Farmaceutische, Biomedische en Dier­geneeskundige Wetenschappen

Doctoraten 2020

Departement Farmaceutische Wetenschappen

Spray-dried formulation development of novel upper respiratory tract lactobacilli - Department Pharmaceutical Sciences - Katarina Jokicevic (12/01/2020)

Public Defence Katarina Jokicevic - Spray-dried formulation development of novel upper respiratory tract lactobacilli - Department Pharmaceutical Sciences

Promotors: Prof. Filip Kiekens - Prof. Sarah Lebeer

ONLINE DEFENCE: https://eu.bbcollab.com/guest/572cf8aeb5c140d69763715539df0eef

ABSTRACT:


With the discovery of penicillin a revolution in chemotherapy and medicine, modern ‘antibiotic era’, has started and lives of millions of people have been saved from death and suffering caused by bacterial infections. However, the overuse of antibiotics within and outside the healthcare sector has led to an emerging, big threat to the well-being of the humankind called the antimicrobial resistance (AMR). The upper respiratory tract (URT) is the main entrance point for many viral and bacterial pathogens, and URT infections are among the most common infections in the world followed by high and often unjustified antibiotic use. Recent evidences imply the importance of lactobacilli as gatekeepers of a healthy URT. However, the benefits of putting health-promoting microbes or potential probiotics, such as these URT lactobacilli, in function of URT disease control and prevention is underestimated, among others because of the absence of adequate formulation modalities.

Probiotics are ‘live microorganisms that, when administered in adequate amounts, confer a health benefit on the host’ (Hill et al. 2014). The incorporation of probiotics into pharmaceutical formulations or functional foods is very challenging. Key problems in the development are maintaining high viability including long-term stability (preferably measured in years), functionality of probiotic cultures, quality and process monitoring and an adequate demonstration of positive benefit-risk ratios. Drying, i.e. lowering water content, is a common strategy used to enhance the short shelf-life of beneficial bacteria. Nevertheless, drying is linked with another set of challenges, such as dehydration, heat or cold stress, osmotic stresses, mechanical stresses, significantly impacting cell viability and functionality. Spray drying is a fast, continuous, cost-effective method with end product being powder with tunable characteristics. Yet, it is an underexplored alternative to the conventional freeze drying method of probiotic preservation due to high process temperatures and insufficient literature data on storage stability, functionality and application feasibility of spray-dried cultures.

Therefore, the goal of this project was to investigate the potential of spray drying for production of viable, functional and long-term stable URT (potential) probiotics. Key process characteristics exhibiting most significant damaging effects on bacterial cells were unravelled and minimised. Different protection strategies were envisaged resulting in high viability both after spray drying and storage (over a year). Novel probiotic nasal sprays and oral powders with uncompromised cell functionality have been developed. In vitro and in vivo testing including human volunteer study confirmed the effectiveness of developed probiotic formulations and the validity of postulated principles.

Finally, it can be concluded that the age of probiotics is yet to come and this research paves the way to new treatment opportunities to ease the battle against URT and other microbial-based diseases and fast-evolving AMR.

Development of new recombinase polymerase amplification (RPA) and antigen-based diagnostic tools for the detection of Trypanosoma evansi infections - Li Zeng (11/12/2020)

Public defence Li Zeng 11/12/2020 - Development of new recombinase polymerase amplification (RPA) and antigen-based diagnostic tools for the detection of Trypanosoma evansi infections - Department Pharmaceutical Sciences

Promotors: Prof. Yann Sterckx - Prof. Stefan Magez

ONLINE DEFENCE

Abstract:

