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- J-8841-2015 (1)
In several countries, a decline in mortality, case-fatality and recurrence rates of stroke was observed. However, studies investigating sex-specific and subtype-specific (pathological and etiological) time trends in stroke mortality, case-fatality and recurrence rates are scarce, especially in Germany. The decline in ischemic stroke mortality and case-fatality might be associated with the high quality of acute care of ischemic stroke, but the exact determinants of early outcome remains unknown for Germany.
Therefore, as first step of this thesis, we investigated the time trends of subtype- and sex-specific age- standardized stroke mortality rates in Germany from 1998 to 2015, by applying joinpoint regression on official causes of death statistics, provided by the Federal Statistical Office. Furthermore, a regional comparison of the time trends in stroke mortality between East and West was conducted. In the second step, time trends in case-fatality and stroke recurrence rates were analyzed using data from a population- based stroke register in Germany between 1996 and 2015. The analysis was stratified by sex and etiological subtype of ischemic stroke. In the third step, quality of stroke care and the association between adherence to measures of quality of acute ischemic stroke care and in-hospital mortality was estimated based on data from nine regional hospital-based stroke registers in Germany from the years 2015 and 2016.
We showed that in Germany, age-standardized stroke mortality declined by over 50% from 1998 to 2015 both, in women and men. Stratified by the pathological subtypes of stroke, the decrease in mortality was larger in ischemic stroke compared to hemorrhagic stroke. Different patterns in the time trends of stroke were observed for stroke subtypes, regions in Germany (former Eastern part of Germany (EG), former Western part of Germany (WG)) and sex, but in all strata a decline was found. By applying joinpoint regression, the number of changes in time trend differed between the regions and up to three changes in the trend in ischemic stroke mortality were detected. Trends in hemorrhagic stroke were in parallel between the regions with up to one change (in women) in joinpoint regression. Comparing the regions, stroke mortality was higher in EG compared to WG throughout the whole observed time period, however the differences between the regions started to diminish from 2007 onwards.
Further it was found that, based on the population-based Erlangen Stroke Project (ESPro), case-fatality and recurrence rates in ischemic stroke patients are still high in Germany. 46% died and 20% got a recurrent stroke within the first five years after stroke. Case-fatality rates declined statistically significant from 1996 to 2015 across all ischemic stroke patients and all etiological subtypes of ischemic stroke. Based on Cox regression no statistically significant decrease in stroke recurrence was observed.
Based on the pooled data of nine regional hospital-based stroke registers from the years 2015 and 2016 covering about 80% of all hospitalized stroke patients in Germany, a high quality of care of acute ischemic stroke patients, measured via 11 evidence-based quality indicators (QI) of process of care, was observed. Across all registers, most QI reached the predefined target values for good quality of stroke care. 9 out of 11 QI showed a significant association with 7-day in-hospital mortality. An inverse linear association between overall adherence to QI and 7-day in-hospital mortality was observed.
In conclusion, stroke mortality and case-fatality showed a favorable development over time in Germany, which might partly be due to improvements in acute treatment. This is supported by the association between overall adherence to quality of care and in-hospital mortality. However, there might be room for improvements in long-term secondary prevention, as no clear reduction in recurrence rates was observed.
