@phdthesis{ElBashir2017, author = {ElBashir, Rasha}, title = {Development of New Mass Spectrometry-based Methods for the Analysis of Posttranslational Modifications}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-153731}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2017}, abstract = {Posttranslational modifications (PTMs) play a crucial role in many cellular processes. They are reversible, dynamic, and highly regulated events that alter the properties of proteins and increase their functional diversity. The identification and quantification of PTMs are critical for deciphering the molecular mechanisms of PTMs-related biological processes and disease treatment and prevention. Two of the most common and important PTMs that regulate many protein functions are acetylation and phosphorylation. An important role of acetylation is the regulation of DNA/RNA-protein interactions. A prominent example for this are histones, whose tail regions are lysine-rich and can be highly acetylated at their N-terminal domain. In spite of the utmost importance of this PTM, methods that allow the accurate measuring the site-specific acetylation degree are missing. One of the challenges in quantifying the acetylation degree at an individual lysine residue of the histones N-termini is the occurrence of multiple lysines in close proximity. Herein, we describe the development of the "Fragment Ion Patchwork Quantification," a new mass spectrometry-based approach for the highly accurate quantification of sites-pecific acetylation degrees. This method combines 13C1-acetyl derivatization on the protein level, proteolysis by low-specificity proteases and quantification on the fragment ion level. Acetylation degrees are determined from the isotope patterns of acetylated b and y ions. We have shown that this approach allows determining the site-specific acetylation degrees of all lysine residues for all core histones of Trypanosoma brucei. In addition, we demonstrate the use of this approach to identify the substrate sites of histone acetyltransferases and to monitor the changes in acetylation of the histones of canonical nucleosome and transcription start site nucleosomes. Phosphorylation is one of the most common and most important PTMs. The analysis of the human genome showed that there are about 518 kinases and more than 500,000 phosphorylation sites are believed to exist in the cellular proteome. Protein phosphorylation plays a crucial role in signaling many different cell processes, such as intercellular communication, cell growth, differentiation of proliferation and apoptosis. Whereas MS-based identification and relative quantification of singly phosphorylated peptides have been greatly improved during the last decade, and large-scale analysis of thousands of phosphopeptides can now be performed on a routine-base, the analysis of multi-phosphorylated peptides is still lagging vastly behind. The low pKa value of phosphate group and the associated negative charge are considered the major source of the problems with the analysis of multi-phosphorylated peptides. These problems include the formation of phosphopeptide-metal complexes during liquid chromatography (e.g. Fe 3+), which leads to a drastic deterioration of the chromatographic properties of these peptides (peak tailing), the decreased ionization efficiencies of phosphorylated peptides compared to their unphosphorylated counterparts, the labile nature of phosphate during CID/HCD fragmentation, and the unsuitability of low-charged phosphopeptides for ETD fragmentation are the most important factors that hinder phosphorylation analysis by LC-MS/MS. Here we aimed to develop a method for improving the identification of multi-phosphorylated peptides as well as the localization of phosphorylation sites by charge-reversal derivatization of the phosphate groups. This method employs a carbodiimide-mediated phosphoramidation to converted the phosphates to stable aromatic phosphoramidates. This chemical modification of phosphosite(s) reversed the negative charge of the phosphate group(s) and increased the number of the positive charges within the phosphopeptide. This modification prevented the formation of phosphopeptide-metal ion complexes that dramatically decreases or completely diminishes the signal intensity of protonated phosphopeptides, specifically multi-phosphorylated peptides. Furthermore, the increased net charge the (phospho-)peptides made them suitable for ETD fragmentation, which generated a high number of fragment ions with high intensities that led to a better phosphopeptide identification and localization of phosphosite(s) with high confidence.}, subject = {LC-MS}, language = {en} } @phdthesis{Endres2024, author = {Endres, Leo Maximilian}, title = {Development of multicellular \(in\) \(vitro\) models of the meningeal blood-CSF barrier to study \(Neisseria\) \(meningitidis\) infection}, doi = {10.25972/OPUS-34621}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-346216}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2024}, abstract = {Neisseria meningitidis (the meningococcus) is one of the major causes of bacterial meningitis, a life-threatening inflammation of the meninges. Traversal of the meningeal blood-cerebrospinal fluid barrier (mBCSFB), which is composed of highly specialized brain endothelial cells (BECs), and subsequent interaction with leptomeningeal cells (LMCs) are critical for disease progression. Due to the human-exclusive tropism of N. meningitidis, research on this complex host-pathogen interaction is mostly limited to in vitro studies. Previous studies have primarily used peripheral or immortalized BECs alone, which do not retain relevant barrier phenotypes in culture. To study meningococcal interaction with the mBCSFB in a physiologically more accurate context, BEC-LMC co-culture models were developed in this project using BEC-like cells derived from induced pluripotent stem cells (iBECs) or hCMEC/D3 cells in combination with LMCs derived from tumor biopsies. Distinct BEC and LMC layers as well as characteristic expression of cellular markers were observed using transmission electron microscopy (TEM) and immunofluorescence staining. Clear junctional expression of brain endothelial tight and adherens junction proteins was detected in the iBEC layer. LMC co-culture increased iBEC barrier tightness and stability over a period of seven days, as determined by sodium fluorescein (NaF) permeability and transendothelial electrical resistance (TEER). Infection experiments demonstrated comparable meningococcal adhesion and invasion of the BEC layer in all models tested, consistent with previously published data. While only few bacteria crossed the iBEC-LMC barrier initially, transmigration rates increased substantially over 24 hours, despite constant high TEER. After 24 hours of infection, deterioration of the barrier properties was observed including loss of TEER and altered expression of tight and adherens junction components. Reduced mRNA levels of ZO-1, claudin-5, and VE-cadherin were detected in BECs from all models. qPCR and siRNA knockdown data suggested that transcriptional downregulation of these genes was potentially but not solely mediated by Snail1. Immunofluorescence staining showed reduced junctional coverage of occludin, indicating N. meningitidis-induced post-transcriptional modulation of this protein, as previous studies have suggested. Together, these results suggest a potential combination of transcellular and paracellular meningococcal traversal of the mBCSFB, with the more accessible paracellular route becoming available upon barrier disruption after prolonged N. meningitidis infection. Finally, N. meningitidis induced cellular expression of pro-inflammatory cytokines and chemokines such as IL-8 in all mBCSFB models. Overall, the work described in this thesis highlights the usefulness of advanced in vitro models of the mBCSFB that mimic native physiology and exhibit relevant barrier properties to study infection with meningeal pathogens such as N. meningitidis.}, subject = {Bakterielle Hirnhautentz{\"u}ndung}, language = {en} } @phdthesis{Zhang2024, author = {Zhang, Tengyu}, title = {Development of Modified polylysine based antibody conjugated nanoparticles with tumor-restricted, FcγR-independent stimulatory activity by targeting Fn14}, doi = {10.25972/OPUS-35865}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-358650}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2024}, abstract = {In this study, we developed an innovative nanoparticle formulation to facilitate the delivery of antitumor antibodies to tumor sites. The study commenced with the utilization of 13 bispecific antibody fusion proteins, which targeted the Fn14 receptor, thereby validating the pivotal role of crosslinking in Fn14 receptor activation. Subsequently, gold nanoparticles were activated using COOH-PEG-SH in combination with EDC/NHS, and subsequently conjugated with two Fn14-targeting antibodies, PDL192 and 5B6. Following this, a pH-sensitive shell was generated on the outer layer of the antibody-coupled gold nanoparticles through the application of chemically modified polylysine. The resultant complexes, termed MPL-antibody-AuNP, demonstrated a release profile reminiscent of the tumor microenvironment (TME). Notably, these complexes released antibody-AuNPs only in slightly acidic conditions while remaining intact in neutral or basic environments. Functionality analysis further affirmed the pH-sensitive property of MPL-antibody-AuNPs, demonstrating that the antibodies only initiated potent Fn14 activation in slightly acidic environments. This formulation holds potential for applicability to antibodies or ligands targeting the 80 TNFRSF family, given that gold nanoparticles successfully served as platforms for antibody crosslinking, thereby transforming these antibodies into potent agonists. Moreover, the TME disintegration profile of MPL mitigates the potential cytotoxic effects of antibodies, thereby circumventing associated adverse side effects. This study not only showcases the potential of nanoparticle formulations in targeted therapy, but also provides a solid foundation for further investigations on their clinical application in the context of targeting category II TNFRSF receptors with antibodies or ligands.