Abstract (ENG)Animal trypanosomosis (AT) is a neglected tropical disease affecting domestic and wild animals, which severely impairs the socio-economic development of endemic areas. The most widespread causative agent of AT is Trypanosoma evansi. This parasite has a lifecycle that allows transmission by a range of different insect vectors and has spread beyond the African continent to large parts of the world including the Middle East, the Mediterranean, Asia and South America. Disease control mainly relies on chemotherapy, as there are no effective vaccines available. In order to optimize treatment, reliable and sensitive point-of-care (POC) tests are needed to detect infected animals. Hence, the work presented in this thesis has focused on two diagnostic development approaches. First, a new technology called Recombinase Polymerase Amplification (RPA) was adopted for the molecular diagnosis of T. evansi infections by targeting the gene encoding T. evansi specific RoTat1.2 VSG. The technique is an isothermal nucleic acid amplification approach that is simple, fast, cost-effective and is suitable for use in minimally equipped laboratories. RPA was subsequently combined with lateral flow (LF) technology, providing a simple test readout suitable for use in resource-limited settings and field conditions. Secondly, the PhD work focused on the development of a serological antigen-based assay format, which would be suitable for the detection of active infections and could be used as a test-of-cure device to monitor anti-trypanosome treatment success in animals. The test we developed is based on Nanobody technology, as this allows antigen capturing through cryptic epitopes on parasite molecules that are hard to target with much larger conventional monoclonal or polyclonal antibodies. The use of an “unbiased” alpaca immunization strategy with T. evansi secretome resulted in the identification of the glycolytic enzyme enolase (TevENO) as a potential biomarker for the Nb-based detection of T. evansi infections. As our approach indicated that TevENO is actively released by the parasite in detectable quantitates, we initiated work that aims at the future elucidation of the role this secreted enzyme could play in the context of the host-pathogen interface. Initial results show the ability of TevENO to bind to human plasminogen (PLG), the precursor of plasmin, which itself an important factor in fibrinolysis and the prevention is of blood clotting.

Stabilization of atherosclerotic plaques via inhibition of regulated necrosis - Department Pharmaceutical Sciences - Isabelle Coornaert (08/12/2020)

Public Defence Isabelle Coornaert 08/12/2020 - Stabilization of atherosclerotic plaques via inhibition of regulated necrosis - Department Pharmaceutical Sciences

Promotors: Prof. Guido De Meyer - Prof. Wim Martinet

ONLINE DEFENCE: https://eu.bbcollab.com/guest/a63fa2826f754532b67783f03218bf85

ABSTRACT:


Stabilization of atherosclerotic plaques via inhibition of regulated necrosis

Atherosclerosis is a progressive inflammatory disease of the large and medium-sized arteries and is characterized by the formation of plaques in the vessel wall. Rupture of these plaques may cause life-threatening complications such as a myocardial infarct and stroke. A plaque that is prone to rupture is characterized by the presence of a large necrotic core, which is formed by cells within the plaque that undergo necrosis. Accordingly, targeting necrotic cell death may be a therapeutic strategy to stabilize atherosclerotic plaques. The main goal of this dissertation was to investigate whether inhibition of two different forms of regulated necrosis, necroptosis and ferroptosis, reduces formation of atherosclerotic plaques.

To investigate the role of the necroptosis mediator RIPK1, we investigated the impact of a myeloid RIPK1 deletion on the development of atherosclerotic plaques in ApoE-/- mice. Myeloid-specific RIPK1-deficient mice (RIPK1F/FLysMC-Cre+) were crossbred with ApoE-/- mice and fed a western-type diet (WD) for 16 or 24 weeks. After 16 weeks WD, plaque size and necrotic core were reduced in RIPK1F/FLysMC-Cre+ApoE-/- mice. However, after 24 weeks WD, plaque size and necrotic core were no longer different between RIPK1F/FLysMC-Cre+ApoE-/- mice and the control mice. Taken together, myeloid RIPK1 has divergent effects on atherosclerosis depending on the lesion stage. Next, we evaluated the effect of a RIPK1 inhibitor in a mouse model of advanced atherosclerosis, the ApoE-/-Fbn1C1039G+/-mouse. Unexpectedly, plaque size and necrotic core were not different between the control mice and the mice treated with the RIPK1 inhibitor. Additional experiments using distinct RIPK1 inhibitors are needed to further unravel the role of necroptosis in atherosclerosis.