Interleukin 2 (IL-2) was the first cytokine applied for cancer treatment in human history. It has been approved as monotherapy for renal cell carcinoma and melanoma by the FDA and does mediate the regression of the tumors in patients. One of the possible mechanisms is that the administration of IL-2 led to T lymphocytes expansion, including CD4+ and CD8+ T cells. In addition, a recent study demonstrated that antigen-specific T cells could also be expanded through the induction of IL-2, which plays a crucial role in mediating tumor regression. However, despite the long-term and extensive use of IL-2 in the clinic, the ratio of patients who get a complete response was still low, and only about one-fifth of patients showed objective tumor regression. Therefore, the function of IL-2 in cancer treatment should continue to be optimized and investigated. A study by Franz O. Smith et al. has shown that the combination treatment of IL-2 and tumor-associated antigen vaccine has a strong trend to increased objective responses compared to patients with melanoma receiving IL-2 alone. Peptide vaccines are anti-cancer vaccines able to induce a powerful tumor antigenspecific immune response capable of eradicating the tumors. According to the type of antigens, peptide vaccines can be classified into two distinct categories: Tumor-associated antigens (TAA) vaccine and tumor-specific neoantigens (TSA) vaccine. Currently, Peptide vaccines are mainly investigated in phase I and phase II clinical trials of human cancer patients with various advanced cancers such as lung cancer, gastrointestinal tumors, and breast cancers. Vaccinia virus (VACV) is one of the safest viral vectors, which has been wildly used in cancer treatment and pathogen prevention. As an oncolytic vector, VACV can carry multiple large foreign genes, which enable the virus to introduce diagnostic and therapeutic agents without dramatically reducing the viral replication. Meanwhile, the recombinant vaccinia virus (rVACV) can be easily generated by homologous recombination. Here, we used the vaccinia virus as the therapeutic cancer vector, expressing mouse Interleukin 2 (IL-2) and tumor-associated antigens simultaneously to investigate the combined effect of anti-tumor immune response in the 4T1 mouse tumor model. As expected, the VACV driven mIL-2 expression remarkably increased both CD4+ and CD8+ populations in vivo, and the virus-expressed tumor-associated peptides successfully elicited theantigen-specific T cell response to inhibit the growth of tumors. Furthermore, the experiments with tumor-bearing animals showed that the mIL-2 plus tumor antigens expressing VACV vector gave a better anti-cancer response than the mIL-2 alone expressing vector. The combinations did significantly more inhibit tumor growth than mIL-2 treatment alone. Moreover, the results confirmed our previous unpublished data that the mIL-2 expression driven by synthetic early/late promoter in the Lister strain VACV could enhance the tumor regression in the 4T1 mouse model.
The internal transcribed spacer 2 (ITS2) of the ribosomal gene repeat is an increasingly important phylogenetic marker whose RNA secondary structure is widely conserved across eukaryotic organisms. The ITS2 database aims to be a comprehensive resource on ITS2 sequence and secondary structure, based on direct thermodynamic as well as homology modelled RNA folds. Results: (a) A rebuild of the original ITS2 database generation scripts applied to a current NCBI dataset reveal more than 60,000 ITS2 structures. This more than doubles the contents of the original database and triples it when including partial structures. (b) The end-user interface was rewritten, extended and now features user-defined homology modelling. (c) Other possible RNA structure discovery methods (namely suboptimal and shape folding) prove helpful but are not able to replace homology modelling. (d) A use case of the ITS2 database in conjunction with other tools developed at the department gave insight into molecular phylogenetic analysis with ITS2.