}, subject = {Immuntherapie}, language = {en} } @phdthesis{Heydarian2021, author = {Heydarian, Motaharehsadat}, title = {Development of human 3D tissue models for studying \(Neisseria\) \(gonorrhoeae\) infection}, doi = {10.25972/OPUS-20496}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-204967}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {Gonorrhea is the second most common sexually transmitted infection worldwide and is caused by Gram-negative, human-specific diplococcus Neisseria gonorrhoeae. It colonizes the mucosal surface of the female reproductive tract and the male urethra. A rapid increase in antibiotic resistance makes gonorrhea a serious threat to public health worldwide. Since N. gonorrhoeae is a human-specific pathogen, animal infection models are not able to recapitulate all the features of infection. Therefore, a realistic in vitro cell culture model is urgently required for studying the gonorrhea infection. In this study, we established and characterized three independent 3D tissue models based on the porcine small intestinal submucosa (SIS) scaffold by co-culturing human dermal fibroblasts with human colorectal carcinoma, endometrial epithelial, and male uroepithelial cells. The histological, immunohistochemical, and ultra-structural analysis showed that the 3D SIS scaffold-based models closely mimic the main characteristics of the site of gonococcal infection in the human host including the formation of epithelial monolayer, underlying connective tissue, mucus production, tight junction (TJ), and microvilli. In addition, functional analysis such as transepithelial electrical resistance (TEER) and barrier permeability indicated high barrier integrity of the cell layer. We infected the established 3D tissue models with different N. gonorrhoeae strains and derivatives presenting various phenotypes regarding adhesion and invasion. The results showed disruption of TJs and growing the interleukins production in response to the infection, which depends on the type of strain and cell. In addition, the 3D tissue models supported bacterial survival, which provided an appropriate in vitro model for long-term infection study. This could be mainly because of the high resilience of the 3D tissue models based on the SIS scaffold to the infection in terms of alteration in permeability, cell destruction, and bacterial transmigration. During gonorrhea infection, a high level of neutrophils migrates to the site of infection. The studies also showed that N. gonorrhoeae can survive or even replicate inside the neutrophils. Therefore, studying the interaction between neutrophils and N. gonorrhoeae is substantially under scrutiny. For this purpose, we generated a 3D tissue model by triple co-culturing of human primary fibroblast cells, human colorectal carcinoma cells, and human umbilical vein endothelial cells. The tissue model was subsequently infected by N. gonorrhoeae. A perfusion-based bioreactor system was employed to recreate blood flow in the side of endothelial cells and consequently study human neutrophils transmigration to the site of infection. We observed neutrophils activation upon the infection. Furthermore, we demonstrated the uptake of N. gonorrhoeae by human neutrophils and reverse transmigration of neutrophils to the basal side carrying N. gonorrhoeae. In summary, the introduced 3D tissue models in this research represent a promising tool to investigate N. gonorrhoeae infections under close-to-natural conditions.}, subject = {3D-Gewebemodell}, language = {en} } @phdthesis{Rossi2017, author = {Rossi, Angela Francesca}, title = {Development of functionalized electrospun fibers as biomimetic artificial basement membranes}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-137618}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2017}, abstract = {The basement membrane separates the epithelium from the stroma of any given barrier tissue and is essential in regulating cellular behavior, as mechanical barrier and as structural support. It further plays an important role for new tissue formation, homeostasis, and pathological processes, such as diabetes or cancer. Breakdown of the basement membrane is believed to be essential for tumor invasion and metastasization. Since the basement membrane is crucial for many body functions, the development of artificial basement membranes is indispensable for the ultimate formation of engineered functional tissue, however, challenging due to their complex structure. Electrospinning enables the production of fibers in the nano- or microscale range with morphological similarities to the randomly orientated collagen and elastic fibers in the basement membrane. However, electrospun fibers often lack the functional similarity to guide cells and maintain tissue-specific functions. Hence, their possible applications as matrix structure for tissue engineering are limited. Herein, the potential of polyester meshes, modified with six armed star-shaped pre-polymers and cell-adhesion-mediating peptides, was evaluated to act as functional isotropic and bipolar artificial basement membranes. Thereby, the meshes were shown to be biocompatible and stable including under dynamic conditions, and the degradation profile to correlate with the rate of new tissue formation. The different peptide sequences did not influence the morphology and integrity of the fibers. The modified membranes exhibited protein-repellent properties over 12 months, indicating the long-term stability of the cross-linked star-polymer surfaces. Cell culture experiments with primary fibroblasts and a human keratinocyte cell line (HaCaT) revealed that cell adhesion and growth strongly depends on the peptide sequences and their combinations employed. HaCaT cells grew to confluence on membranes modified with a combination of laminin/collagen type IV derived binding sequences and with a combination of fibronectin/laminin/collagen type IV derived peptide sequences. Fibroblasts strongly adhered to the fibronectin derived binding sequence and to membranes containing a combination of fibronectin/laminin/collagen type IV derived peptide sequences. The adhesion and growth of fibroblasts and HaCaT cells were significantly reduced on membranes modified with laminin, as well as collagen IV derived peptide sequences. HaCaT cells and fibroblasts barely adhered onto meshes without peptide sequences. Co-culture experiments at the air-liquid interface with fibroblasts and HaCaT cells confirmed the possibility of creating biocompatible, biofunctional and biomimetic isotropic and bipolar basement membranes, based on the functionalized fibers. HaCaT cells grew in several layers, differentiating towards the surface and expressing cytokeratin 10 in the suprabasal and cytokeratin 14 in the basal layers. Migration of fibroblasts into the electrospun membrane was shown by vimentin staining. Moreover, specific staining against laminin type V, collagen type I, III, IV and fibronectin illustrated that cells started to remodel the electrospun membrane and produced new extracellular matrix proteins following the adhesion to the synthetic surface structures. The culturing of primary human skin keratinocytes proved to be difficult on electrospun fibers. Cells attached to the membrane, but failed to form a multilayered, well-stratified, and keratinized epidermal layer. Changing the fiber composition and fixation methods did not promote tissue development. Further investigations of the membrane demonstrated the tremendous influence of the pore size of the membrane on epithelial formation. Furthermore, primary keratinocytes reacted more sensitive to pH changes in the medium than HaCaT cells did. Since primary keratinocytes did not adequately develop on the functionalized meshes, polycarbonate membranes were used instead of electrospun meshes to establish oral mucosa models. The tissue-engineered models represented important features of native human oral mucosa. They consisted of a multilayered epithelium with stratum basale, stratum spinosum, stratum granulosum, and stratum corneum. The models formed a physical barrier and the expression of characteristic cell markers was comparable with that in native human oral mucosa. The results from the ET-50 assay and the irritation study reflected the reproducibility of the tissue equivalents. Altogether, electrospinning enables the production of fibers with structural similarity to the basement membrane. Incorporating extracellular matrix components to mimic the functional composition offers a safe and promising way to modify the fibers so that they can be used for different tissue engineering applications. The resultant biomimetic membranes that can be functionalized with binding sequences derived from widely varying proteins can be used as a toolbox to study the influence of isotropic and bipolar basement membranes on tissue formation and matrix remodeling systematically, with regards to the biochemical composition and the influence and importance of mono- and co-culture. The oral mucosa models may be useful for toxicity and permeation studies, to monitor the irritation potential of oral health care products and biomaterials or as a disease model.}, subject = {Tissue Engineering}, language = {en} } @phdthesis{Stumpf2015, author = {Stumpf, Anette D.