Apart from necroptosis, the role of ferroptosis in atherosclerosis has been studied. Ferroptosis is characterized by an iron-dependent accumulation of lipid hydroperoxides. Because GPX4 reduces lipid hydroperoxides thereby preventing ferroptosis, we evaluated the impact of GPX4 overexpression in ApoE-/- mice. Although GPX4 overexpression inhibited lipid peroxidation in atherosclerotic plaques of ApoE-/- mice fed a WD, we could not demonstrate that GPX4 overexpression reduces atherosclerosis in ApoE-/- mice. Given that iron contributes to the development of advanced atherosclerotic plaques, we performed a pharmacological study to inhibit ferroptosis using a-tocopherol (vitamin E) in a mouse model of advanced atherosclerosis, namely the ApoE-/-Fbn1C1039G+/- mouse. A high dose of a-tocopherol improved cardiac function and decreased plaque thickness and necrotic core size. Because mice treated with a high dose of a-tocopherol showed reduced plaque formation, we assume that a-tocopherol limits plaque progression before ferroptosis could occur. Moreover, mice treated with a high dose of a-tocopherol showed increased lipid peroxidation. Overall, this study demonstrates that a-tocopherol inhibits atherogenesis independent of its antioxidant properties.

Taken together, this study highlighted the role of RIPK1 in atherosclerosis. However, treatment with a RIPK1 inhibitor did not yield therapeutic benefits in atherosclerosis. The complex role of RIPK1 should be taken into account when using RIPK1 as a therapeutic tool for the treatment of atherosclerosis. Additionally, we were not able to demonstrate whether ferroptosis inhibition using GPX4-overexpressing mice or the pharmacological inhibitor a-tocopherol affects atherosclerosis. Nonetheless, a-tocopherol decreased plaque size and improved cardiac function independent of its antioxidant properties.

Humane blootstelling aan bisfenol A en alternatieven: bronnen, concentraties, patronen en determinanten - Celine Gys (30/11/2020)

  • ​Celine Gys 
  • 30/11/2020
  • 17-19 uur 
  • Online defence
  • Promotoren: Adrian Covaci, Hugo Neels

Abstract

De toxiciteit en het voorkomen van bisfenol A (BPA) zijn uitgebreid bestudeerd, maar ondanks de hoeveelheid beschikbaar onderzoek is er geen consensus over de gevolgen voor de menselijke gezondheid en het milieu. BPA wordt gebruikt in verschillende toepassingen, zoals elektronische apparatuur, bouwmaterialen, voedselverpakkingen en thermaal papier. De groeiende bezorgdheid over BPA leidt wereldwijd tot restricties, met als gevolg dat het vermoedelijk toxische maar goed gedocumenteerde BPA wordt vervangen door minder bestudeerde bisfenolen. We hebben enkele van de hiaten in de kennis over BPA en de meest toegepaste alternatieve bisfenolen aangepakt.

Met behulp van hoge-resolutie massaspectrometrie in combinatie met suspect en non-target screeningsworkflows werden biotransformatieproducten, gegenereerd door het menselijk metabolisme en door afbraak van BPA-analogen in het milieu, en nieuwe alternatieven voor BPA in thermaal papier geïdentificeerd. Met behulp van een humaan in vitro metabolisatie experiment werd een volledige screening en structurele opheldering van in vitro metabolieten van BPS uitgevoerd. De transformatie van bisfenolen in waterig milieu werd onderzocht door middel van fotochemische en biologische afbraakprocessen en de gegenereerde transformatieproducten werden geïdentificeerd. Aangezien huidcontact met thermaal papier (bv. kassabonnen en tickets) als een substantiële bron van blootstelling aan BPA en zijn alternatieven wordt beschouwd, hebben we > 300 monsters van thermaal papier uit 14 verschillende landen gescreend. Hoewel BPA nog steeds de belangrijkste ontwikkelaar was, werden acht alternatieven, o.a. BPS gedetecteerd. Er werden verschillende trends waargenomen tussen de landen, die waarschijnlijk van invloed zullen zijn op de blootstelling van de mens.

Om de totale interne blootstelling aan bisfenolen te meten via biomonitoring, werd een analytische methode op basis van gaschromatografie gekoppeld aan tandem-massaspectrometrie ontwikkeld en gevalideerd. Concentraties van bepaalde BPA-analogen werden voor het eerst gemeten in meerdere urinestalen die gedurende vijf opeenvolgende dagen bij tien individuen werden verzameld en vertoonden een hoge interne variabiliteit. Op basis van deze resultaten werden aanbevelingen geformuleerd voor toekomstige bemonsteringsstrategieën. Determinanten van urinaire bisfenolconcentraties werden onderzocht in onderzoekspopulaties van Japanse schoolkinderen en Vlaamse adolescenten, met behulp van vragenlijstgegevens. Hoewel de gemeten niveaus van bisfenolen onderhevig zijn aan regionale verschillen, was het duidelijk dat de blootstelling aan BPA de afgelopen tien jaar significant afnam, waarschijnlijk als gevolg van beperkingen en vervanging. Als risico-evaluatie werden de geschatte dagelijkse inname en de gemeten urinaire concentraties vergeleken met de beschikbare health-based richtwaarden. Zelfs in scenario's met een hoge blootstelling werden voor de onderzochte populaties geen gezondheidsproblemen verwacht. Verdere studies zijn nodig om de blootstelling aan bisfenolen in de algemene bevolking en hun mogelijke schadelijke gezondheidseffecten te karakteriseren.

