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Die alveoläre Echinokokkose (AE), die durch den Fuchsbandwurm Echinococcus multilocularis verursacht wird, ist eine seltene jedoch schwere und oft tödlich verlaufende Erkrankung. Aufgrund der späten Diagnosestellung sind kurative Behandlungsmethoden häufig nicht durchführbar und als einzige Behandlungsmöglichkeit bleibt eine lebenslange und nebenwirkungsreiche Therapie mit Benzimidazolen. Verbesserte Therapieoptionen durch die Entwicklung neuer Medikamente sind dringend notwendig. Hierfür kann es hilfreich sein die Biologie des Fuchsbandwurmes und die Kommunikationswege zwischen Parasit und Wirt zu verstehen. Bereits in vorherigen Arbeiten als auch in dieser Arbeit erwiesen sich evolutionsgeschichtlich konservierte Signalwege als Kommunikationsweg zwischen dem Fuchsbandwurm und seinem Wirt von zentraler Rolle.
Die Entschlüsselung des Echinococcus-Genoms gab Hinweise darauf, dass ein Mitglied der Tumornekrosefaktor-Rezeptor-Superfamilie, jedoch kein endogener TNF α ähnlicher Ligand im Genom kodiert wird. Ein Mitglied der TNFR-Superfamilie des Fuchsbandwurmes (EmTNFR) wurde in dieser Arbeit als membranständiger Rezeptor mit einer intrazellulären Todesdomäne (DD) und hoher Ähnlichkeit zum humanen Typ 16 der TNF-Rezeptor-Superfamilie, auch 〖p75〗^NTR genannt, charakterisiert. Sowohl in bioinformatischen als auch in Sequenzanalysen wurden drei alternative Splicing-Formen von emtnfr (emtnfr, emtnfr-v2 und emtnfr-v3) nachgewiesen. emtnfr-v2 entsteht durch Alternatives Splicing und kodiert ein Protein, das keine intrazelluläre Todesdomäne besitzt. emtnfr-v3 verwendet einen alternativen Transkriptionstart und wird von den letzten 3 Exons von emtnfr kodiert. emtnfr-v3, kodiert ein Protein ohne extrazelluläre Region, aber mit intrazellulärer Todesdomäne. Ein löslicher TNF-Rezeptor konnte auf Proteinebene nicht nachgewiesen werden. Aufgrund von phylogenetischen Analysen und der Rezeptor-Struktur ist zu vermuten, dass EmTNFR ein p75NTR Homolog ist und damit der ursprünglichen Form der TNF-Rezeptoren entspricht. Mitglieder eines intrazellulären TNF-Signalweges wurden in bioinformatischen Analysen beim Fuchsbandwurm E. multilocularis identifiziert.
Expressionsuntersuchungen zeigten sowohl in Trankriptomdaten als auch auf Proteinebene eine starke Expression von EmTNFR in Primärzellen und im Metazestoden (MZ), dem pathogenen Stadium für den Zwischenwirt. Echinococcus-Stammzellkulturen zeigten nach RNA-Interferenz-basiertem Knockdown des EmTNFR-kodierenden Gens deutliche Entwicklungsdefekte. Des Weiteren zeigten Echinococcus-Stammzellkulturen nach einer Behandlung mit TNF-α, einem potentiellen Liganden des TNF-Rezeptors und einem zentralen Zytokin in der Immunabwehr des Zwischenwirtes, Entwicklungsfortschritte, wie eine verbesserte Bildung von MZ aus Stammzellen. Zusätzlich wurde in whole-mount in situ Hybridisierungs-Versuchen eine ubiquitäre Expression von emtnfr in der Germinalschicht des MZ sowie eine Spezifität von emtnfr für den MZ, welcher ursächlich für die AE ist, nachgewiesen. Somit scheinen sowohl EmTNFR als auch TNF-α eine wichtige Funktion bei der Entwicklung und Etablierung des Fuchsbandwurmes während der frühen Phase der Infektion des Zwischenwirtes zu haben. TNF-α könnte ein weiterer Faktor für den ausgeprägten Organtropismus des Parasiten zur Leber sein, denn dort bestehen durch Kupfferzellen produzierte hohe lokale Konzentration von TNF-α.
Zusammenfassend deuten die hier erarbeiteten Daten darauf hin, dass EmTNFR über die Bindung von Wirts-TNF-α bei der frühen Entwicklung des Echincoccus-Metazestoden eine Rolle spielt.