}, title = {Development of fluorescent FRET receptor sensors for investigation of conformational changes in adenosine A1 and A2A receptors}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-125469}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2015}, abstract = {Adenosine receptors that belong to the rhodopsin-like G protein-coupled receptors (GPCRs) are involved in a lot of regulatory processes and are widely distributed throughout the body which makes them an attractive target for drugs. However, pharmacological knowledge of these receptors is still limited. A big advance regarding the structural knowledge of adenosine receptors was the development of the first crystal structure of the adenosine A2A receptor in 2008. The crystal structure revealed the amino acids that form the ligand binding pocket of the receptor and depicted the endpoint of receptor movement in the ligand binding process. Within the scope of this work two members of the adenosine receptor family were investigated, namely the adenosine A1 and the A2A receptor (A1R, A2AR). A1R was generated on base of the previously developed A2AR. Receptors were tagged with fluorophores, with the cyan fluorescent protein (CFP) at the C-terminal end of receptor and the Fluorescein Arsenical Hairpin binder (FlAsH) binding sequence within the third intracellular loop of receptors. Resulting fluorescent receptor sensors A1 Fl3 CFP and A2A Fl3 CFP were investigated with help of Fluorescence Resonance Energy Transfer (FRET) measurements within living cells. FRET experiments enable the examination of alteration in the distance of two fluorophores and thus the observation of receptor dynamical movements. For comparison of A1R and A2AR regarding receptor dynamical movement upon ligand binding, fluorescent receptor sensors A1 Fl3 CFP and A2A Fl3 CFP were superfused with various ligands and the outcomes of FRET experiments were compared regarding signal height of FRET ratio evoked by the distinct ligand that is correlated to the conformational change of receptor upon ligand binding. Beside the different direction of FRET ratio upon ligand binding at A1R and A2AR sensor, there were differences observable when signal height and association and dissociation kinetics of the various ligands investigated were compared to each other. Differences between the adenosine receptor subtypes were especially remarkable for the A1R subtype selective agonist CPA and the A2AR subtype selective agonist CGS 21680. Another part of the project was to investigate the influence of single amino acids in the ligand binding process within the fluorescent A1R sensor. Amino acid positions were derived from the crystal structure of the A2AR forming the ligand binding pocket and these amino acids were mutated in the A1R structure. Investigation of the A1R sensor and its mutants regarding confocal analysis showed involvement of some amino acids in receptor localization. When these amino acids were mutated receptors were not expressed in the plasma membrane of cells. Some amino acids investigated were found to be involved in the ligand binding process in general whereas other amino acids were found to have an influence on the binding of distinct structural groups of the ligands investigated. In a further step, A1R and A2AR were N-terminally tagged with SNAP or CLIP which allowed to label receptor sensors with multiple fluorophores. With this technique receptor distribution in cells could be investigated with help of confocal analysis. Furthermore, ligand binding with fluorescent adenosine receptor ligands and their competition with help of a non-fluorescent antagonist was examined at the SNAP tagged A1R and A2AR. Finally the previously developed receptor sensors were combined to the triple labeled receptor sensors SNAP A1 Fl3 CFP and SNAP A2A Fl3 CFP which were functional regarding FRET experiments and plasma membrane expression was confirmed via confocal analysis. In the future, with the help of this technique, interaction between fluorescent ligand and SNAP tagged receptor can be monitored simultaneously with the receptor movement that is indicated by the distance alteration between FlAsH and CFP. This can lead to a better understanding of receptor function and its dynamical movement upon ligand binding which may contribute to the development of new and more specific drugs for the A1R and A2AR in the future.}, subject = {Adenosinrezeptor}, language = {en} } @phdthesis{ElMesery2014, author = {El-Mesery, Mohamed}, title = {Development of CD40-targeted bifunctional scFv-TRAIL fusion proteins that induce TRAILR1- and TRAILR2-specifc cell death and dendritic cells activation}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-100114}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2014}, abstract = {TRAIL is a member of TNF superfamily and mediates apoptosis by binding to two DRs, TRAILR1 and TRAILR2. Despite the fact that there are other TRAILRs, TRAILR1 and TRAILR2 receive the major research interest due to their ability to trigger apoptosis and their possible use as targets in tumor therapy. Due to the potential advantages of TRAILR1- or TRAILR2-specific targeting, we investigated recently published TRAIL DR-specific mutants, one conferring specificity for TRAILR1 (TRAILmutR1) and one for TRAILR2 (TRAILmutR2). It was well proved in this work that TRAILmutR1 shows specific binding to TRAILR1 and no specific binding to TRAILR2. TRAILmutR2 vice versa shows specific binding to TRAILR2 and no significant binding to TRAILR1. Moreover, these mutants were able to induce caspase activation and cell death in a TRAILR1/2-specific manner. Moreover, the enhancement of TRAILR2-induced apoptosis by secondary oligomerization of soluble wild-type TRAIL was confirmed for the TRAILR2-specifc TRAIL mutant and similar findings were made with the TRAILR1-specific TRAIL mutant. The soluble form of TRAIL exhibits weak apoptotic activity as compared to transmembrane TRAIL. Therefore, there is the challenge in clinical research to improve the activity of soluble TRAIL. A second strategy besides the above mentioned oligomerization to improve soluble TRAIL activity is anchoring of the molecule to the cell surface, e.g. through the genetic fusion with a scFv domain recognizing a cell surface antigen. In this work, we generated fusion proteins of TRAIL, TRAILmutR1 and TRAILmutR2 with a scFv recognizing CD40 (scFv:G28). Initially, we analyzed the functionality of both the TRAIL domain and the scFv:G28 domain of the corresponding fusion proteins. TRAIL functionality was well proved through its ability to induce cell death in TRAIL sensitive cells such as Jurkat cells, provided that scFv:G28-TRAIL fusion proteins were oligomerized by anti-Flag mAb M2. Concerning the scFv:G28 domain, the fusion proteins showed enhanced binding affinity to cell lines expressing CD40 as compared to their parental CD40-negative cells. Consistent with previous studies investigating TRAIL fusion proteins with other cell surface antigen-targeting scFvs, the scFv:G28 fusion proteins with TRAIL, TRAILmutR1 and TRAILmutR2 showed enhanced induction of cell death in a CD40-dependent manner. Moreover, our results revealed that these fusion proteins have a significant paracrine apoptotic effect on CD40-negative bystander cells upon anchoring to CD40-positive cells which are TRAIL resistant. Thus, the current work provides for the first time scFv fusion proteins of TRAIL and TRAILR1- and TRAILR2-specific TRAIL mutants with CD40-restricted activity. These fusion proteins provide the advantage of attenuating the off-target effects and the potential side effects of per se highly active TRAIL variants on one hand due to the CD40-binding dependent enhancement of activity and on the other hand due to the differential use of TRAILR1 and TRAILR2. CD40 represents a tumor associated marker which is expressed on many tumor cells but also on immune cells. Therefore, the last part of this work focused on the analysis of the ability of scFv:G28-TRAIL fusion proteins to induce CD40 signaling both in tumor cells and also in immune cells. It turned out that the scFv:G28-TRAIL fusion proteins are able to induce CD40 signaling in CD40-positive tumor cells but especially also in immune cells such as iDCs leading to their maturation and further activation of immune responses. Taken together, this work provides novel bifunctional scFv-TRAIL fusion proteins which combine the induction of apoptosis via TRAIL DR with stimulation of CD40 signaling which possibly enhances antitumor immunity.}, subject = {Tumor-Nekrose-Faktor}, language = {en} } @phdthesis{MathewSchmitt2024, author = {Mathew-Schmitt, Sanjana}, title = {Development of blood-brain barrier spheroid models based on human induced pluripotent stem cells (hiPSCs) and investigation of shear stress on hiPSC-derived brain capillary endothelial-like cells}, doi = {10.25972/OPUS-32247}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-322475}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2024}, abstract = {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, 7 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. 8 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.