Das Multiple Myelom (MM) ist eine unheilbare Erkrankung, die aus einer klonalen Proliferation maligner Plasmazellen im Knochenmark hervorgeht. Dabei liegt ein komplexes Signalnetzwerk vor, das zum Überleben und Wachstum der MM-Zellen führt. Das MM ist durch eine enorme genetische und phänotypische Heterogenität gekennzeichnet. Die konstitutive Aktivierung des PI3K/Akt-Signalwegs spielt bei ungefähr der Hälfte der Patienten mit MM eine wichtige Rolle für das Überleben der MM-Zellen und ist daher ein potentieller therapeutischer Ansatzpunkt. Isoform-spezifische Untersuchungen der katalytischen Untereinheiten der Klasse I-PI3K (p110α, p110β, p110γ, p110δ) sollten zur Erkenntnis führen, welche dieser Isoformen für das MM Zellüberleben wichtig sind, um spezifischere Behandlungen mit möglichst geringen Nebenwirkungen zu erlauben. Dafür wurden zunächst Isoform-spezifische Knockdown-Experimente mit MM Zelllinien durchgeführt und sowohl deren Überleben als auch die Aktivierung der nachgeschalteten Komponenten im PI3K Signalweg untersucht. Zur Verifizierung der Ergebnisse wurden sowohl MM Zelllinien als auch Primärzellen mit Isoform-spezifischen PI3K-Inhibitoren behandelt (BYL 719 für p110α, TGX 221 für p110β, CAY10505 für p110γ und CAL 101 für p110δ) und in gleicher Weise untersucht. In beiden Versuchsansätzen stellte sich die katalytische Untereinheit p110α als wichtigste Isoform für das Überleben von MM Zellen mit konstitutiv phosphoryliertem Akt Signal heraus. Weder der Knockdown noch die pharmakologische Inhibition der anderen drei Isoformen (p110β, p110γ, p110δ) führten in MM-Zelllinien zur Beeinträchtigung des Zellüberlebens. Auch reagierten die Primärzellen von MM Patienten größtenteils nicht mit Apoptose auf eine Behandlung mit TGX 221, CAY10505 oder CAL 101. Aufbauend auf der postulierten Bedeutung von p110α, wurde der dafür spezifische Inhibitor BYL 719 mit bereits klinisch etablierten Therapeutika in Kombination verwendet, woraus eine im Vergleich zur Einzelbehandlung verstärkte Apoptose resultierte. Insgesamt deuten diese Daten darauf hin, dass PI3K/p110α eine therapeutisch nutzbare Zielstruktur zur Behandlung des Multiplen Myeloms darstellt. Daher scheinen weitergehende prä-klinische Studien mit p110α Inhibitoren erfolgversprechend.
Der Meniskus, ein scheibenförmiger Faserknorpel, spielt im Kniegelenk eine bedeutende Rolle, weil er Kräfte und Druck im Kniegelenk gleichmäßig verteilt, Stöße dämpft sowie der Kraftübertragung und Stabilisierung dient. Durch die Entfernung des Gewebes, der sogenannten Totalmeniskektomie, nach einer Meniskusverletzung oder einem Riss, verändern sich die mechanischen Eigenschaften des Gelenks stark und verursachen durch die erhöhte Belastung der Gelenkflächen Arthrose. Arthrose ist weltweit die Häufigste aller Gelenkerkrankungen. Der Erhalt der körperlichen Leistungsfähigkeit und Mobilität bis ins hohe Alter sowie die Bewahrung der Gesundheit von Herz-Kreislauf- und Stoffwechselorganen zählen aufgrund des demografischen Wandels zu den großen medizinischen Herausforderungen. Die Erkrankung des muskuloskelettalen Systems stellte 2010 im Bundesgebiet die am häufigsten vorkommende Krankheitsart dar.
Während Risse in den äußeren Teilen des Meniskus aufgrund des Anschlusses an das Blutgefäßsystem spontan heilen können, können sie dies in tieferen Zonen nicht. Durch die begrenzte Heilungsfähigkeit des Knorpels bleibt langfristig der Einsatz eines Ersatzgewebes die einzige therapeutische Alternative.
In der vorliegenden Arbeit wurde als therapeutische Alternative erfolgreich ein vaskularisiertes Meniskusersatzgewebe mit Methoden des Tissue Engineering entwickelt. Es soll in Zukunft als Implantat Verwendung finden. Tissue Engineering ist ein interdisziplinäres Forschungsfeld, in dem Gewebe außerhalb des Körpers generiert werden. Schlüsselkomponenten sind Zellen, die aus einem Organismus isoliert werden, und Trägerstrukturen, die mit Zellen besiedelt werden. Die Biomaterialien geben den Zellen eine geeignete Umgebung, die die Extrazelluläre Matrix (EZM) ersetzen soll, um die Funktion der Zellen beizubehalten, eigene Matrix zu bilden. Zum Erhalt eines funktionelles Gewebes werden oftmals dynamische Kultursysteme, sogenannte Bioreaktoren, verwendet, die natürliche Stimuli wie beispielsweise den Blutfluss oder mechanische Kompressionskräfte während der in vitro Reifungsphase des Gewebes, zur Verfügung stellen. Das Gewebekonstrukt wurde auf Basis natürlicher Biomaterialien aufgebaut, unter Verwendung ausschließlich primärer Zellen, die später direkt vom Patienten gewonnen werden können und damit Abstoßungsreaktionen auszuschließen sind. Da der Meniskus teilvaskularisiert ist und die in vivo Situation des Gewebes bestmöglich nachgebaut werden sollte, wurden Konstrukte mit mehreren Zelltypen, sogenannte Ko-Kulturen aufgebaut. Neben mikrovaskulären Endothelzellen (mvEZ) und Meniskuszellen (MZ) erfolgten Versuche mit mesenchymalen Stammzellen (MSZ).