Laut Statistischem Bundesamt (Destatis) starben allein im Jahr 2020 zirka 985.500 Menschen. Die häufigsten Todesursachen waren Herz-Kreislauf- und Krebs-Erkrankungen (vgl. Destatis 2020). Die meisten Menschen haben den Wunsch zuhause zu sterben, doch die Mehrheit stirbt in Krankenhäusern, Alten- und Pflegeheimen (vgl. DHPV 2017; Dasch et al. 2015). Der Tod eines nahestehenden Menschen kann bei Hinterbliebenen zu großen Belastungen, gesundheitlichen Problemen sowie einer gesteigerten Mortalität führen (vgl. Stroebe et al. 2007). Ziel dieser Arbeit war es, mit Hilfe von halbstandardisierten Interviews mit 30 Trauernden Faktoren herauszuarbeiten, die sich förderlich oder hinderlich auf den Trauerprozess auswirken können. Die Interviews wurden mit der Transkriptionssoftware f4transkript verschriftlicht und mittels qualitativer Inhaltsanalyse nach Mayring ausgewertet. Es entstand ein Kategoriensystem mit je vier Oberkategorien innerhalb der zwei Hauptkategorien, Förderliche und Hinderliche Faktoren. Folgende Faktoren konnten identifiziert werden: Förderliche Faktoren in der Oberkategorie Betreuung der erkrankten und trauernden Person sind eine gute Symptomkontrolle sowie der verständnisvolle Umgang mit den Nahestehenden, während mangelhafte Kommunikation wiederum hinderlich für eine positive Trauerbewältigung ist. In der Oberkategorie Intrapersonale Faktoren sind die Antizipation des Todes sowie die Auseinandersetzung mit der Trauer förderlich, während negative Gefühle (z.B. Schuldgefühle, Hilfslosigkeit) sich in besagter Hinsicht hinderlich auswirken. In der Oberkategorie Beziehung zur verstorbenen Person können die optimale Nutzung der verbliebenen Zeit sowie der offene Umgang mit der Erkrankung förderliche Faktoren darstellen, während ein “schwieriger“ Abschied sowie ungeklärte Konflikte oder offene Fragen Hindernisse für den Trauerprozess sein können. In der Oberkategorie Soziales Umfeld sind die unaufgeforderte Unterstützung, die emotionale Begleitung sowie ein flexibler Arbeitgeber förderlich. Streitigkeiten innerhalb der Familie und Unverständnis der Mitmenschen dagegen sind hinderlich. Eine gute und würdevolle Sterbebegleitung, wie sie in der Palliativmedizin in der Regel gewährleistet ist, ist von großer Bedeutung für einen gelingenden Trauerprozess. Daher sollte eine palliative Haltung disziplinübergreifend vorangebracht und ausgebaut werden. In der Gesellschaft sollte Trauernden mehr Toleranz und Verständnis entgegengebracht und offen mit dem Thema Tod und Sterben umgegangen werden.
Site-directed bioorthogonal conjugation techniques have substantially advanced research in numerous areas. Their exceptional value reflects in the extent of applications, that have been realized with spacial-controlled bioorthogonal reactions. Specific labeling of surfaces, proteins, and other biomolecule allows for new generations of drug delivery, tracking, and analyzing systems. With the continuous advance and refinement of available methods, this field of research will become even more relevant in the time to come. Yet, as individual as the desired purpose is, as different can be the most suitable modification strategy. In this thesis, two different bioconjugation approaches, namely CuAAC and factor XIIIa mediated ligation, are used in distinct application fields, featuring eGFP as a model protein showcasing the advantages as well as the challenges of each technique.
The introduction of a unique accessible functionality is the most critical feature of a site-specific reaction, and the first considerable hurdle to clear. While most surfaces, peptides, or small molecules might require less expenditure to modulate, equipping large biomolecules like proteins with additional traits requires careful consideration to preserve the molecule’s stability and function. Therefore, the first section of this project comprises the engineering of eGFP via rational design. Initially, wild-type eGFP was subcloned, expressed, and characterized to serve as a reference value for the designed variants. Subsequently, eGFP was mutated and expressed to display a recognition site for factor XIIIa. Additionally, a second mutant harbored a TAG-codon to enable amber codon suppression and consequently the incorporation of the alkyne bearing unnatural amino acid Plk to support a CuAAC reaction. Fluorescence spectroscopy was used to confirm that the fluorescent properties of all expressed muteins were identically equal to wild-type eGFP, which is a reliable marker for the intact barrel structure of the protein. Trypsin digestion and HPLC were deployed to confirm each protein variant's correct sequence and mass.
The second part of this work focuses on the conjugation of cargo molecules deploying the chosen approaches. Solid-phase peptide synthesis was used to create a peptide that served as a lysine donor substrate in the crosslinking mechanism of FXIIIa. Additionally, the peptide was provided with a cysteine moiety to allow for highly flexible and simple loading of desired cargo molecules via conventional thiol-Michael addition, thus establishing an adaptive labeling platform. The effective ligation was critically reviewed and confirmed by monitoring the exact mass changes by HPLC. Protocols for attaching payloads such as biotin and PEG to the linker peptide were elaborated. While the biotin construct was successfully conjugated to the model protein, the eGFP-PEG linkage was not achieved judging by SDS-PAGE analysis. Furthermore, featuring isolated peptide sequences, the properties of the FXIIIa-mediated reaction were characterized in detail. Relative substrate turnover, saturation concentrations, by-product formation, and incubation time were comprehensively analyzed through HPLC to identify optimal reaction conditions. CuAAC was successfully used to label the Plk-eGFP mutein with Azide-biotin, demonstrated by western blot imaging.
Within the last part of this study, the application of the conjugation systems was extended to different surfaces. As regular surfaces do not allow for immediate decoration, supplementary functionalization techniques like gold-thiol interaction and silanization on metal oxides were deployed. That way gold-segmented nanowires and Janus particles were loaded with enoxaparin and DNA, respectively. Nickel and cobalt nanowires were modified with silanes that served as linker molecules for subsequent small molecule attachment or PEGylation. Finally, the eGFP muteins were bound to a particle surface in a site-specific manner. Beads displaying amino groups were utilized to demonstrate the effective use of FXIIIa in surface modification. Moreover, the bead’s functional moieties were converted to azides to enable CuAAC “Click Chemistry” and direct comparison. Each modification was analyzed and confirmed through fluorescence microscopy.
The aim of the present work was to improve drug monitoring in patients with various diseases in the context of precision medicine. This was pursued through the development and validation of mass spectrometric methods for determining the drug concentrations of kinase inhibitors and their clinical application. Besides conventional approaches to determine plasma level concentrations, the focus was also on alternative sampling techniques using volumetric absorptive microsampling (VAMS).
A conventional LC-MS/MS method was developed for the determination of cabozantinib in human EDTA plasma and validated according to the guidelines of the European and United States drug authorities (EMA, FDA). The method met the required criteria for linearity, accuracy and precision, selectivity, sensitivity, and stability of the analyte. Validation was also performed for dilution integrity, matrix effect, recovery, and carry-over, with results also in accordance with the requirements. The importance of monitoring the exposure of cabozantinib was demonstrated by a clinical case report of a 34-year-old female patient with advanced adrenocortical carcinoma who also required hemodialysis due to chronic kidney failure. Expected cabozantinib plasma concentrations were simulated for this off-label use based on a population pharmacokinetic model. It was shown that the steady state trough levels were much lower than expected but could not be explained by hemodialysis. Considering the critical condition and potential drug-drug interaction with metyrapone, a substance the patient had taken among several others during the observation period, individual pharmacokinetics could consequently not be estimated without drug monitoring.