}, subject = {Blut-Hirn-Schranke}, language = {en} } @phdthesis{Aido2024, author = {Aido, Ahmed}, title = {Development of anti-TNF antibody-gold nanoparticles (anti-TNF-AuNPs)}, doi = {10.25972/OPUS-34921}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-349212}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2024}, abstract = {Gold nanoparticles of diameter ca. 60 nm have been synthesized based on Turkevich and Frens protocols. We have demonstrated that the carboxyl-modified gold nanoparticles can be coupled covalently with antibodies (Ab) of interest using the EDC/NHS coupling procedure. Binding studies with Ab-grafted AuNPs and GpL fusion proteins proved that conjugation of AuNPs with antibodies enables immobilization of antibodies with preservation of a significant antigen binding capacity. More importantly, our findings showed that the conjugation of types of anti-TNF receptors antibodies such as anti-Fn14 antibodies (PDL192 and 5B6) (Aido et al., 2021), anti-CD40, anti-4-1BB and anti-TNFR2 with gold nanoparticles confers them with potent agonism. Thus, our results suggest that AuNPs can be utilized as a platform to immobilize anti-TNFR antibodies which, on the one hand, helps to enhance their agonistic activity in comparison to "free" inactive antibodies by mimicking the effect of cell-anchored antibodies or membrane-bound TNF ligands and, on the other hand, allows to develop new generations of drug delivery systems. These constructs are characterized with their biocompatibility and their tunable synthesis process. In a further work part, we combined the benefits of the established system of Ab-AuNPs with materials used widely in the modern biofabrication approaches such as the photo-crosslinked hydrogels, methacrylate-modified gelatin (GelMA), combined with embedded variants of human cell lines. The acquired results demonstrated clearly that the attaching of proteins like antibodies to gold nanoparticles might reduce their release rate from the crosslinked hydrogels upon the very low diffusion of gold nanoparticles from the solid constructs to the surrounding medium yielding long-term local functioning proteins-attached particles. Moreover, our finding suggests that hydrogel-embedded AuNP-immobilized antibodies, e.g. anti-TNFα-AuNPs or anti-IL1-AuNPs enable local inhibitory functions, To sum up, our results demonstrate that AuNPs can act as a platform to attach anti-TNFR antibodies to enhance their agonistic activity by resembling the output of cell-anchoring or membrane bounding. Gold nanoparticles are considered, thus, as promising tool to develop the next generation of drug delivery systems, which may contribute to cancer therapy. On top of that, the embedding of anti-inflammatory-AuNPs in the biofabricated hydrogel presents new innovative strategy of the treatment of autoinflammatory diseases.}, subject = {Nanopartikel}, language = {en} } @phdthesis{Schwab2017, author = {Schwab, Andrea}, title = {Development of an osteochondral cartilage defect model}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-155617}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2017}, abstract = {The limited intrinsic self-healing capability of articular cartilage requires treatment of cartilage defects. Material assisted and cell based therapies are in clinical practice but tend to result in formation of mechanical inferior fibro-cartilage in long term follow up. If a lesion has not been properly restored degenerative diseases are diagnosed as late sequela causing pain and loss in morbidity. Complex three dimensional tissue models mimicking physiological situation allow investigation of cartilage metabolism and mechanisms involved in repair. A standardized and reproducible model cultured under controllable conditions ex vivo to maintain tissue properties is of relevance for comparable studies. Topic of this thesis was the establishment of an cartilage defect model that allows for testing novel biomaterials and investigate the effect of defined defect depths on formation of repair tissue. In part I an ex vivo osteochondral defect model was established based on isolation of porcine osteochondral explants (OCE) from medial condyles, 8 mm in diameter and 5 mm in height. Full thickness cartilage defects with 1 mm to 4 mm in diameter were created to define ex vivo cartilage critical size after 28 days culture with custom developed static culture device. In part II of this thesis hydrogel materials, namely collagen I isolated from rat tail, commercially available fibrin glue, matrix-metalloproteinase clevable poly(ethylene glycol) polymerized with heparin (starPEGh), methacrylated poly(N-(2-hydroxypropyl) methacrylamide mono-dilactate-poly(ethylene glycol) triblock copolymer/methacrylated hyaluronic acid (MP/HA), thiol functionalized HA/allyl functionalized poly(glycidol) (P(AGE/G)-HA-SH), were tested cell free and chondrocyte loaded (20 mio/ml) as implant in 4 mm cartilage defects to investigate cartilage regeneration. Reproducible chondral defects, 8 mm in diameter and 1 mm in height, were generated with an artificial tissue cutter (ARTcut®) to investigate effect of defect depth on defect regeneration in part III. In all approaches OCE were analyzed by Safranin-O staining to visualize proteoglycans in cartilage and/or hydrogels. Immuno-histological and -fluorescent stainings (aggrecan, collagen II, VI and X, proCollagen I, SOX9, RUNX2), gene expression analysis (aggrecan, collagen II and X, SOX9, RUNX2) of chondrocyte loaded hydrogels (part II) and proteoglycan and DNA content (Part I \& II) were performed for detailed analysis of cartilage regeneration. Part I: The development of custom made static culture device, consisting of inserts in which OCE is fixed and deep well plate, allowed tissue specific media supply without supplementation of TGF � . Critical size diameter was defined to be 4 mm. Part II: Biomaterials revealed differences in cartilage regeneration. Collagen I and fibrin glue showed presence of cells migrated from OCE into cell free hydrogels with indication of fibrous tissue formation by presence of proCollagen I. In chondrocyte loaded study cartilage matrix proteins aggrecan, collagen II and VI and transcription factor SOX9 were detected after ex vivo culture throughout the two natural hydrogels collagen I and fibrin glue whereas markers were localized in pericellular matrix in starPEGh. Weak stainings resulted for MP/HA and P(AGE/G)-HA-SH in some cell clusters. Gene expression data and proteoglycan quantification supported histological findings with tendency of hypertrophy indicated by upregulation of collagen X and RunX2 in MP/HA and P(AGE/G)-HA-SH. Part III: In life-dead stainings recruitment of cells from OCE into empty or cell free collagen I treated chondral defects was seen. Separated and tissue specific media supply is critical to maintain ECM composition in cartilage. Presence of OCE stimulates cartilage matrix synthesis in chondrocyte loaded collagen I hydrogel and reduces hypertrophy compared to free swelling conditions and pellet cultures. Differences in cartilage repair tissue formation resulted in preference of natural derived polymers compared to synthetic based materials. The ex vivo cartilage defect model represents a platform for testing novel hydrogels as cartilage materials, but also to investigate the effect of cell seeding densities, cell gradients, cell co-cultures on defect regeneration dependent on defect depth. The separated media compartments allow for systematic analysis of pharmaceutics, media components or inflammatory cytokines on bone and cartilage metabolism and matrix stability.}, subject = {Hyaliner Knorpel}, language = {en} } @phdthesis{Martens2020, author = {Martens, Johannes}, title = {Development of an In-Silico Model of the Arterial Epicardial Vasculature}, doi = {10.25972/OPUS-18247}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-182478}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2020}, abstract = {In dynamic CE MR perfusion imaging the passage of an intravenously injected CA bolus through tissue is monitored to assess the myocardial pefusion state. To enable this, knowledge of the shape of CA wash-in through upstream epicardial vessels is required, the so-called AIF. For technical reasons this cannot be quantified directly in the supplying vessels and is thus measured in the left ventricle, which introduces the risk of systematic errors in quantification of MBF due to bolus dispersion in coronary vessels. This means occuring CA dispersion must be accounted in the quantification process in order to produce reliable and reproducible results. In order to do this, CFD simulations are performed to analyze and approximate these errors and deepen insights and knowledge gained from previous CFD analyses on both idealized as well as realistic and pathologically altered 3D geometries. In a first step, several different procedures and approaches are undertaken in order to accelerate the performed workflow, however, maintaining a sufficient degree of numerical accuracy. In the end, the implementation of these steps makes the analysis of the cardiovascular 3D model of unprecedented detail including vessels at pre-arteriolar level feasible at all. The findings of the Navier-Stokes simulations are thus validated with regard to different aspects of cardiac blood flow. These include the distribution of VBF into the different myocardial regions, the areals, which can be associated to the large coronary arteries as well as the fragmentation of VBF into vessels of different diameters. The subsequently performed CA transport simulations yield results on the one hand confirming previous studies. On the other hand, interesting additional knowledge about the behavior of CA dispersion in coronary arteries is obtained both regarding travelled distance as well as vessel diameters. The relative dispersion of the so-called vascular transport function, a characterizing feature of vascular networks, shows a linear decrease with vessel diameter. This results in asymptotically decreased additional dispersion of the CA time curve towards smaller and more distal vessels. Nonetheless, perfusion quantification errors are subject to strong regional variability and reach an average value of \$(-28\pm16)\$ \\% at rest across the whole myocardium. Depending on the distance from the inlet and the considered coronary tree, MBF errors up to 62 \\% are observed.