Zur Bereitstellung einer zelltypspezifischen Matrixumgebung, diente den mvEZ ein Stück Schweinedarm mit azellularisierten Gefäßstrukturen (BioVaSc®) und den MZ diente eine geeig- nete Kollagenmatrix (Kollagen Typ I Hydrogel). Die Validierung und Charakterisierung des aufgebauten 3D Meniskuskonstrukts, welches in einem dynamischen Perfusions-Bioreaktorsystem kultiviert wurde, erfolgte mit knorpeltypischen Matrixmarkern wie Aggrekan, Kollagen Typ I, II und X sowie mit den Transkriptionsfaktoren RunX2 und Sox9, die in der Knorpelentstehung von großer Bedeutung sind. Zusätzlich erfolgten Auswertungen mit endothelzellspezifischen Markern wie vWF, CD31 und VEGF, um die Vaskularisierung im Konstrukt nachzuweisen. Analysiert wurden auch die Zellvitalitäten in den Konstrukten.
Aufgrund einer nur geringen Verfügbarkeit von MZ wurden Kulturansätze mit alternativen Zellquellen, den MSZ, durchgeführt. Dafür erfolgte zunächst deren Isolation und Charakterisierung und die Auswahl einer geeigneten 3D Kollagenmatrix. Die beste Zellintegration der MSZ konnte auf einer eigens hergestellten elektrogesponnenen Matrix beobachtet werden. Die Matrix besteht aus zwei unterschiedlichen Kollagentypen, die auf insgesamt fünf Schichten verteilt sind. Die Fasern besitzen weiter unterschiedliche Ausrichtungen. Während die Kollagen Typ I Fasern in den äußeren Schichten keiner Ausrichtung zugehören, liegen die Kollagen Typ II Fasern in der mittleren Schicht parallel zueinander. Der native Meniskus war für den Aufbau einer solchen Kollagen-Trägerstruktur das natürliche Vorbild, das imitiert werden sollte. Nach der Besiedelung der Matrix mit MSZ, konnte eine Integration der Zellen bereits nach vier Tagen bis in die Mittelschicht sowie eine spontane chondrogene Differenzierung nach einer insgesamt dreiwöchigen Kultivierung gezeigt werden. Das Biomaterial stellt in Hinblick auf die Differenzierung der Zellen ohne die Zugabe von Wachstumsfaktoren eine relevante Bedeutung für klinische Studien dar.
Zur Kultivierung des 3D Meniskuskonstrukts wurde ein Bioreaktor entwickelt. Mit diesem können neben Perfusion der Gefäßsysteme zusätzlich Kompressionskräfte sowie Scherspannungen auf das Ersatzgewebe appliziert und die Differenzierung von MZ bzw. MSZ während der in vitro Kultur über mechanische Reize stimuliert werden. Ein anderes Anwendungsfeld für den neuartigen Bioreaktor ist seine Verwendung als Prüftestsystem für die Optimierung und Qualitätssicherung von Gewebekonstrukten.
Malaria is a challenging infection with increasing and wide-spread treatment failure risk due to resistance. With a estimated death toll of 1-3 Million per year, most cases of Malaria affect children under the age of five years in Sub-Saharan Africa. In this thesis, I analyse the current status of malaria control (focussing on diagnosis and therapy) in Burkina Faso to show how this disease burdens public health in endemic countries and to identify possible approaches to improvement. MB is discussed as a therapeutic option under these circumstances.
Burkina Faso is used as a representative example for a country in Sub-Saharan Africa with high endemicity for malaria and is here portrayed, its health system characterised and discussed under socioeconomic aspects.