In addition, a VAMS method for simultaneous determination of ten kinase inhibitors from capillary blood was developed. This microsampling technique was mainly characterized by the collection of a defined volume of blood, which could be dried and subsequently analyzed. The guidelines for bioanalytical method validation of the EMA and FDA were also used for this evaluation. As the nature of dried blood samples differs from liquid matrices, further parameters were investigated. These include the investigation of the hematocrit effect, process efficiency, and various stability conditions, for example at increased storage temperatures. The validation showed that the developed method is suitable to analyze dried matrix samples accurate, precise, and selective for all analytes. Apart from the stability tests, all acceptance criteria were met. The decreased stability of two analytes was probably due to the reproducible but reduced recovery. In vitro studies provided results on the VAMS-to-plasma correlation to predict the analyte distribution between both matrices, at least in an exploratory manner. It revealed a heterogeneous picture of analytes with different VAMS-to-plasma distributions. Furthermore, the analysis of 24 patient samples indicated the applicability of at-home VAMS. Both should be confirmed later as part of the clinical validation.
The clinical investigation of the VAMS method pursued two objectives. On the one hand, the simultaneous collection of VAMS and serum samples should enable a conversion of the determined concentrations and, on the other hand, the feasibility of autonomous microsampling at home should be examined more closely. For the former, it could be shown that different conversion methods are suitable for converting VAMS concentrations into serum levels. The type of conversion was secondary for the prediction. However, the previously defined criteria could not be fulfilled for all five kinase inhibitors investigated. The framework conditions of the study led to increased variability, especially for analytes with short half-life. A low and varying hematocrit, caused by the underlying disease, also made prediction difficult for a specific patient collective. For the second objective, investigating the feasibility of VAMS, different aspects were considered. It could be shown that the majority of patients support home-based microsampling. The acceptance is likely to increase even further when microsampling is no longer part of a non-interventional study, but participation is accompanied by targeted monitoring and subsequent adjustment of the therapy. The fact that additional training increases understanding of the correct sampling procedure is also a source of confidence. Demonstrated stability during storage under real-life conditions underlines the practicality of this sampling technique.
Taken together, mass spectrometric methods for both plasma and VAMS could be developed and validated, and their clinical application could be successfully demonstrated. The availability of simple bioanalytical methods to determine kinase inhibitor exposure could improve access to prospective studies and thus facilitate the implementation of routine therapeutic drug monitoring.
The relationship between a farmer and their cultivated crops in agriculture is multifaceted, with pathogens affecting both the farmer and crop, and weeds that take advantage of resources provided by farmers. For my doctoral thesis, I aimed to gain a comprehensive understanding of the ecology and symbiosis of fungus farming ambrosia beetles.
Through my research, I discovered that the microbial composition of fungus gardens, particularly the mutualists, is significantly influenced by the presence of both adults and larvae. The recognition of both beneficial and harmful symbionts is crucial for the success of ambrosia beetles, who respond differently depending on their life stage and the microbial species they encounter, which can contribute to the division of labour among family groups. The presence of antagonists and pathogens in the fungus garden depends on habitat and substrate quality, and beetle response to their introduction results in behavioural and developmental changes. Individual and social immunity measures, as well as changes in bacterial and fungal communities, were detected as a result of pathogen introduction. Additionally, the ability of ambrosia beetles to establish two nutritional fungal species depends on several factors. These insects must strike a balance between their essential functions and adapt to the constantly changing ecological and social conditions, which demonstrates their adaptive flexibility. However, interpreting data from laboratory studies should be approached with caution, as the natural environment allows for more flexibility and the potential for other beneficial symbionts to become more prominent if required.
To aid in my research, I designed primers that use the ‘fungal large subunit’ (LSU) as genetic marker to identify and differentiate mutualistic and antagonistic fungi in X. saxesenii. The primers were able to distinguish closely related species of the Ophiostomataceae and other fungal symbionts. This allowed me to associate the abundance of key fungal taxa with factors such as the presence of beetles, the nest's age and condition, and the various developmental stages present. My primers are a valuable tool for understanding fungal communities, including their composition and the identification of previously unknown functional symbionts. However, some aspects should be approached with caution due to the exclusion of non-amplified taxa in the relative fungal community compositions.
A highly regulated microenvironment is essential in maintaining normal functioning of the central nervous system (CNS). The existence of a biological barrier, termed as the blood-brain barrier (BBB), at the blood to brain interface effectively allows for selective passage of substances and pathogens into the brain (Kadry, Noorani et al. 2020). The BBB chiefly serves in protecting the brain from extrinsic toxin entry and pathogen invasions. The BBB is formed mainly by brain capillary endothelial cells (BCECs) which are responsible for excluding ∼ 100% of large-molecule neurotherapeutics and more than 98% of all small-molecule drugs from entry into the brain. Minimal BBB transport of major potential CNS drugs allows for attenuated effective treatments for majority of CNS disorders (Appelt-Menzel, Oerter et al. 2020). Animals are generally used as model systems to study neurotherapeutic delivery into the brain, however due to species based disparity, experimental animal models lead to several false positive or false negative drug efficacy predictions thereby being unable to fully predict effects in humans (Ruck, Bittner et al. 2015). An example being that over the last two decades, much of the studies involving animals lead to high failure rates in drug development with ~ 97% failure in cancers and ~ 99% failure for Alzheimer´s disease (Pound 2020). Widespead failures in clinical trials associated with neurological disorders have resulted in questions on whether existing preclinical animal models are genuinely reflective of the human condition (Bhalerao, Sivandzade et al. 2020). Apart from high failure rates in humans, the costs for animal testings is extremely high. According to the Organisation for Economic Co-operation and Development (OECD), responsible for determining animal testing guidelines and methodology for government, industry, and independent laboratories the average cost of a single two-generation reproductive animal toxicity study worldwide is 318,295 € and for Europe alone is ~ 285,842 € (Van Norman 2019). Due to these reasons two separate movements exist within the scientific world, one being to improve animal research and the other to promote new approach methodologies with the European government setting 2025 - 2035 as a deadline for gradually disposing the use of animals in pharmaceutical testing (Pound 2020).