}, subject = {Computerunterst{\"u}tztes Verfahren}, language = {en} } @phdthesis{Choi2024, author = {Choi, Jihyoung}, title = {Development of an Add-On Electrode for Non-Invasive Monitoring in Bioreactor Cultures and Medical Devices}, doi = {10.25972/OPUS-35823}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-358232}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2024}, abstract = {Electrochemical impedance spectroscopy (EIS) is a valuable technique analyzing electrochemical behavior of biological systems such as electrical characterization of cells and biomolecules, drug screening, and biomaterials in biomedical field. In EIS, an alternating current (AC) power signal is applied to the biological system, and the impedance of the system is measured over a range of frequencies. In vitro culture models of endothelial or epithelial barrier tissue can be achieved by culturing barrier tissue on scaffolds made with synthetic or biological materials that provide separate compartments (apical and basal sides), allowing for further studies on drug transport. EIS is a great candidate for non-invasive and real-time monitoring of the electrical properties that correlate with barrier integrity during the tissue modeling. Although commercially available transendothelial/transepithelial electrical resistance (TEER) measurement devices are widely used, their use is particularly common in static transwell culture. EIS is considered more suitable than TEER measurement devices in bioreactor cultures that involve dynamic fluid flow to obtain accurate and reliable measurements. Furthermore, while TEER measurement devices can only assess resistance at a single frequency, EIS measurements can capture both resistance and capacitance properties of cells, providing additional information about the cellular barrier's characteristics across various frequencies. Incorporating EIS into a bioreactor system requires the careful optimization of electrode integration within the bioreactor setup and measurement parameters to ensure accurate EIS measurements. Since bioreactors vary in size and design depending on the purpose of the study, most studies have reported using an electrode system specifically designed for a particular bioreactor. The aim of this work was to produce multi-applicable electrodes and established methods for automated non-invasive and real-time monitoring using the EIS technique in bioreactor cultures. Key to the electrode material, titanium nitride (TiN) coating was fabricated on different substrates (materials and shape) using physical vapor deposition (PVD) and housed in a polydimethylsiloxane (PDMS) structure to allow the electrodes to function as independent units. Various electrode designs were evaluated for double-layer capacitance and morphology using EIS and scanning electron microscopy (SEM), respectively. The TiN-coated tube electrode was identified as the optimal choice. Furthermore, EIS measurements were performed to examine the impact of influential parameters related to culture conditions on the TiN-coated electrode system. In order to demonstrate the versatility of the electrodes, these electrodes were then integrated into in different types of perfusion bioreactors for monitoring barrier cells. Blood-brain barrier (BBB) cells were cultured in the newly developed dynamic flow bioreactor, while human umblical vascular endothelial cells (HUVECs) and Caco-2 cells were cultured in the miniature hollow fiber bioreactor (HFBR). As a result, the TiN-coated tube electrode system enabled investigation of BBB barrier integrity in long-term bioreactor culture. While EIS measurement could not detect HUVECs electrical properties in miniature HFBR culture, there was the possibility of measuring the barrier integrity of Caco-2 cells, indicating potential usefulness for evaluating their barrier function. Following the bioreactor cultures, the application of the TiN-coated tube electrode was expanded to hemofiltration, based on the hypothesis that the EIS system may be used to monitor clotting or clogging phenomena in hemofiltration. The findings suggest that the EIS monitoring system can track changes in ion concentration of blood before and after hemofiltration in real-time, which may serve as an indicator of clogging of filter membranes. Overall, our research demonstrates the potential of TiN-coated tube electrodes for sensitive and versatile non-invasive monitoring in bioreactor cultures and medical devices.}, subject = {Monitoring}, language = {en} } @phdthesis{Schweinlin2016, author = {Schweinlin, Matthias Oliver}, title = {Development of advanced human intestinal in vitro models}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-142571}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2016}, abstract = {The main function of the small intestine is the absorption of essential nutrients, water and vitamins. Moreover, it constitutes a barrier protecting us from toxic xenobiotics and pathogens. For a better understanding of these processes, the development of intestinal in vitro models is of great interest to the study of pharmacological and pathological issues such as transport mechanisms and barrier function. Depending on the scientific questions, models of different complexity can be applied. In vitro Transwell® systems based on a porous PET-membrane enable the standardized study of transport mechanisms across the intestinal barrier as well as the investigation of the influence of target substances on barrier integrity. However, this artificial setup reflects only limited aspects of the physiology of the native small intestine and can pose an additional physical barrier. Hence, the applications of this model for tissue engineering are limited. Previously, tissue models based on a biological decellularized scaffold derived from porcine gut tissue were demonstrated to be a good alternative to the commonly used Transwell® system. This study showed that preserved biological extracellular matrix components like collagen and elastin provide a natural environment for the epithelial cells, promoting cell adhesion and growth. Intestinal epithelial cells such as Caco-2 cultured on such a scaffold showed a confluent, tight monolayer on the apical surface. Additionally, myofibroblasts were able to migrate into the scaffold supporting intestinal barrier formation. In this thesis, dendritic cells were additionally introduced to this model mimicking an important component of the immune system. This co-culture model was then successfully proven to be suitable for the screening of particle formulations developed as delivery system for cancer antigens in peroral vaccination studies. In particular, nanoparticles based on PLGA, PEG-PAGE-PLGA, Mannose-PEG-PAGE-PLGA and Chitosan were tested. Uptake studies revealed only slight differences in the transcellular transport rate among the different particles. Dendritic cells were shown to phagocytose the particles after they have passed the intestinal barrier. The particles demonstrated to be an effective carrier system to transport peptides across the intestinal barrier and therefore present a useful tool for the development of novel drugs. Furthermore, to mimic the complex structure and physiology of the gut including the presence of multiple different cell types, the Caco-2 cell line was replaced by primary intestinal cells to set up a de novo tissue model. To that end, intestinal crypts including undifferentiated stem cells and progenitor cells were isolated from human small intestinal tissue samples (jejunum) and expanded in vitro in organoid cultures. Cells were cultured on the decellularized porcine gut matrix in co-culture with intestinal myofibroblasts. These novel tissue models were maintained under either static or dynamic conditions. Primary intestinal epithelial cells formed a confluent monolayer including the major differentiated cell types positive for mucin (goblet cells), villin (enterocytes), chromogranin A (enteroendocrine cells) and lysozyme (paneth cells). Electron microscopy images depicted essential functional units of an intact epithelium, such as microvilli and tight junctions. FITC-dextran permeability and TEER measurements were used to assess tightness of the cell layer. Models showed characteristic transport activity for several reference substances. Mechanical stimulation of the cells by a dynamic culture system had a great impact on barrier integrity and transporter activity resulting in a tighter barrier and a higher efflux transporter activity. In Summary, the use of primary human intestinal cells combined with a biological decellularized scaffold offers a new and promising way to setup more physiological intestinal in vitro models. Maintenance of primary intestinal stem cells with their proliferation and differentiation potential together with adjusted culture protocols might help further improve the models. In particular, dynamic culture systems and co culture models proofed to be a first crucial steps towards a more physiological model. Such tissue models might be useful to improve the predictive power of in vitro models and in vitro in vivo correlation (IVIVC) studies. Moreover, these tissue models will be useful tools in preclinical studies to test pharmaceutical substances, probiotic active organisms, human pathogenic germs and could even be used to build up patient-specific tissue model for personalized medicine.