More than half of this country’s population live in absolute poverty. The burden that malaria, especially treatment cost, poses on these people cannot be under-estimated.
A retrospective study of case files from the university pediatric hospital in Burkina Faso’s capital, Ouagadougou, shows that the case load is huge, and especially the specific diagnosis of severe malaria is difficult to apply in the hospital’s daily routine. Treatment policy as proposed by WHO is not satisfactorily implemented neither in home treatment nor in health services, as data for pretreatment clearly show.
In the face of growing resistance in malaria parasites, pharmacological combination therapies are important. Artemisinins currently are the last resort of malaria therapy. As I show with homology models, even this golden bullet is not beyond resistance development. Inconsidered mass use has rendered other drugs virtually useless before. Artemisinins should thus be protected similar to reserve antibiotics against multi-resistant bacteria.
There is accumulating evidence that MB is an effective drug against malaria. Here the biological effects of both MB alone and in combination therapy is explored via modeling and experimental data. Several different lines of MB attack on Plasmodium redox defense were identified by analysis of the network effects. Next, CQ resistance based on Pfmdr1 and PfCRT transporters as well as SP resistance were modeled in silico. Further modeling shows that MB has a favorable synergism on antimalarial network effects with these commonly used antimalarial drugs, given their correct application.
Also from the economic point of view MB shows great potential: in terms of production price, it can be compared to CQ, which could help to diminuish the costs of malaria treatment to affordable ranges for those most affected and struk by poverty.
Malaria control is feasible, but suboptimal diagnosis and treatment are often hindering the achievment of this goal. In order to achieve malaria control, more effort has to be made to implement better adjusted and available primary treatment strategies for uncomplicated malaria that are highly standardised. Unfortunately, campaigns against malaria are chronically underfinanced. In order to maximize the effect of available funds, a cheap treatment option is most important, especially as pharmaceuticals represent the biggest single matter of expense in the fight against malaria.
Dynamic interactions and their changes are at the forefront of current research in bioinformatics and systems biology. This thesis focusses on two particular dynamic aspects of cellular adaptation: miRNA and metabolites.
miRNAs have an established role in hematopoiesis and megakaryocytopoiesis, and platelet miRNAs have potential as tools for understanding basic mechanisms of platelet function. The thesis highlights the possible role of miRNAs in regulating protein translation in platelet lifespan with relevance to platelet apoptosis and identifying involved pathways and potential key regulatory molecules. Furthermore, corresponding miRNA/target mRNAs in murine platelets are identified. Moreover, key miRNAs involved in aortic aneurysm are predicted by similar techniques. The clinical relevance of miRNAs as biomarkers, targets, resulting later translational therapeutics, and tissue specific restrictors of genes expression in cardiovascular diseases is also discussed.
In a second part of thesis we highlight the importance of scientific software solution development in metabolic modelling and how it can be helpful in bioinformatics tool development along with software feature analysis such as performed on metabolic flux analysis applications. We proposed the “Butterfly” approach to implement efficiently scientific software programming. Using this approach, software applications were developed for quantitative Metabolic Flux Analysis and efficient Mass Isotopomer Distribution Analysis (MIDA) in metabolic modelling as well as for data management. “LS-MIDA” allows easy and efficient MIDA analysis and, with a more powerful algorithm and database, the software “Isotopo” allows efficient analysis of metabolic flows, for instance in pathogenic bacteria (Salmonella, Listeria). All three approaches have been published (see Appendices).
Platelets are important players in haemostasis and their activation is essential to limit post-traumatic blood loss upon vessel injury. On the other hand, pathological platelet activation may lead to thrombosis resulting in myocardial infarction and stroke. Platelet activation and subsequent thrombus formation are, therefore, tightly regulated and require a well-defined interplay of platelet surface receptors, intracellular signalling molecules, cytoskeletal rearrangements and the activation of the coagulation cascade.