The discovery of human induced pluripotent stem cell (hiPSC) technology in 2006 (Takahashi and Yamanaka 2006, Takahashi, Tanabe et al. 2007) revolutionized the field of drug discovery in-vitro. HiPSCs can be differentiated into various tissue types that mimic disease phenotypes, thereby offering the possibility to deliver humanized in-vitro test systems. With respect to the BBB, several strategies to differentiate hiPSCs to BCECs (iBCECs) are reported over the years (Appelt-Menzel, Oerter et al. 2020). However, iBCECs are said to possess an epithelial or undifferentiated phenotype causing incongruity in BBB lineage specifications (Lippmann,
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Azarin et al. 2020). Therefore, in order to identify a reliable differentiation strategy in deriving iBCECs possessing hallmark BBB characteristics, which can be used for downstream applications, the work in this thesis compared two methods, namely the co-differentiation (CD) and the directed differentiation (DD). Briefly, CD mimics a brain like niche environment for iBCEC specification (Lippmann, Al-Ahmad et al. 2014), while DD focuses on induction of the mesoderm followed by iBCEC specification (Qian, Maguire et al. 2017). The results obtained verified that while iBCECs derived via CD, in comparison to human BCEC cell line hCMEC/D3 showed the presence of epithelial transcripts such as E-Cadherin (CDH1), and gene level downregulation of endothelial specific platelet endothelial cell adhesion molecule-1 (PECAM-1) and VE-cadherin (CDH5) but demonstrated higher barrier integrity. The CD strategy essentially presented iBCECs with a mean trans-endothelial electrical resistance (TEER) of ~ 2000 – 2500 Ω*cm2 and low permeability coefficients (PC) of < 0.50 μm/min for small molecule transport of sodium fluorescein (NaF) and characteristic BCEC tight junction (TJ) protein expression of claudin-5 and occludin. Additionally, iBCECs derived via CD did not form tubes in response to angiogenic stimuli. DD on the other hand resulted in iBCECs with similar down regulations in PECAM-1 and CDH5 gene expression. They were additionally characterized by lower barrier integrity, measured by mean TEER of only ~ 250 – 450 Ω*cm2 and high PC of > 5 μm/min in small molecule transport of NaF. Although iBCECs derived via DD formed tubes in response to angiogenic stimuli, they did not show positive protein expression of characteristic BCEC TJs such as claudin-5 and occludin. These results led to the hypothesis that maturity and lineage specification of iBCECs could be improved by incorporating in-vivo like characteristics in-vitro, such as direct co-culture with neurovascular unit (NVU) cell types via spheroid formation and by induction of shear stress and fluid flow. In comparison to standard iBCEC transwell mono-cultures, BBB spheroids showed enhanced transcript expression of PECAM-1 and reduced expression of epithelial markers such as CDH1 and claudin-6 (CLDN6). BBB spheroids showed classical BCEC-like ultrastructure that was identified by TJ particles on the protoplasmic face (P-face) and exoplasmic face (E-face) of the plasma membrane. TJ strands were organized as particles and particle-free grooves on the E-face, while on the P-face, partly beaded particles and partly continuous strands were identified. BBB spheroids also showed positive protein expression of claudin-5, VE-cadherin, PECAM-1, glucose transporter-1 (GLUT-1), P-glycoprotein (P-gp) and transferrin receptor-1 (Tfr-1). BBB spheroids demonstrated higher relative impedance percentages in comparison to spheroids without an iBCEC barrier. Barrier integrity assessments additionally corresponded with lower permeability to small molecule tracer NaF, with spheroids containing iBCECs showing higher relative fluorescence unit percentages (RFU%) of ~ 90% in apical compartments, compared to ~ 80% in spheroids without iBCECs. In summary, direct cellular contacts in the complex spheroid model resulted in enhanced maturation of iBCECs.
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A bioreactor system was used to further assess the effect of shear stress. This system enabled inclusion of fluidic flow and shear stress conditions in addition to non-invasive barrier integrity measurements (Choi, Mathew et al. 2022). iBCECs were cultured for a total of seven days post differentiation (d17) within the bioreactor and barrier integrity was non-invasively monitored. Until d17 of long-term culture, TEER values of iBCECs steadily dropped from ~ 1800 Ω*cm2 ~ 400 Ω*cm2 under static conditions and from ~ 2500 Ω*cm2 to ~ 250 Ω*cm2 under dynamic conditions. Transcriptomic analyses, morphometric analyses and protein marker expression showed enhanced maturation of iBECs under long-term culture and dynamic flow. Importantly, on d10 claudin-5 was expressed mostly in the cytoplasm with only ~ 5% iBCECs showing continuous staining at the cell borders. With increase in culture duration, iBCECs at d17 of static culture showed ~ 18% of cells having continuous cell border expression, while dynamic conditions showed upto ~ 30% of cells with continuous cell-cell border expression patterns. Similarly, ~ 33% of cells showed cell-cell border expression of occludin on d10 with increases to ~ 55% under d17 static and up to ~ 65% under d17 dynamic conditions, thereby indicating iBCEC maturation.