Formulation and process research to safeguard Lacticaseibacillus rhamnosus GG during tablet manufacturing - Eline Byl (27/10/2020)

Eline Byl

  • 27 oktober 2020
  • Promotoren: Filip Kiekens en Sarah Lebeer

Abstract

The morbidity rate of respiratory infections is high. Viral infections are cured with over-the-counter products that treat the symptoms. However, a virus is a disruptive occupant of the respiratory tract that can cause opportunistic infections. Bacterial infections are treated with antibiotics. Although, an extensive and incorrect use, can lead to antibiotic resistance, which is one of the biggest threats to global health. Therefore an alternative strategy for the prevention and treatment of infectious diseases is highly necessary. Recent years, the knowledge in the human microbiome and its functionalities has been increasing continuously, resulting in a growing awareness of the potential application of probiotic bacteria for acute respiratory prophylaxis and treatment.

Tablets are easy to use, have a good patient acceptance and are suitable for large-scale production. Therefore, tablets are an interesting dosage form for oral probiotics. However, due to the complex nature of the probiotic bacteria, the manufacturing of a stable and high-quality probiotic tablet remains a challenge. Specifically, the probiotic cells are exposed to high mechanical stresses during tablet manufacturing, which may affect probiotic survival, efficacy and safety. Therefore, extensive research is needed to develop a successful probiotic tablet. This PhD-project aimed to investigate which process and formulation parameters have a major impact on the survival of the prototype probiotic strain Lacticaseibacillus rhamnosus GG during tablet manufacturing.

The results demonstrated that the survival rate is highly affected by the applied pressure and the tableting properties of the probiotic powder blend. Specifically, the survival rate was significantly better when L. rhamnosus GG was compacted with a powder blend that needs time and pressure to deform and which, after some deformation, fractures. Additionally, it may be important that the powder particles show elastic recovery during decompression. Furthermore, the results suggest that the large galactose-rich cell wall polysaccharides act as shielding molecules. This implies that bacterial cells that lack these molecules, may experience more stress and may be therefore more sensitive to the tableting process. The results also suggest that larger bacterial cells are better protected within plastically deforming powders.

These results are promising and show that the probiotic cells can be protected during tablet production by selecting the appropriate manufacturing determinants, resulting in a better survival and therefore in a probiotic tablet with a sufficient amount of viable cells.

Role of autophagy in the cardiovascular system and implications for the development of age-related vascular pathologies - Dorien De Munck (01/10/2020)

​Dorien De Munck

  • 1 oktober 2020
  • Promotoren: Guido De Meyer en Wim Martinet

Abstract

Recent evidence showed that autophagy, a catabolic cellular mechanism responsible for nutrient recycling, plays a major role in the physiology of vascular cells such as endothelial cells (ECs) and vascular smooth muscle cells (VSMCs). Moreover, dysregulation of this process is associated with the development of age-related cardiovascular disorders including heart failure, atherosclerosis, hypertension and arterial stiffness. To increase the understanding about the role of autophagy on the vasculature, with implications for the development of age-related pathologies we investigated in this project the role of VSMC and EC autophagy on the vasculature using mouse models with a knockout of the essential autophagy gene Atg7 in VSMCs or ECs. 