In vivo thrombosis and haemostasis models mimic thrombus formation at sites of vascular lesions and are frequently used to assess thrombotic and haemostatic functions of platelets. In this dissertation, different in vivo models were used in mice to address the question at what level a reduced platelet count (PC) compromises stable thrombus formation. To study this, mice were rendered thrombocytopenic by low-dose anti-GPIbα antibody treatment and subjected to a tail bleeding time assay as well as to four different in vivo thrombosis models. Haemostasis and occlusive thrombus formation in small vessels were only mildly affected even at severe reductions of the PC. In contrast, occlusive thrombus formation in larger arteries required higher PCs demonstrating that considerable differences in the sensitivity for PC reductions exist between these models.
In a second part of this study, mice were rendered thrombocytopenic by injection of high-dose anti-GPIbα antibody which led to the complete loss of all platelets from the circulation for several days. During recovery from thrombocytopenia, the newly generated platelet population was characterised and revealed a defect in immunoreceptor tyrosine-based activation motif (ITAM)-signalling. This defect translated into impaired arterial thrombus formation.
To further investigate ITAM-signalling in vivo, genetically modified mice were analysed which display a positive or negative regulation of platelet ITAM-signalling in vitro. Whereas mice lacking the adapter Grb2 in platelets showed a delayed thrombus formation in vivo after acetylsalicylic acid treatment, Clp36ΔLIM bone marrow chimeric mice and SLAP/SLAP2-deficient mice displayed pro-thrombotic properties in vivo. Finally, mice lacking the adapter protein EFhd2 were analysed in vitro and in vivo. However, EFhd2-deficient platelets showed only a minor increase in the procoagulant activity compared to control.
Staphylococcus aureus (SA) causes nosocomial infections including life threatening sepsis by multi-resistant strains (MRSA). It has the ability to form biofilms to protect it from the host immune system and from anti staphylococcal drugs. Biofilm and planctonic life style is regulated by a complex Quorum-Sensing (QS) system with agr as a central regulator. To study biofilm formation and QS mechanisms in SA a Boolean network was build (94 nodes, 184 edges) including two different component systems such as agr, sae and arl. Important proteins such as Sar, Rot and SigB were included as further nodes in the model. System analysis showed there are only two stable states biofilm forming versus planctonic with clearly different subnetworks turned on. Validation according to gene expression data confirmed this. Network consistency was tested first according to previous knowledge and literature. Furthermore, the predicted node activity of different in silico knock-out strains agreed well with corresponding micro array experiments and data sets. Additional validation included the expression of further nodes (Northern blots) and biofilm production compared in different knock-out strains in biofilm adherence assays. The model faithfully reproduces the behaviour of QS signalling mutants. The integrated model allows also prediction of various other network mutations and is supported by experimental data from different strains. Furthermore, the well connected hub proteins elucidate how integration of different inputs is achieved by the QS network. For in silico as well as in vitro experiments it was found that the sae-locus is also a central modulator of biofilm production. Sae knock-out strains showed stronger biofilms. Wild type phenotype was rescued by sae complementation. To elucidate the way in which sae takes influence on biofilm formation the network was used and Venn-diagrams were made, revealing nodes regulated by sae and changed in biofilms. In these Venn-diagrams nucleases and extracellular proteins were found to be promising nodes. The network revealed DNAse to be of great importance. Therefore qualitatively the DNAse amount, produced by different SA mutants was measured, it was tried to dissolve biofilms with according amounts of DNAse and the concentration of nucleic acids, proteins and polysaccharides were measured in biofilms of different SA mutants.
With its thorough validation the network model provides a powerful tool to study QS and biofilm formation in SA, including successful predictions for different knock-out mutant behaviour, QS signalling and biofilm formation. This includes implications for the behaviour of MRSA strains and mutants. Key regulatory mutation combinations (agr–, sae–, sae–/agr–, sigB+, sigB+/sae–) were directly tested in the model but also in experiments. High connectivity was a good guide to identify master regulators, whose detailed behaviour was studied both in vitro and in the model. Together, both lines of evidence support in particular a refined regulatory role for sae and agr with involvement in biofilm repression and/or SA dissemination. With examination of the composition of different mutant biofilms as well as with the examination of the reaction cascade that connects sae to the biofilm forming ability of SA and also by postulating that nucleases might play an important role in that, first steps were taken in proving and explaining regulatory links leading from sae to biofilms. Furthermore differences in biofilms of different mutant SA strains were found leading us in perspective towards a new understanding of biofilms including knowledge how to better regulate, fight and use its different properties.