In conclusion, the data presented within this thesis demonstrates the maturation of iBCECs in BBB spheroids, obtained via direct cellular contacts and by the application of flow and shear stress. Both established novel models need to be further validated for pharmaceutical drug applications together with in-vitro-in-vivo correlations in order to exploit their full potential.
Die vorliegende Arbeit befasst sich mit der Darstellung und Reaktivität neutraler Diboraarene, wobei im ersten Teil die Synthese neuer Metallkomplexe eines cAAC-stabilisierten (cAAC = cyclisches Alkyl(amino)carben) 1,4-Diborabenzols sowie deren Folgereaktivität im Fokus steht. Im zweiten Abschnitt wird die Reaktivität des Diborabenzols und eines cAAC-stabilisierten 9,10-Diboraanthracens gegenüber Hauptgruppenelementverbindungen untersucht und vergleichend gegenübergestellt. Darauffolgend werden neben der Synthese neuer Metallkomplexe des Diboraanthracens auch weitere Reaktivitätsuntersuchungen der Verbindung behandelt. Der letzte Teil der Arbeit befasst sich mit der Darstellung neuartiger neutraler und cAAC-stabilisierter Diboraacene über eine Modulation des π-Systems. Dabei wird der synthetische Zugang zu einem 1,4-Diboranaphthalin und einem 6,13-Diborapentacen ermöglicht und ausgewählte Reaktivitäten beider Verbindungen demonstriert.
In dieser Arbeit wurde der Einfluss sozialer Stresserfahrung sowie des 5-Htt-Genotyps auf die neuronale Morphologie bestimmter Hirnregionen anhand eines Mausmodells untersucht. Es wurde in mit Golgi-Cox gefärbten Gehirnen der 5-HTT-KO-Linie in der lateralen Amygdala (LA) die Apikal- und Basaldendriten pyramidenzellähnlicher Neurone und die Apikaldendriten der Pyramidenzellen der Cornu ammonis (CA)3-Region des Hippocampus mithilfe des Neurolucidasystems rekonstruiert und die so gewonnenen Daten anschließend statistisch ausgewertet.
Die erzielten Ergebnisse belegen, dass vor allem die Erfahrung von sozialem Verteidigungsstress aber auch der 5-Htt-Genotyp (WT, HET, KO) im Mausmodell signifikanten Einfluss auf die Morphologie der Neurone der LA und der CA3-Region besitzen. Um die in dieser Arbeit mit allen drei 5-Htt-Genotypen erzielten Ergebnisse der LA-Neurone besser mit den Ergebnissen von Nietzer und Bonn (nur WT, KO) vergleichen zu können (Nietzer et al., 2011), wurden die von mir erhobenen Daten nicht nur in einem 3er-Vergleich, sondern auch einem 2er-Vergleich (WT vs. KO) statistisch analysiert. Untersuchungen der LA-Neurone aller drei 5-Htt-Genotypen zeigen, dass sozialer Stress zu einer Zunahme der Komplexität der Dendritenbäume durch längere und auch stärker verzweigte Dendriten vor allem in der Gruppe der WT-Mäuse führt. HET- und KO-Mäuse zeigten keinen entsprechenden Stress-Effekt. Darüber hinaus zeigten sich deutliche Genotypeffekte. Unabhängig vom Stresserleben besitzen HET-Mäuse längere Dendriten als WT-Mäuse sowie eine höhere Spinedichte als WT- und KO-Mäuse. Die Hypothese, die in der Arbeit von Nietzer et al. aufgestellt wurde, dass eine vollständige 5-HTT-Defizienz zu mehr Spines führt, ließ sich hier weder durch den 3er- noch durch den 2er-Vergleich replizieren. Die Pyramidenzellen der CA3-Region, die in dieser Studie zum ersten Mal analysiert wurden, zeigen in Bezug auf die durch den Stress ausgelösten Veränderungen ein im Vergleich zu den LA-Neuronen entgegengesetzten Effekt. Der soziale Stress führt hier zu einer Dendritenatrophie in der WT-Gruppe mit kürzeren und weniger komplexen Dendriten. Außerdem führte er zu einer geringeren Spinedichte bei den HET-Mäusen. Es zeigten sich klare Genotypeffekte, unabhängig von der Stresserfahrung, mit einer reduzierten Spinedichte der KO-Mäuse gegenüber den WT-Mäusen und einer nur in den Kontrollen detektierten, reduzierten Spinedichte der KO-Mäuse im Vergleich zu den WT- und HET-Mäusen. Sowohl in der LA als auch in der CA3-Region lassen sich Kompensationsmechanismen des 5-HTT-Defizits der HET-Tiere vermuten, über die die KO-Tiere nicht verfügen.
Die in LA und CA3 gezeigten gegensätzlichen Auswirkungen des sozialen Stresses weisen auf die unterschiedlichen Funktionen dieser beiden Regionen im Furchtkreislauf und/oder bei der Verarbeitung von Stress hin. Darüber hinaus deutet diese Arbeit darauf hin, dass Arbeiten mit ähnlichen Untersuchungsmethoden und sogar gleichem Untersuchungsmaterial unterschiedliche Ergebnisse liefern können.
Introduction: The German PID-NET registry was founded in 2009, serving as the first national registry of patients with primary immunodeficiencies (PID) in Germany. It is part of the European Society for Immunodeficiencies (ESID) registry. The primary purpose of the registry is to gather data on the epidemiology, diagnostic delay, diagnosis, and treatment of PIDs.
Methods: Clinical and laboratory data was collected from 2,453 patients from 36 German PID centres in an online registry. Data was analysed with the software Stata® and Excel.