First we focused on the role of VSMC autophagy on the vascular structure and its, reactivity and biomechanics using a traditional organ bath setup, wire myograph and an in-house developed Rodent Oscillatory Tension Set-up to study Arterial Compliance (ROTSAC). Vascular reactivity measurements at 2 months of age showed enhanced voltage gated calcium channel (VGCC)-mediated contraction in the aorta and femoral artery segments of mice with an VSMC autophagy defect, which resulted in increased sensitivity to depolarization induced contractions. In addition, femoral artery segments showed a large increase in IP3-mediated contraction at the age of 2 months while this was absent in the aorta. Surprisingly, deletion of Atg7 in VSMCs also affected the relaxing capacities of the aorta and the femoral artery. In aortic segments basal unstimulated nitric oxide (NO) release as well as stimulated NO release was enhanced, while femoral artery segments display increased VSMC sensitivity to exogenous NO.

Aortic segments of mice with a VSMC autophagy defect also displayed attenuated compliance and higher arterial stiffness, which was more evident at higher distention pressures. At the age of 3.5 months, passive aortic wall remodeling, rather than differences in VSMC tone, was responsible for these phenomena, since differences in compliance and stiffness were more pronounced when VSMCs were completely relaxed by the addition of exogenous NO. These observations are supported by histological data showing extracellular matrix remodeling. Short-term adaptations in the aorta, measured at 2 months, also included changes in the active modulation of arterial stiffness since depolarization induced contraction significantly increased vascular stiffness more in aortic segments of mice with an VSMC autophagy defect. As an increase of the focal adhesion protein vinculin was observed, we speculate that the enhanced active component is possibly due to an increase in focal adhesion sites.

Similar to VSMC autophagy, we also investigated the effect of an EC selective Atg7 deficiency on the aortic reactivity and biomechanical properties using a ROTSAC setup and a traditional isometric organ bath setup. Short-term as well as long-term consequences were evaluated by measurements at the age of 2 and 3 months and 1 year. Surprisingly, no differences were seen between the EC autophagy deficient mice and control mice at the age of 1 year. This was in contrast to younger mice where isometric tension measurements of EC-specific autophagy deficient aorta segments showed features of endothelial dysfunction such as decreased acetylcholine sensitivity. Moreover, isobaric measurements however, showed a decline in basal NO bioavailability, which was mostly pronounced at the age of 3 months. In addition, a higher stiffness was present in aortic segments of mice with an EC autophagy defect as compared to control mice in unstimulated conditions. Blocking of basal NO with the eNOS inhibitor L-NAME eliminated this difference indicating this effect of EC autophagy on arterial stiffness were solely dependent on effects on basal NO bioavailability. This was in contrast with stimulated conditions where aortic segments of mice with an EC autophagy defect showed increased stiffness, independent on the presence of L-NAME.

Overall we can conclude that autophagy in VSMCs and ECs plays a major role in normal vascular function since both the contractile and relaxing capacities of large elastic arteries as well as the smaller muscular arteries in mice are affected by a selective Atg7 knockout. Moreover, disruption of this homeostatic process results in an increased development of arterial stiffness as measured by an ex-vivo experimental setup.

Natural products as potential inhibitors of Advanced Glycation Endproducts (AGEs) and modulators of autophagy - Stefaniya Velichkova (25/09/2020)

​Stefaniya Velichkova

  • 25 september 2020
  • Promotoren: Luc Pieters en Kenn Foubert

Abstract

Al eeuwenlang voorzien geneeskrachtige planten de menselijke beschaving van remedies voor het behoud van de gezondheid en de bestrijding van ziekten. Tegenwoordig zijn natuurlijke bronnen nog steeds een startpunt voor het onderzoek naar geneesmiddelen. Ze trekken de aandacht voor hun potentiële toepassing als nieuwe therapeutische middelen bij de behandeling van hedendaagse ziekten met grote sociale en economische gevolgen, zoals diabetes type 2, hart- en vaatziekten). Er zijn verschillende mechanismen voorgesteld om de oorzaak van chronische aandoeningen te verklaren, en op biochemisch niveau correleert de eiwitglycatie (vorming van geavanceerde glycatie-eindproducten (AGEs)) met vele pathologische complicaties. Net als bij AGEs is autofagie in verband gebracht met een groot aantal pathologieën, waaronder hartziekten, kanker, neurodegeneratie, infectieziekten, diabetes en auto-immuunziekten. Daarom vertegenwoordigen AGEs en autofagie nieuwe therapeutische doelwitten in het onderzoek naar natuurlijke producten. Het doel van dit doctoraatsproject was het isoleren en identificeren van verschillende geselecteerde klassen van natuurlijke producten (polymethoxyflavonoïden (PMFs), biflavonoïden, quinazolinealkaloïden) uit vier verschillende plantensoorten: Citrus sinensis, Citrus depressa, Ginkgo biloba en Adhatoda vasica. Vervolgens werden hun eigenschappen als AGEs remmers en autofagie modulatoren geëvalueerd door middel van een reeks experimentele procedures.