}, subject = {Tissue Engineering}, language = {en} } @phdthesis{Reuter2023, author = {Reuter, Christian Steffen}, title = {Development of a tissue-engineered primary human skin infection model to study the pathogenesis of tsetse fly-transmitted African trypanosomes in mammalian skin}, doi = {10.25972/OPUS-25114}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-251147}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2023}, abstract = {Many arthropods such as mosquitoes, ticks, bugs, and flies are vectors for the transmission of pathogenic parasites, bacteria, and viruses. Among these, the unicellular parasite Trypanosoma brucei (T. brucei) causes human and animal African trypanosomiases and is transmitted to the vertebrate host by the tsetse fly. In the fly, the parasite goes through a complex developmental cycle in the alimentary tract and salivary glands ending with the cellular differentiation into the metacyclic life cycle stage. An infection in the mammalian host begins when the fly takes a bloodmeal, thereby depositing the metacyclic form into the dermal skin layer. Within the dermis, the cell cycle-arrested metacyclic forms are activated, re-enter the cell cycle, and differentiate into proliferative trypanosomes, prior to dissemination throughout the host. Although T. brucei has been studied for decades, very little is known about the early events in the skin prior to systemic dissemination. The precise timing and the mechanisms controlling differentiation of the parasite in the skin continue to be elusive, as does the characterization of the proliferative skin-residing trypanosomes. Understanding the first steps of an infection is crucial for developing novel strategies to prevent disease establishment and its progression. A major shortcoming in the study of human African trypanosomiasis is the lack of suitable infection models that authentically mimic disease progression. In addition, the production of infectious metacyclic parasites requires tsetse flies, which are challenging to keep. Thus, although animal models - typically murine - have produced many insights into the pathogenicity of trypanosomes in the mammalian host, they were usually infected by needle injection into the peritoneal cavity or tail vein, bypassing the skin as the first entry point. Furthermore, animal models are not always predictive for the infection outcome in human patients. In addition, the relatively small number of metacyclic parasites deposited by the tsetse flies makes them difficult to trace, isolate, and study in animal hosts. The focus of this thesis was to develop and validate a reconstructed human skin equivalent as an infection model to study the development of naturally-transmitted metacyclic parasites of T. brucei in mammalian skin. The first part of this work describes the development and characterization of a primary human skin equivalent with improved mechanical properties. To achieve this, a computer-assisted compression system was designed and established. This system allowed the improvement of the mechanical stability of twelve collagen-based dermal equivalents in parallel through plastic compression, as evaluated by rheology. The improved dermal equivalents provided the basis for the generation of the skin equivalents and reduced their contraction and weight loss during tissue formation, achieving a high degree of standardization and reproducibility. The skin equivalents were characterized using immunohistochemical and histological techniques and recapitulated key anatomical, cellular, and functional aspects of native human skin. Furthermore, their cellular heterogeneity was examined using single-cell RNA sequencing - an approach which led to the identification of a remarkable repertoire of extracellular matrix-associated genes expressed by different cell subpopulations in the artificial skin. In addition, experimental conditions were established to allow tsetse flies to naturally infect the skin equivalents with trypanosomes. In the second part of the project, the development of the trypanosomes in the artificial skin was investigated in detail. This included the establishment of methods to successfully isolate skin-dwelling trypanosomes to determine their protein synthesis rate, cell cycle and metabolic status, morphology, and transcriptome. Microscopy techniques to study trypanosome motility and migration in the skin were also optimized. Upon deposition in the artificial skin by feeding tsetse, the metacyclic parasites were rapidly activated and established a proliferative population within one day. This process was accompanied by: (I) reactivation of protein synthesis; (II) re-entry into the cell cycle; (III) change in morphology; (IV) increased motility. Furthermore, these observations were linked to potentially underlying developmental mechanisms by applying single-cell parasite RNA sequencing at five different timepoints post-infection. After the initial proliferative phase, the tsetse-transmitted trypanosomes appeared to enter a reversible quiescence program in the skin. These quiescent skin-residing trypanosomes were characterized by very slow replication, a strongly reduced metabolism, and a transcriptome markedly different from that of the deposited metacyclic forms and the early proliferative trypanosomes. By mimicking the migration from the skin to the bloodstream, the quiescent phenotype could be reversed and the parasites returned to an active proliferating state. Given that previous work has identified the skin as an anatomical reservoir for T. brucei during disease, it is reasonable to assume that the quiescence program is an authentic facet of the parasite's behavior in an infected host. In summary, this work demonstrates that primary human skin equivalents offer a new and promising way to study vector-borne parasites under close-to-natural conditions as an alternative to animal experimentation. By choosing the natural transmission route - the bite of an infected tsetse fly - the early events of trypanosome infection have been detailed with unprecedented resolution. In addition, the evidence here for a quiescent, skin-residing trypanosome population may explain the persistence of T. brucei in the skin of aparasitemic and asymptomatic individuals. This could play an important role in maintaining an infection over long time periods.}, subject = {Trypanosoma brucei}, language = {en} } @phdthesis{Behne2024, author = {Behne, Robert Stefan Friedrich}, title = {Development Of A Human iPSC-Derived Cortical Neuron Model Of Adaptor- Protein-Complex-4-Deficiency}, doi = {10.25972/OPUS-35139}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-351390}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2024}, abstract = {Adaptor-protein-4-deficiency (AP-4-deficiency) is an autosomal-recessive childhood- onset form of complicated hereditary spastic paraplegia (HSP) caused by bi-allelic loss- of-function mutations in one of the four subunits of the AP-4-complex. These four conditions are named SPG47 (AP4B1, OMIM \#614066), SPG50 (AP4M1, OMIM \#612936), SPG51 (AP4E1, OMIM \#613744) and SPG52 (AP4S1, OMIM \#614067), respectively and all present with global developmental delay, progressive spasticity and seizures. Imaging features include a thinning of the corpus callosum, ventriculomegaly and white matter changes. AP-4 is a highly conserved heterotetrameric complex, which is responsible for polarized sorting of transmembrane cargo including the autophagy- related protein 9 A (ATG9A). Loss of any of the four subunits leads to an instable complex and defective sorting of AP-4-cargo. ATG9A is implicated in autophagosome formation and neurite outgrowth. It is missorted in AP-4-deficient cells and CNS-specific knockout of Atg9a in mice results in a phenotype reminiscent of AP-4-deficiency. However, the AP-4-related cellular phenotypes including ATG9A missorting have not been investigated in human neurons. Thus, the aim of this study is to provide the first human induced pluripotent stem cell- derived (iPSC) cortical neuron model of AP-4-deficiency to explore AP-4-related phenotypes in preparation for a high-content screening. Under the hypothesis that AP-4- deficiency leads to ATG9A missorting, elevated ATG9A levels, impaired autophagy and neurite outgrowth in human iPSC-derived cortical neurons, in vitro biochemical and imaging assays including automated high-content imaging and analysis were applied. First, these phenotypes were investigated in fibroblasts from three patients with compound heterozygous mutations in the AP4B1 gene and their sex-matched parental controls. The same cell lines were used to generate iPSCs and differentiate them into human excitatory cortical neurons. This work shows that ATG9A is accumulating in the trans-Golgi-network in AP-4- deficient human fibroblasts and that ATG9A levels are increased compared to parental controls and wild type cells suggesting a compensatory mechanism. Protein levels of the AP4E1-subunit were used as a surrogate marker for the AP-4-complex and were decreased in AP-4-deficient fibroblasts with co-immunoprecipitation confirming the instability of the complex. Lentiviral re-expression of the AP4B1-subunit rescues this corroborating the fact that a stable AP-4-complex is needed for ATG9A trafficking. Surprisingly, autophagic flux was present in AP-4-deficient fibroblasts under nutrient- rich and starvation conditions. These phenotypic markers were evaluated in iPSC-derived cortical neurons and here, a robust accumulation of ATG9A in the juxtanuclear area was seen together with elevated ATG9A protein levels. Strikingly, assessment of autophagy markers under nutrient-rich conditions showed alterations in AP-4-deficient iPSC- derived cortical neurons indicating dysfunctional autophagosome formation. These findings point towards a neuron-specific impairment of autophagy and need further investigation. Adding to the range of AP-4-related phenotypes, neurite outgrowth and branching are impaired in AP-4-deficient iPSC-derived cortical neurons as early as 24h after plating and together with recent studies point towards a distinct role of ATG9A in neurodevelopment independent of autophagy. Together, this work provides the first patient-derived neuron model of AP-4-deficiency and shows that ATG9A is sorted in an AP-4-dependent manner. It establishes ATG9A- related phenotypes and impaired neurite outgrowth as robust markers for a high-content screening. This disease model holds the promise of providing a platform to further study AP-4-deficiency and to search for novel therapeutic targets.