Results: The minimum prevalence of PID in Germany is 2.72 per 100,000 inhabitants. Among patients aged 1-25, there was a clear predominance of males. The median age of living patients ranged between 7 and 40 years, depending on the respective PID. Predominantly antibody disorders were the most prevalent group with 57% of all 2,453 PID patients (including 728 CVID patients). A gene defect was identified in 36% of patients. Familial cases were observed in 21% of patients. The age of onset for presenting symptoms ranged from birth to late adulthood (range 0-88 years). Presenting symptoms comprised infections (74%) and immune dysregulation (22%). Ninety-three patients were diagnosed without prior clinical symptoms. Regarding the general and clinical diagnostic delay, no PID had undergone a slight decrease within the last decade. However, both, SCID and hyper IgE-syndrome showed a substantial improvement in shortening the time between onset of symptoms and genetic diagnosis. Regarding treatment, 49% of all patients received immunoglobulin G (IgG) substitution (70%-subcutaneous; 29%-intravenous; 1%-unknown). Three-hundred patients underwent at least one hematopoietic stem cell transplantation (HSCT). Five patients had gene therapy.
Conclusion: The German PID-NET registry is a precious tool for physicians, researchers, the pharmaceutical industry, politicians, and ultimately the patients, for whom the outcomes will eventually lead to a more timely diagnosis and better treatment.
Heterozygous mutations in the glucocerebrosidase gene (GBA1) represent the most common genetic risk factor for Parkinson's disease (PD) and are histopathologically associated with a widespread load of alpha-synuclein in the brain. Therefore, PD patients with GBA1 mutations are a cohort of high interest for clinical trials on disease-modifying therapies targeting alpha-synuclein. There is evidence that detection of phospho-alpha-synuclein (p-syn) in dermal nerve fibers might be a biomarker for the histopathological identification of PD patients even at premotor or very early stages of disease. It is so far unknown whether dermal p-syn deposition can also be found in PD patients with GBA1 mutations and may serve as a biomarker for PD in these patients. Skin biopsies of 10 PD patients with different GBA1 mutations (six N3705, three E326K, one L444P) were analyzed by double-immunofluorescence labeling with anti-p-syn and anti-protein gene product 9.5 (PGP9.5, axonal marker) to detect intraaxonal p-syn deposition. Four biopsy sites (distal, proximal leg, paravertebral Th10, and C7) per patient were studied. P-syn was found in six patients (three N370S, three E326K). P-syn deposition was mainly detected in autonomic nerve fibers, but also in somatosensory fibers and was not restricted to a certain GBA1 mutation. In summary, dermal p-syn in PD patients with GBA1 mutations seems to offer a similar distribution and frequency as observed in patients without a known mutation. Skin biopsy may be suitable to study p-syn deposition in these patients or even to identify premotor patients with GBA1 mutations.
In young (n = 36, mean +/- SD: 24.8 +/- 4.5 years) and older (n = 34, mean +/- SD: 65.1 +/- 6.5 years) healthy participants, we employed a modified version of the Serial Reaction Time task to measure procedural learning (PL) and consolidation while providing monetary and social reward. Using voxel-based morphometry (VBM), we additionally determined the structural correlates of reward-related motor performance (RMP) and PL. Monetary reward had a beneficial effect on PL in the older subjects only. In contrast, social reward significantly enhanced PL in the older and consolidation in the young participants. VBM analyses revealed that motor performance related to monetary reward was associated with larger grey matter volume (GMV) of the left striatum in the young, and motor performance related to social reward with larger GMV of the medial orbitofrontal cortex in the older group. The differential effects of social reward in young (improved consolidation) and both social and monetary rewards in older (enhanced PL) healthy subjects point to the potential of rewards for interventions targeting aging-associated motor decline or stroke-induced motor deficits.
Background: Non-alcoholic steatohepatitis (NASH) is a chronic liver disease with severe complications and without approved therapies. Currently, there is limited data on the overall burden of the disease for patients or on patient needs and preferences. This study investigates patient preferences in relation to potential future therapies for NASH. In addition, the factors that are relevant to patients and their importance in relation to future treatment options are explored.
Method: Telephone in-depth interviews (TDIs) preceded an online 30-min quantitative survey. The online survey included (1) multiple choice questions (MCQs) on NASH diagnosis and disease background. (2) An exercise to determine patients' satisfaction levels with information provided at diagnosis, and to explore symptomatology in detail. (3) Exercises to evaluate potential new products and product attributes, including a "drag and drop" ranking exercise, and an adaptive choice-based conjoint exercise (ACBC). (4) The EQ-5D-5L questionnaire and the Visual Analog Scale (VAS), which measures patients' health status. (5) Collection of socio-demographic data, and (6) Questions to measure patient satisfaction with the survey.
Results: There were 166 patients included in this study from Canada [n = 36], Germany [n = 50], the UK [n = 30], and USA [n = 50]. Fifty seven percent of patients [n = 94] had had a liver biopsy for confirmation of NASH. Patients were often unable to link their symptoms to NASH or other conditions. ACBC results showed that efficacy, defined as "impact on liver status" was the single most important attribute of a potential future NASH therapy. Other attributes considered to have secondary importance included impact on weight, symptom control and the presence of side effects. The EQ-5D utility score was 0.81 and VAS = 67.2.
Conclusion: "Impact on liver status" is the primary outcome sought. Patients demonstrate a general lack of understanding of their disease and appeared to be unfamiliar with longer-term consequences of NASH. It is necessary to improve patient understanding of NASH and its progressive nature, and there is a need for improving confirmatory diagnosis and monitoring.
In the recent two decades, LIM and SH3 protein 1 (LASP1) has been developed from a simple actin-binding structural protein to a tumor biomarker and subsequently to a complex, nuclear transcriptional regulator. Starting with a brief historical perspective, this review will mainly compare and contrast LASP1 and LASP2 from the angle of the newest data and importantly, examine their role in transcriptional regulation. We will summarize the current knowledge through pictorial models and tables including the roles of different microRNAs in the differential regulation of LASP1 levels and patient outcome rather than specify in detail all tumor entities. Finally, the novel functional roles of LASP1 in secretion of vesicles, expression of matrix metalloproteinases and transcriptional regulation as well as the activation of survival and proliferation pathways in different cancer types are described.