Het project omvatte gedetailleerd fytochemisch onderzoek door middel van verschillende chromatografische technieken: open kolom, flash en semi-preparatieve vloeistofchromatografie. De structuur van de verbindingen werd opgehelderd door 1D- en 2D- NMR-spectroscopie en massaspectrometrie. De preliminaire tests voor AGEs remmende eigenschappen van de verkregen zuivere verbindingen omvatten de runderserumalbumine (BSA) / glucose test, en evaluatie van de vorming van fructosamine-adducten en alfa-dicarbonylverbindingen. Omwille van bepaalde beperkingen van deze standaardmethoden werd een methode ontwikkeld om van niet-selectieve colorimetrische  /fluorimetrische technieken te evolueren naar een meer betrouwbare methode op basis van chromatografie. Een HILIC UPLC/MS-methode moest worden ontwikkeld en gevalideerd, en werd daarna gebruikt om de AGEs remmende eigenschappen van enkel zuivere verbindingen en commercieel beschikbare standaarden te onderzoeken . Om de autofagie modulatie door enkele geïsoleerde zuivere verbindingen en commerciële standaarden te evalueren, werden verschillende testen gebruikt: LC3 detectie en kwantificering door western blot analyse, en de Cyto-ID autofagie detectie kit. Als onderdeel van het analytische werk werd een methode ontwikkeld en gevalideerd voor de kwantificering van vasicine - het belangrijkste chinazolinealkaloïde in de bladeren van Adhatoda vasica. Daarnaast werd de vastgestelde methode toegepast voor de kwaliteitscontrole van in de handel verkrijgbare kruidenproducten die Adhatoda poeder of -extract bevatten. In het algemeen zijn geavanceerde glycatie en modulatie van autofagie belangrijke oorzaken voor de progressie en pathogenese van veel chronische ziekten, en daarom kan het gebruik van gevalideerde methoden en technieken bijdragen tot de ondubbelzinnige ontdekking van nieuwe krachtige anti-AGEs en autofagie­modulerende middelen.

Role of non-myocyte NRG1/ERBB4 signaling in cardiac remodeling - Lindsey Dugaucquier (22/09/2020)

​Lindsey Dugaucquier

  • 22 september 2020
  • Promotoren: Gilles De Keulenaer en Vincent Segers

Abstract

​In this thesis, we aimed to study ERBB4 signaling in non-myocytes, specifically in ECs and inflammatory cells, and challenge the current concept that the cardiac effects of NRG1 are mediated by paracrine activation of ERBB4/ERBB2 receptors on cardiomyocytes. We studied how NRG1/ERBB4 signaling in ECs or inflammatory cells could contribute to the beneficial effects of NRG1 during cardiac remodeling. The central hypothesis of this thesis is that NRG1/ERBB4 receptor signaling in non-myocytes contributes to the beneficial effects of NRG1 during cardiac remodeling.

​Therefore, we first generated mice with myeloid-specific deletion of Erbb4 and tested the effects of myeloid-specific Erbb4 deletion in 2 models of cardiac remodeling—MI and pressure overload. We observed that myeloid-specific Erbb4 deletion accentuated the early increase of myocardial macrophage density in the viable myocardium after MI, but that subsequent ventricular dilation and dysfunction, CM hypertrophy, or interstitial myocardial fibrosis remained unaffected. Interestingly, myeloid-specific Erbb4 deletion reduced infarct scar. In the TAC model, pressure overload–induced myocardial inflammation remained absent, and LV remodeling progressed independently of myeloid ERBB4. Overall, this study shows a modulatory role of NRG1/ERBB4 signaling in myeloid cells during the early inflammatory phase of MI.