}, subject = {Adaptorproteine}, language = {en} } @phdthesis{Bakirci2024, author = {Bakirci, Ezgi}, title = {Development of \(In\) \(vitro\) Models for Tissue Engineering Applications Using a High-Resolution 3D Printing Technology}, doi = {10.25972/OPUS-25164}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-251645}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2024}, abstract = {In vitro models mimic the tissue-specific anatomy and play essential roles in personalized medicine and disease treatments. As a sophisticated manufacturing technology, 3D printing overcomes the limitations of traditional technologies and provides an excellent potential for developing in vitro models to mimic native tissue. This thesis aims to investigate the potential of a high-resolution 3D printing technology, melt electrowriting (MEW), for fabricating in vitro models. MEW has a distinct capacity for depositing micron size fibers with a defined design. In this thesis, three approaches were used, including 1) extending the MEW polymer library for different biomedical applications, 2) developing in vitro models for evaluation of cell growth and migration toward the different matrices, and 3) studying the effect of scaffold designs and biochemical cues of microenvironments on cells. First, we introduce the MEW processability of (AB)n and (ABAC)n segmented copolymers, which have thermally reversible network formulation based on physical crosslinks. Bisurea segments are combined with hydrophobic poly(dimethylsiloxane) (PDMS) or hydrophilic poly(propylene oxide)-poly(ethylene oxide)-poly(propylene oxide) (PPO-PEG-PPO) segments to form the (AB)n segmented copolymers. (ABAC)n segmented copolymers contain all three segments: in addition to bisurea, both hydrophobic and hydrophilic segments are available in the same polymer chain, resulting in tunable mechanical and biological behaviors. MEW copolymers either support cells attachment or dissolve without cytotoxic side effects when in contact with the polymers at lower concentrations, indicating that this copolymer class has potential in biological applications. The unique biological and surface properties, transparency, adjustable hydrophilicity of these copolymers could be beneficial in several in vitro models. The second manuscript addresses the design and development of a melt electrowritten competitive 3D radial migration device. The approach differs from most of the previous literature, as MEW is not used here to produce cell invasive scaffolds but to fabricate an in vitro device. The device is utilized to systematically determine the matrix which promotes cell migration and growth of glioblastoma cells. The glioblastoma cell migration is tested on four different Matrigel concentrations using a melt electrowritten radial device. The glioblastoma U87 cell growth and migration increase at Matrigel concentrations 6 and 8 mg mL-1 In the development of this radial device, the accuracy, and precision of melt electrowritten circular shapes were investigated. The results show that the printing speed and design diameter are essential parameters for the accuracy of printed constructs. It is the first instance where MEW is used for the production of in vitro devices. The influence of biochemical cues and scaffold designs on astrocytes and glioblastoma is investigated in the last manuscript. A fiber comprising the box and triangle-shaped pores within MEW scaffolds are modified with biochemical cues, including RGD and IKVAV peptides using a reactive NCO-sP(EO-stat-PO) macromer. The results show that astrocytes and glioblastoma cells exhibit different phenotypes on scaffold designs and peptide-coated scaffolds.}, subject = {3D-Druck}, language = {en} } @phdthesis{Wallstabe2022, author = {Wallstabe, Lars}, title = {Development and preclinical evaluation of tumour-reactive T cells expressing a chemically programmable chimeric antigen receptor}, doi = {10.25972/OPUS-17907}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-179071}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2022}, abstract = {The genetic modification of T cells for the expression a chimeric antigen receptor (CAR) endows them with a new specificity for an antigen. Adoptive immunotherapy with CD19-CAR T cells has achieved high rates of sustained complete remissions in B cell malignancies. However, the downregulation or loss of the targeted antigen after mono-specific CAR T cell therapy, e.g. against CD19 or CD22, has been reported. Targeting multiple antigens on tumour cells, sequentially or simultaneously, could overcome this limitation. Additionally, targeting multiple antigens with CAR T cells could drive the translation from hematologic malignancies to prevalent solid cancers, which often express tumour-associated antigens heterogeneously. We hypothesised that expression of a universal CAR, which can be programmed with hapten-like molecules, could endow T cells with specificities for multiple antigens. In this study we introduce a novel chemically programmable CAR (cpCAR) based on monoclonal antibody h38C2. Our data show, that cpCARs form a reversible chemical bond to molecules containing a diketone-group and therefore can be programmed to acquire multiple specificities. We programmed cpCAR T cells with hapten-like compounds against integrins αvβ3 and α4β1 as well as the folate receptor. We observed tumour cell lysis, IFN ɣ and IL-2 production and proliferation of programmed cpCAR T cells against tumour cells expressing the respective target antigen in vitro. As a reference to cpCARs programmed against αvβ3, we further introduced novel conventional αvβ3-CARs. These CARs, based on humanised variants of monoclonal antibody LM609 (hLM609), directly bind to integrin αvβ3 via their scFv. The four αvβ3-CAR constructs comprised either an scFv with higher affinity (hLM609v7) or lower affinity (hLM609v11) against αvβ3 integrin and either a long (IgG4 hinge, CH2, CH3) or short (IgG4 hinge) extracellular spacer. We selected the hLM609v7-CAR with short spacer, which showed potent anti-tumour reactivity both in vitro and in a murine xenograft model, for comparison with the cpCAR programmed against αvβ3. Our data show specific lysis of αvβ3-positive tumour cells, cytokine production and proliferation of both hLM609-CAR T cells and cpCAR T cells in vitro. However, conventional hLM609-CAR T cells mediated stronger anti-tumour effects compared to cpCAR T cells in the same amount of time. In line with the in vitro data, complete destruction of tumour lesions in a murine melanoma xenograft model was only observed for mice treated with conventional αvβ3-CAR T cells. Collectively, we introduce a cpCAR, which can be programmed against multiple tumour antigens, and hLM609-CARs specific for the integrin αvβ3. The cpCAR technology bears the potential to counteract current limitations, e.g. antigen loss, of current monospecific CAR T cell therapy. Targeting αvβ3 integrin with CAR T cells could have clinical applications in the treatment of solid malignancies, because αvβ3 is not only expressed on a variety of solid malignancies, but also on tumour-associated vasculature and fibroblast.}, subject = {Tumorimmunologie}, language = {en} } @phdthesis{Weber2024, author = {Weber, Justus C.}, title = {Development and preclinical assessment of ROR2-specific CAR-T cells for the treatment of clear cell renal cell carcinoma and multiple myeloma}, doi = {10.25972/OPUS-31039}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-310399}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2024}, abstract = {Adoptive immunotherapy using chimeric antigen receptor (CAR)-modified T cells is an effective treatment for hematological malignancies that are refractory to conventional chemotherapy. To address a wider variety of cancer entities, there is a need to identify and characterize additional target antigens for CAR-T cell therapy. The two members of the receptor tyrosine kinase-like orphan receptor family, ROR1 and ROR2, have been found to be overexpressed on cancer cells and to correlate with aggressive cancer phenotypes. Recently, ROR1-specific CAR-T cells have entered testing in phase I clinical trials, encouraging us to assess the suitability of ROR2 as a novel target for CAR-T cell therapy. To study the therapeutic potential of targeting ROR2 in solid and hematological malignancies, we selected two representative cancer entities with high unmet medical need: renal cell carcinoma and multiple myeloma. Our data show that ROR2 is commonly expressed on primary samples and cell lines of clear cell renal cell carcinoma and multiple myeloma. To study the efficacy of ROR2-specific CAR T cell therapy, we designed two CAR constructs with 10-fold binding affinity differences for the same epitope of ROR2. We found both cell products to exhibit antigen-specific anti-tumor reactivity in vitro, including tumor cell lysis, secretion of the effector cytokines interleukin-2 (IL-2) and interferon-gamma (IFNγ), and T cell proliferation. In vivo studies revealed ROR2 specific CAR-T cells to confer durable responses, significant survival benefits and long-term persistence of CAR-expressing T cells. Overall, there was a trend towards more potent anti-tumor efficacy upon treatment with T cells that expressed the CAR with higher affinity for ROR2, both in vitro and in vivo. We performed a preclinical safety and toxicology assessment comprising analyses of ROR2 expression in healthy human and murine tissues, cross-reactivity, and adoptive T cell transfer in immunodeficient mice. We found ROR2 expression to be conserved in mice, and low-level expression was detectable in the male and female reproductive system as well as parts of the gastrointestinal tract. CAR-T cells targeting human ROR2 were found to elicit similarly potent reactivity upon recognition of murine ROR2. In vivo analyses showed transient tissue-specific enrichment and activation of ROR2-specific CAR-T cells in organs with high blood circulation, such as lung, liver, or spleen, without evidence for clinical toxicity or tissue damage as determined by histological analyses. Furthermore, we humanized the CAR binding domain of ROR2-specific CAR-T cells to mitigate the risk of adverse immune reactions and concomitant CAR-T cell rejection. Functional analyses confirmed that humanized CARs retained their specificity and functionality against ROR2-positive tumor cells in vitro. In summary, we show that ROR2 is a prevalent target in RCC and MM, which can be addressed effectively with ROR2-specific CAR-T cells in preclinical models. Our preliminary toxicity studies suggest a favorable safety profile for ROR2-specific CAR-T cells. These findings support the potential to develop ROR2-specific CAR-T cells clinically to obtain cell products with broad utility.