected these latter observations. In functional in-vitro-assays it was shown that migration of immune cells was significantly enhanced by soluble factors from CFC and GA-fixed tissue, but not by factors from IFC-tissue. In co-cultures of macrophages on aortic tissue, none of the preserved tissue induced activation. Exclusively GA-fixed tissue triggered the polarization of macrophages towards a M1-phenotype. Moreover, cues from only CFC-tissue but not IFC-tissue amplified T cell activation and proliferation. In conclusion, IFC selectively modulates the characteristics of tissues resulting in an attenuated activation of the human immune system. Therefore, IFC treatment is a promising strategy for improved tissue preservation and storage of cardiovascular allografts for clinical use.

Das humorale Immungedächtnis wird von reifen Plasmazellen des Knochenmarks vermittelt, welche bei Immunreaktionen aus aktivierten B-Lymphozyten gebildet werden. Dabei sind im Blut Plasmablasten als unmittelbare Vorläufer der Plasmazellen nachweisbar, die von dort aus in das Knochenmark einwandern. Anhand der durchflusszytometrischen Detektion spezifischer Plasmablasten gelang es hier, das simultane Auftauchen von Wellen neu generierter, migratorischer Plasmablasten und reifer, nicht-migratorischer Plasmazellen im Blut eine Woche nach einer Tetanusimpfung nachzuweisen. Plasmablasten und Plasmazellen lagen stets im Gleichgewicht vor, wodurch auf die stöchiometrische Mobilisierung reifer Plasmazellen des Knochenmarks durch systemisch induzierte Plasmablasten geschlossen wurde. Ein solcher Verdrängungsmechanismus wird hier erstmalig als Anpassungsmechanismus des humoralen Immungedächtnisses dargestellt, der die Aufnahme neuer Spezifitäten in das Gedächtnis unter Wahrung der Stabilität präexistierender Spezifitäten erlaubt. Anders als systemisch induzierte Plasmablasten, weisen Plasmablasten, die im immunologischen Ruhephase zirkulieren, Kennzeichen mukosaler Immunreaktionen auf: sie exprimieren IgA sowie die mukosalen Zellmigrationsrezeptoren alpha4beta7-Integrin und CCR10. Wahrscheinlich wandern sie in mukosale Plasmazelldepots ein und interferieren nicht mit den Plasmazellen des Knochenmarks, sodass die Stabilität des humoralen Gedächtnisses in der Ruhephase gewahrt bleibt. Eine Anpassung des humoralen Gedächtnisses findet somit nur im Rahmen systemischer Immunreaktionen statt. Bei splenektomierten Patienten und unter der B-Zell-Depletionstherapie bei Rheumapatienten bleiben mukosale Plasmablasten im Blut nachweisbar. Dies belegt deren autonome Bildung aus mukosalen, therapie-refraktären B-Zellen. Insgesamt wird hier eine bisher unbeachtete Komplexität menschlicher peripherer Plasmablasten und Plasmazellen und ihren Beziehungen zum humoralen Immungedächtnis dargestellt. / Humoral memory, i.e. persistence of specific antibody titers, is provided by plasma cells in the bone marrow, which are generated from activated B cells during immune responses. At this, immediate plasma cell precursors, the plasmablasts, migrate via the blood to the bone marrow. Using cytometric detection of antigen-specific plasmablasts, synchronous circulation of waves of recently generated, migratory plasmablasts and non migratory plasma cells with a mature phenotype was demonstrated one week after tetanus vaccination. Circulating plasmablast and plasma cell numbers were always in homeostasis, so that the stoichiometric mobilization of old bone marrow plasma cells by recently generated plasmablasts was hypothesized. This plasma cell replacement mechanism is herein described for the first time as an adaption mechanism of the humoral memory that allows incorporation of new antibody specificities while maintaining pre-existing ones. In immunological steady state, very low numbers of plasmablasts are detectable in any donor. These express IgA and receptors for mucosal homing, alpha4beta7 integrin and CCR10, and therefore most likely migrate into mucosal plasma cell depots and do not interfere with plasma cells of the bone marrow, preserving the stability of humoral memory during steady state. Hence, adaption of humoral memory is only possible during systemic immune reactions. Circulating mucosal plasmablasts produced during steady state remain detectable in patients with rheumatoid arthritis during B cell depletion therapy as well as in asplenic patients. Hence, this type of plasmablasts is self-sufficiently generated from mucosal B cells that are refractory to B cell depletion therapy. This work demonstrates a hitherto disregarded complexity of peripheral plasmablast and plasma cell subsets in healthy humans, with implications for the regulation of induction and maintenance of humoral memory.

Hemocompatibility tuning was adopted to explore and refine an innovative, GA-free preparation strategy combining decellularization, riboflavin/UV crosslinking, and low-energy electron irradiation (SULEEI) procedure. A SULEEI-protocol was established to avoid GA-dependent deterioration that results in insufficient long-term aortic valve bioprosthesis durability. Final SULEEI-pericardium, intermediate steps and GA-fixed reference pericardium were exposed in vitro to fresh human whole blood to elucidate effects of preparation parameters on coagulation and inflammation activation and tissue histology. The riboflavin/UV crosslinking step showed to be less efficient in inactivating extracellular matrix (ECM) protein activity than the GA fixation, leading to tissue-factor mediated blood clotting. Intensifying the riboflavin/UV crosslinking with elevated riboflavin concentration and dextran caused an enhanced activation of the complement system. Yet activation processes induced by the previous protocol steps were quenched with the final electron beam treatment step. An optimized SULEEI protocol was developed using an intense and extended, trypsin-containing decellularization step to inactivate tissue factor and a dextran-free, low riboflavin, high UV crosslinking step. The innovative and improved GA-free SULEEI-preparation protocol results in low coagulant and low inflammatory bovine pericardium for surgical application.