Bone represents a common site of metastases for several solid tumors. However, the ability of neuroendocrine neoplasms (NENs) to localize to bone has always been considered a rare and late event. Thanks to the improvement of therapeutic options, which results in longer survival, and of imaging techniques, particularly after the introduction of positron emission tomography (PET) with gallium peptides, the diagnosis of bone metastases (BMs) in NENs is increasing. The onset of BMs can be associated with severe skeletal complications that impair the patient's quality of life. Moreover, BMs negatively affect the prognosis of NEN patients, bringing out the lack of curative treatment options for advanced NENs. The current knowledge on BMs in gastro-entero-pancreatic (GEP) and bronchopulmonary (BP) NENs is still scant and is derived from a few retrospective studies and case reports. This review aims to perform a critical analysis of the evidence regarding the role of BMs in GEP- and BP-NENs, focusing on the molecular mechanisms underlining the development of BMs, as well as clinical presentation, diagnosis, and treatment of BMs, in an attempt to provide suggestions that can be used in clinical practice.
The probiotic escherichia coli strain Nissle 1917 combats lambdoid bacteriophages stx and lambda
(2018)
Shiga toxin (Stx) producing E. coli (STEC) such as Enterohemorrhagic E. coli (EHEC) are the major cause of foodborne illness in humans. In vitro studies showed the probiotic Escherichia coil strain Nissle 1917 (EcN) to efficiently inhibit the production of Stx. Life threatening EHEC strains as for example the serotype 0104:H4, responsible for the great outbreak in 2011 in Germany, evolutionary developed from certain E. coll strains which got infected by stx2-encoding lambdoid phages turning the E. coil into lysogenic and subsequently Stx producing strains. Since antibiotics induce stx genes and Stx production, EHEC infected persons are not recommended to be treated with antibiotics. Therefore, EcN might be an alternative medication. However, because even commensal E. coli strains might be converted into Stx-producers after becoming host to a stx encoding prophage, we tested EcN for stx-phage genome integration. Our experiments revealed the resistance of EcN toward not only stx-phages but also against lambda-phages. This resistance was not based on the lack of or by mutated phage receptors. Rather it involved the expression of a phage repressor (pr) gene of a defective prophage in EcN which was able to partially protect E. coli K-12 strain MG1655 against stx and lambda phage infection. Furthermore, we observed EcN to inactivate phages and thereby to protect E. coli K-12 strains against infection by stx- as well as lambda-phages. Inactivation of lambda-phages was due to binding of lambda-phages to LamB of EcN whereas inactivation of stx-phages was caused by a thermostable protein of EcN. These properties together with its ability to inhibit Stx production make EcN a good candidate for the prevention of illness caused by EHEC and probably for the treatment of already infected people.
Glycine receptors (GlyRs) are important mediators of fast inhibitory neurotransmission in the mammalian central nervous system. Their function is controlled by multiple cellular mechanisms, including intracellular regulatory processes. Modulation of GlyR function by protein kinases has been reported for many cell types, involving different techniques, and often yielding contradictory results. Here, we studied the effects of protein kinase C (PKC) and cAMP-dependent protein kinase A (PKA) on glycine induced currents in HEK293 cells expressing human homomeric \(\alpha\)1 and heteromeric \(\alpha\)1-\(\beta\) GlyRs using whole-cell patch clamp techniques as well as internalization assays. In whole-cell patch-clamp measurements, modulators were applied in the intracellular buffer at concentrations between 0.1 \(\mu\)M and 0.5 \(\mu\)M. EC50 of glycine increased upon application of the protein kinase activators Forskolin and phorbol-12-myristate-13-acetate (PMA) but decreased in the presence of the PKC inhibitor Staurosporine aglycon and the PKA inhibitor H-89. Desensitization of recombinant \(\alpha\)1 receptors was significantly increased in the presence of Forskolin. Staurosporine aglycon, on the other hand decreased desensitization of heteromeric \(\alpha\)1-\(\beta\) GlyRs. The time course of receptor activation was determined for homomeric \(\alpha\)1 receptors and revealed two simultaneous effects: cells showed a decrease of EC50 after 3-6 min of establishing whole-cell configuration. This effect was independent of protein kinase modulators. All modulators of PKA and PKC, however, produced an additional shift of EC50, which overlay and eventually exceeded the cells intrinsic variation of EC50. The effect of kinase activators was abolished if the corresponding inhibitors were co-applied, consistent with PKA and PKC directly mediating the modulation of GlyR function. Direct effects of PKA-and PKC-modulators on receptor expression on transfected HEK cells were monitored within 15 min of drug application, showing a significant increase of receptor internalization with PKA and PKC activators, while the corresponding inhibitors had no significant effect on receptor surface expression or internalization. Our results confirm the observation that phosphorylation via PKA and PKC has a direct effect on the GlyR ion channel complex and plays an important role in the fine-tuning of glycinergic signaling.