​Secondly, we generated EC–specific Erbb4 KO mice to specifically eliminate endothelial autocrine ERBB4 signaling without affecting paracrine NRG1/ERBB4 signaling in the heart. First, we observed no basal cardiac phenotype in these mice up to 32 weeks. We next studied these mice following TAC, exposure to Ang II or MI in terms of cardiac performance, myocardial hypertrophy, myocardial fibrosis and capillary density. In general, no major differences between EC–specific Erbb4 KO mice and control littermates were observed. However, 8 weeks following TAC both myocardial hypertrophy and fibrosis were attenuated by EC–specific Erbb4 deletion, albeit these responses were normalized after 20 weeks. Similarly, 4 weeks after Ang II treatment myocardial fibrosis was less pronounced compared to control littermates. These observations were supported by RNA-sequencing experiments on cultured ECs showing that NRG1 controls the expression of various hypertrophic and fibrotic pathways. Overall, these data contribute to our understanding of myocardial cell-cell communication during cardiac remodeling, and to the role of autocrine NRG1/ERBB4 signaling specifically. NRG1 has direct effects on human ECs in vitro, and endothelial autocrine NRG1/ERBB4 signaling plays a role in the modulation of hypertrophic and fibrotic responses during early cardiac remodeling.​

The carboxypeptidase U system in acute ischemic stroke: translation from bench to bedside - Joachim Mertens (07/09/2020)

​Joachim Mertens

  • 7 september 2020
  • Promotoren: Dirk Hendriks en Anne-Marie Lambeir

Abstract

The carboxypeptidase U system in acute ischemic stroke: translation from bench to bedside

Currently, only 20% of acute ischemic stroke (AIS) patients can be successfully treated. New treatment strategies are therefore urgently needed. The antifibrinolytic enzyme carboxypeptidase U (CPU) is an appealing target to improve stroke therapy. CPU circulates in plasma as an inactive zymogen, proCPU, that can be activated by thrombin, the thrombin-thrombomodulin complex, or plasmin. We aimed to assess the CPU system as a potential target to improve ischemic stroke treatment.

Activity-based, immunologic and functional assays were optimized and further characterize  for application in observational and clinical studies as well as in preclinical animal models of ischemic stroke.  Special attention was paid to the pre-analytical phase which is key to accurate CPU measurement. Especially the impact of in vitro hemolysis on the measurement of the CPU system was assessed. Assay-specific cut-off values reflecting maximal allowable oxyhemoglobin levels were determined as significant inhibition of CPU activity was observed.

In the preclinical part, a CPU inhibition strategy for the treatment of AIS was tested in a rat model of transient middle cerebral artery occlusion. Clear activation of the CPU system was observed in both saline- and rtPA-treated animals. Administration of the CPU inhibitor AZD9884 resulted in complete inhibition and reduced fibrinogen levels in the brain, which is a parameter of microvascular thrombosis (MT).

Two observational studies were performed on AIS patients. CPU activity and CPU+CPUi antigen increased in patients upon arrival in the hospital compared to controls. In AIS patients receiving either rtPA or rtPA with endovascular thrombectomy (EVT), the CPU system was clearly activated. Maximum CPU and CPU+CPUi levels tended to be higher in patients undergoing additional EVT compared to those that received rtPA alone. Some findings point towards a potential role of MT in these patient populations but, large inter-individual variation was observed in CPU and CPU+CPUi kinetics.

ProCPU levels were also quantified in the cerebrospinal fluid (CSF) of stroke patients that did not receive thrombolytic treatment and were increased compared to controls Patients with progressive stroke or poor outcome had higher proCPU CSF levels. The proCPU levels were also associated with blood-brain barrier dysfunction, which was found to be the likely cause of the increase.

In the last part of this project, we performed the pharmacodynamic assessment of a novel CPU inhibitor in a first-in-man trial. The combined use of the CPU activity assay and the two in vitro clot lysis assays proved to be valuable to confirm the target engagement of the inhibitor.     