}, subject = {CAR-T-Zell-Therapie}, language = {en} } @phdthesis{Reuter2020, author = {Reuter, Isabel}, title = {Development and function of monoaminergic systems in the brain of zebrafish}, doi = {10.25972/OPUS-20408}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-204089}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2020}, abstract = {This thesis explores the development of monoaminergic systems in the central nervous system (CNS) of zebrafish. The serotonergic cells of the hypothalamus pose the main focus of the present work. Most vertebrates except for mammals possess serotonin (5-HT) synthesising cells in more than one region of the CNS. In zebrafish such regions are, e.g. the hypothalamus, the raphe nuclei and the spinal cord. Serotonin functions as a neurotransmitter and neuromodulator in the CNS. Presumably due to its neuromodulatory tasks hypothalamic serotonergic cells are in contact with the cerebrospinal fluid (CSF), which expands the field of potential serotonergic targets tremendously. This highlights that serotonergic CSF-contacting (CSF-c) cells are vital for the execution of many functions and behaviours. Further, the hypothalamic serotonergic clusters constitute the largest population of serotonergic cells in the CNS of zebrafish. Together, these facts emphasise the need to understand the development and function of serotonergic CSF-c cells in the hypothalamus. Few studies have dealt with this subject, hence, information about the development of these cells is scarce. The zinc-finger transcription factor fezf2, and Fibroblast growth factor (Fgf)-signalling via the ETS-domain transcription factor etv5b are known to regulate serotonergic cell development in the hypothalamus (Bosco et al., 2013; Rink and Guo, 2004). However, the main Fgf ligand responsible for this mediation has not been determined prior to this work. The present thesis identifies Fgf3 as a crucial Fgf ligand. To achieve this result three independent strategies to impair Fgf3 activity have been applied to zebrafish embryos: the fgf3t24152 mutant, an fgf3 morpholino-based knock-down and the CRISPR/Cas9 technique. The investigations show that Fgf3 regulates the development of monoaminergic CSF-c cells in the hypothalamus. Additionally, Fgf3 impacts on cells expressing the peptide hormone arginine vasopressin (avp). Most interestingly, the requirement for Fgf3 by these cells follows a caudo-rostral gradient with a higher dependence on Fgf3 by caudal cells. This also seems to be the case for dopaminergic CSF-c cells in the hypothalamus (Koch et al., 2014). Moreover, etv5b a downstream target of Fgf-signalling is demonstrated to be under the control of Fgf3. With regard to serotonergic CSF-c cell development, it is shown that fgf3 is expressed several hours before tph1a and 5-HT (Bellipanni et al., 2002; Bosco et al., 2013). Together with the result that the hypothalamus is already smaller before mature serotonergic CSF-c cells appear, this argues for an early impact of Fgf3 on serotonergic specification. This hypothesis is supported by several findings in this study: the universal decrease of proliferating cells in the hypothalamus and simultaneous increase of cell death after fgf3 impairment. Complementary cell fate experiments confirm that proliferating serotonergic progenitors need Fgf3 to commit serotonergic specification. Further, these results corroborate findings of an earlier study stating that hypothalamic serotonergic progenitors require Fgf-signalling via etv5b to maintain the progenitor pool (Bosco et al., 2013). Additionally, the transcriptome of the hypothalamus has been analysed and 13 previously overlooked transcripts of Fgf ligands are expressed at developmental stages. The transcriptome analysis provides evidence for a self-compensatory mechanism of fgf3 since expression of fgf3 is upregulated as a consequence of its own impairment. Moreover, the Fgf-signalling pathway appears to be mildly affected by fgf3 manipulation. Together, Fgf-signalling and especially Fgf3 are established to be of critical importance during hypothalamic development with effects on serotonergic, dopaminergic CSF-c and avp expressing cells. Furthermore, this thesis provides two strategies to impair the tph1a gene. Both strategies will facilitate investigations regarding the function of hypothalamic serotonergic CSF-c cells. Finally, the presented findings in this study provide insights into the emergence of the posterior recess region of the hypothalamus, thereby, contributing to the understanding of the evolution of the vertebrate hypothalamus.}, subject = {Hypothalamus}, language = {en} } @phdthesis{Junker2015, author = {Junker, Markus}, title = {Development and characterization of monoclonal antibodies to GDF-15 for potential use in cancer therapy}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-132424}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2015}, abstract = {Background GDF-15 is a divergent member of the TGF-superfamily, which was first described as macrophage inhibitory cytokine-1 (MIC-1), revealing an immune modulatory function. GDF-15 is a soluble protein which is, under physiological conditions, highly expressed in the placenta and found in elevated levels in blood sera of pregnant women. Apart from the placenta, GDF-15 is expressed in healthy tissue, albeit to a lower extent and overexpressed in many solid tumors. A variety of different functions are attributed to GDF-15 in healthy as well as diseased humans. On the one hand, GDF-15 is required for successful pregnancy and low GDF-15 serum levels during pregnancy correlate with fetal abortion. On the other hand, overexpression of GDF-15, which can be observed in several malignancies is correlated with a poor prognosis. Furthermore, tumor derived GDF-15 leads to cancer associated anorexia-cachexia syndrome in mice. The aim of my PhD thesis was to further investigate the role of GDF-15 as an immune modulatory factor in cancer, in particular, by inhibiting the target molecule in vitro and in vivo. Therefore, the main focus was placed on the generation and characterization of monoclonal GDF-15 specific blocking antibodies, which were tested in vitro and in vivo, which represents a substantial part of my work. Results Here, GDF-15 was shown to be highly expressed in human gynecological cancer and brain tumors. We could then demonstrate that GDF-15 modulates effector immune cells in vitro. GDF-15 mediated a slight downregulation of the activating NKG2D receptor on NK and CD8+ T cells, which is crucial for proper anti-tumoral immune responses. Furthermore, we could demonstrate that GDF-15 reduces the adhesion of CD4+ and CD8+ T cells on endothelial cells in vitro. A negatively affected trans-endothelial migration of leukocytes into inflamed tissue could explain the low T cell infiltration in GDF-15 expressing tumors, which were observed in vivo, where mice bearing (shRNA mediated) GDF-15 deficient glioma cells revealed enhanced immune cell infiltrates in the tumor microenvironment, compared with the GDF-15 expressing control group. Those animals further exhibited a decreased tumor growth and prolonged survival. GDF-15 is a soluble protein, secreted by more than 50 \% of solid tumors and associated with grade of malignancy. Therefore a neutralizing monoclonal antibody to GDF-15 was assumed to be an auspicious therapeutically anti-cancer tool. Such an antibody was thus generated in GDF-15 knock out mice against human GFD-15. Amongst many clones, the GDF-15 antibody clone B1-23 was found to be applicable in Western Blot as well as in ELISA techniques, detecting a three-dimensional epitope of the mature GDF-15 dimer with high affinity and specificity. To enable the humanization for a later administration in humans, the variable regions of antibody B1-23 were identified by a special PCR method using degenerate primers and cloned into a sequencing vector. The sequence obtained thereby enabled the generation of chimeric and humanized B1-23 variants. After further comprehensive characterization, the original mouse antibody B1-23 as well as the chimeric antibody (ChimB1-23) and the humanized B1-23 antibody (H1L5) were applied in a melanoma xenograft study in vivo. None of the antibodies could significantly inhibit tumor growth. .However of utmost importance, body weight loss mediated by tumor derived GDF-15 could be significantly prevented upon administration of all three GDF-15 specific antibodies, which confirmed the antagonizing functionality of the immunoglobulin. Conclusion GDF-15 is a promising cancer target, involved in tumor progression and cancer related cachexia. A monoclonal GDF-15 antibody was generated, which served on one hand as a tool for molecular biological applications (Western Blot, ELISA, etc.) and on the other hand was applied as an antagonizing antibody in vitro and in vivo. Even though tumor growth inhibition by GDF-15 depletion in T cell deficient athymic mice failed using B1-23, the same antibody and derivates thereof (chimeric and humanized) impressively prevented tumor associated cachexia in UACC-257 melanoma bearing nude mice. The missing anti-tumor effect in our own melanoma model in nude mice can only partially be explained by the missing secondary immunity, in particular cytotoxic T cells, in the athymic animals, since in a similar melanoma model, performed by an external company, a tumor reduction in immunocompromised animals was observed, when B1-23 was administered. These findings support the idea that T cells are substantial for an effective tumor immunity and are in line with the results of the syngeneic, T cell comprising, mouse glioma model, where silencing of tumor expressed GDF-15 led to an enhanced intratumoral T cell infiltration and a prolonged survival. Taken together our data allow for the conclusion that tumor associated cachexia can be combatted with the GDF-15 antibody B1-23. Further, B1-23 might elicit direct anti-tumor effects in immune competent models, which contain T cells, rather than in an athymic, T cell deficient nude mouse model.}, subject = {Growth-differentiation Factor 15}, language = {en} }