Voluntary movements induce postural perturbations which are counteracted by anticipatory postural adjustments (APAs). These actions are known to build up long fixation chains toward available support points (inter-limb APAs), so as to grant whole body equilibrium. Moreover, recent studies highlighted that APAs also build-up short fixation chains, within the same limb where a distal segment is moved (intra-limb APAs), aimed at stabilizing the proximal segments. The neural structures generating intra-limb APAs still need investigations; the present study aims to compare focal movement kinematics and intra-limb APA latencies and pattern between healthy subjects and parkinsonian patients, assuming the latter as a model of basal ganglia dysfunction. Intra-limb APAs that stabilize the arm when the index-finger is briskly flexed were recorded in 13 parkinsonian patients and in 10 age-matched healthy subjects. Index-finger movement was smaller in parkinsonian patients vs. healthy subjects (p = 0.01) and more delayed with respect to the onset of the prime mover flexor digitorum superficialis (FDS, p < 0.0001). In agreement with the literature, in all healthy subjects the FDS activation was preceded by an inhibitory intra-limb APA in biceps brachii (BB) and anterior deltoid (AD), and almost simultaneous to an excitatory intra-limb APA in triceps brachii (TB). In parkinsonian patients, no significant differences were found for TB and AD intra-limb APA timings, however only four patients showed an inhibitory intra-limb APA in BB, while other four did not show any BB intra-limb APAs and five actually developed a BB excitation. The frequency of occurrence of normal sign, lacking, and inverted BB APAs was different in healthy vs. parkinsonian participants (p = 0.0016). The observed alterations in index-finger kinematics and intra-limb APA pattern in parkinsonian patients suggest that basal ganglia, in addition to shaping the focal movement, may also contribute to intra-limb APA control.
Introduction: Acute stroke care delivered by interdisciplinary teams is time-sensitive. Simulation-based team training is a promising tool to improve team performance in medical operations. It has the potential to improve process times, team communication, patient safety, and staff satisfaction. We aim to assess whether a multi-level approach consisting of a stringent workflow revision based on peer-to-peer review and 2–3 one-day in situ simulation trainings can improve acute stroke care processing times in high volume neurocenters within a 6 months period.
Methods and Analysis: The trial is being carried out in a pre-test-post-test design at 7 tertiary care university hospital neurocenters in Germany. The intervention is directed at the interdisciplinary multiprofessional stroke teams. Before and after the intervention, process times of all direct-to-center stroke patients receiving IV thrombolysis (IVT) and/or endovascular therapy (EVT) will be recorded. The primary outcome measure will be the “door-to-needle” time of all consecutive stroke patients directly admitted to the neurocenters who receive IVT. Secondary outcome measures will be intervention-related process times of the fraction of patients undergoing EVT and effects on team communication, perceived patient safety, and staff satisfaction via a staff questionnaire.
Interventions: We are applying a multi-level intervention in cooperation with three “STREAM multipliers” from each center. First step is a central meeting of the multipliers at the sponsor's institution with the purposes of algorithm review in a peer-to-peer process that is recorded in a protocol and an introduction to the principles of simulation training and debriefing as well as crew resource management and team communication. Thereafter, the multipliers cooperate with the stroke team trainers from the sponsor's institution to plan and execute 2–3 one-day simulation courses in situ in the emergency department and CT room of the trial centers whereupon they receive teaching materials to perpetuate the trainings.
Clinical Trial Registration: STREAM is a registered trial at https://clinicaltrials.gov/ct2/show/NCT03228251.
The immune suppressants cyclosporin A (CsA) and tacrolimus (FK506) are used worldwide in transplantation medicine to suppress graft rejection. Both CsA and FK506 inhibit the phosphatase calcineurin (CN) whose activity controls the immune receptor-mediated activation of lymphocytes. Downstream targets of CN in lymphocytes are the nuclear factors of activated T cells (NFATs). We show here that the activity of NFATc1, the most prominent NFAT factor in activated lymphocytes supports the acute rejection of heterotopic heart allografts. While ablation of NFATc1 in T cells prevented graft rejection, ectopic expression of inducible NFATc1/αA isoform led to rejection of heart allografts in recipient mice. Acceptance of transplanted hearts in mice bearing NFATc1-deficient T cells was accompanied by a reduction in number and cytotoxicity of graft infiltrating cells. In CD8\(^+\) T cells, NFATc1 controls numerous intracellular signaling pathways that lead to the metabolic switch to aerobic glycolysis and the expression of numerous lymphokines, chemokines, and their receptors, including Cxcr3 that supports the rejection of allogeneic heart transplants. These findings favors NFATc1 as a molecular target for the development of new strategies to control the cytotoxicity of T cells upon organ transplantation.
Staphylococcus epidermidis, the common inhabitant of human skin and mucosal surfaces has emerged as an important pathogen in patients carrying surgical implants and medical devices. Entering the body via surgical sites and colonizing the medical devices through formation of multi-layered biofilms leads to refractory and persistent device-related infections (DRIs). Staphylococci organized in biofilms are more tolerant to antibiotics and immune responses, and thus are difficult-to-treat. The consequent morbidity and mortality, and economic losses in health care systems has strongly necessitated the need for development of new anti-bacterial and anti-biofilm-based therapeutics. In this study, we describe the biological activity of a marine sponge-derived Streptomyces sp. SBT348 extract in restraining staphylococcal growth and biofilm formation on polystyrene, glass, medically relevant titan metal, and silicone surfaces. A bioassay-guided fractionation was performed to isolate the active compound (SKC3) from the crude SBT348 extract. Our results demonstrated that SKC3 effectively inhibits the growth (MIC: 31.25 \(\mu\)g/ml) and biofilm formation (sub-MIC range: 1.95-<31.25 \(\mu\)g/ml) of S. epidermidis RP62A in vitro. Chemical characterization of SKC3 by heat and enzyme treatments, and mass spectrometry (HRMS) revealed its heat-stable and non-proteinaceous nature, and high molecular weight (1258.3 Da). Cytotoxicity profiling of SKC3 in vitro on mouse fibroblast (NIH/3T3) and macrophage (J774.1) cell lines, and in vivo on the greater wax moth larvae Galleria mellonella revealed its non-toxic nature at the effective dose. Transcriptome analysis of SKC3 treated S. epidermidis RP62A has further unmasked its negative effect on central metabolism such as carbon flux as well as, amino acid, lipid, and energy metabolism. Taken together, these findings suggest a potential of SKC3 as a putative drug to prevent staphylococcal DRIs.