Traditionally, ischemic stroke has been regarded as the mere consequence of cessation of cerebral blood flow, e.g. due to the thromboembolic occlusion of a major brain supplying vessel. However, the simple restoration of blood flow via thrombolysis and/or mechanical recanalization alone often does not guarantee a good functional outcome. It appears that secondary detrimental processes are triggered by hypoxia and reoxygenation, which are referred to as ischemia/reperfusion (I/R) injury. During recent years it became evident that, beside thrombosis inflammation and edema formation are key players in the pathophysiology of cerebral ischemia. The contact-kinin system represents an interface between thrombotic, inflammatory and edematous circuits. It connects the intrinsic coagulation pathway with the plasma kallikrein-kinin system (KKS) via coagulation factor FXII.
The serine protease inhibitor C1-inhibitor (C1-INH) has a wide spectrum of inhibitory activities and counteracts activation of the contact-kinin system at multiple levels. The first part of the thesis aimed to multimodally interfere with infarct development by C1-INH and to analyze modes of actions of human plasma derived C1-INH Berinert® P in a murine model of focal cerebral ischemia. It was shown that C57BL/6 mice following early application of 15.0 units (U) C1-INH, but not 7.5 U developed reduced brain infarctions by ~60% and less neurological deficits in the model of transient occlusion of the middle cerebral artery (tMCAO). This protective effect was preserved at more advanced stages of infarction (day 7), without increasing the risk of intracerebral bleeding or affecting normal hemostasis. Less neurological deficits could also be observed with delayed C1-INH treatment, whereas no improvement was achieved in the model of permanent MCAO (pMCAO). Blood-brain-barrier (BBB) damage, inflammation and thrombosis were significantly improved following 15.0 U C1-INH application early after onset of ischemia. Based on its strong antiedematous, antiinflammatory and antithrombotic properties C1-INH constitutes a multifaceted therapeutic compound that protects from ischemic neurodegeneration in ‘clinically meaningful’ settings.
The second part of the thesis addresses the still elusive functional role of macrophages in the early phase of stroke, especially the role of the macrophage-specific adhesion molecule sialoadhesin (Sn). For the first time, sialoadhesin null (Sn-/-) mice, homozygous deficient for Sn on macrophages were subjected to tMCAO to assess the clinical outcome. Neurological and motor function was significantly improved in Sn-/- mice on day 1 after ischemic stroke compared with wildtype (Sn+/+) animals. These clinical improvements were clearly detectable even on day 3 following tMCAO. Infarctions on day 1 were roughly the same size as in Sn+/+ mice and did not grow until day 3. No intracerebral bleeding could be detected at any time point of data acquisition. Twenty four hours after ischemia a strong induction of Sn was detectable in Sn+/+ mice, which was previously observed only on perivascular macrophages in the normal brain. Deletion of Sn on macrophages resulted in less disturbance of the BBB and a reduced number of CD11b+ (specific marker for macrophages/microglia) cells, which, however, was not associated with altered expression levels of inflammatory cytokines. To further analyze the function of macrophages following stroke this thesis took advantage of LysM-Cre+/-/IKK2-/- mice bearing a nuclear factor (NF)-ϰB activation defect in the myeloid lineage, including macrophages. Consequently, macrophages were not able to synthesize inflammatory cytokines under the control of NF-ϰB. Surprisingly, infarct sizes and neurological deficits upon tMCAO were roughly the same in conditional knockout mice and respective wildtype littermates. These findings provide evidence that macrophages do not contribute to tissue damage and neurological deficits, at least, not by release of inflammatory cytokines in the early phase of cerebral ischemia. In contrast, Sn which is initially expressed on perivascular macrophages and upregulated on macrophages/microglia within the parenchyma following stroke, influenced functional outcome.