Proline-specific enzymes in infection and inflammation - Gwendolyn Vliegen (22/06/2020)

​Gwendolyn Vliegen

  • 22 juni 2020
  • Promotoren: I. De Meester en A.M. Lambeir

Abstract

In dit doctoraat werden een aantal proline-specifieke enzymen van de dipeptidyl peptidase (DPP)-familie bestudeerd, zijnde DPP4, fibroblast activation protein α (FAP), DPP8 en DPP9. Deze enzymen knippen preferentieel hun substraten na een proline als deze op de voorlaatste plaats staat aan de N-terminus. Dit doctoraatsonderzoek was tweeledig. Enerzijds werd er getracht om humaan DPP8 en DPP9 recombinant aan te maken, om voldoende voorraad te hebben voor verder onderzoek. Anderzijds werden er ook enkele functionele aspecten van deze enzymen bekeken. Hiervoor werden DPP4 en FAP onderzocht in bloedstalen van patiënten met septische shock (gedefinieerd volgens sepsis-2) op dagen 1, 3, 5 en 7 om na te gaan of ze als biomerker kunnen dienen voor diagnose of prognose en of er associaties zijn met sepsis-gerelateerde parameters. Daarnaast werd een anti-inflammatoir effect gemeld van de DPP8/9-inhibitor 1G244 op humane en muriene macrofagen. Bovendien induceerde deze inhibitor ook celdood in de J774 cellijn. Echter is het niet geweten hoe deze inhibitor deze effecten bewerkstelligt. Er werd getracht om deze achterliggende mechanismen te ontcijferen.

DPP8 en DPP9 werden succesvol tot expressie gebracht en opgezuiverd uit insectcellen. De identiteit van de enzymen werd bevestigd met behulp van PCR, western blot en enzymatische activiteitsmetingen. Bovendien werden hun kenmerken geëvalueerd door hun pH-profiel, de Km- en kcat-waarden van Gly-Pro-pNA en Gly-Pro-AMC en de IC50-waarden voor 1G244 en Val-boroPro te bepalen. Deze experimenten leverden resultaten op binnen de verwachtingen.

Zowel DPP4 als FAP waren significant lager in de septische shock patiënten in vergelijking met een ICU controle groep en dit voor alle dagen. ROC-curves gaven AUC-waarden voor FAP van 0,94 (CI: 0,89-0,99) en voor DPP4 van 0,86 (CI: 0,77-0,95). Een beperkte associatie werd gezien tussen DPP4 en overleving op dag 90. Een hogere DPP4-activiteit ging gepaard met een toename van de overleving. Deze resultaten tonen aan dat FAP en DPP4 verder moeten worden onderzocht als mogelijke diagnostische (en voor DPP4 ook als prognostische) biomerkers.

Om de mechanismen achter de effecten van de DPP8/9-inhibitor te ontcijferen werd er gekeken naar de mogelijke betrokkenheid van kinasen met behulp van het PamGene kinomics platform. In primaire muriene macrofagen werden proteïne kinase C (PKC) en Ca2+/calmodulin-afhankelijk proteïne kinase II (CaMKII) geïdentificeerd als zijnde minder actief na inhibitie. In de J774 muismacrofaag cellijn vertoonden CaMKII en src familie kinasen (SFK’s) minder activiteit in de geïnhibeerde stalen. Deze resultaten moeten nog verder gevalideerd worden met specifieke methoden.

Development of novel chemical tools for bioorthogonal pretargeted PET imaging - Eduardo Figueiredo Pires Ruivo (30/03/2020)

​Eduardo Figueiredo Pires Ruivo

  • 30 maart 2020
  • Promotoren: Koen Augustyns en Pieter Van der Veken

Abstract

Pretargeted in vivo imaging using the bioorthogonal IEDDA reaction between TCO and Tz holds promise to allow imaging of mAbs with shorter living isotopes, to reduce radiation burden to the patient. This strategy can allow the development of a single radiolabeled probe that can be used with many mAbs, facilitating its use in R&D and clinical practice. For application of the TCO-Tz ligation, the radiolabeled probe should be stable and have fast reaction kinetics allowing reaction with the tag within minutes at low μM concentration.

The two-step in vivo labeling approach also facilitates the use of shorter living radioisotopes like 18F (T1/2 = 110 min) that would otherwise not be compatible with the long circulation times of high molecular weight molecules (mAb). Considerable research has been devoted to the development of mAb-TCO conjugates, and their application for pretargeted tumor imaging has been reported using diverse 18F-labelled tetrazines. However, it has been shown that TCO has the tendency to isomerize to its isomer, cis-cyclooctene (CCO), that is several orders of magnitude less reactive with tetrazine after prolonged exposure to physiological conditions.

Therefore, in this thesis we explored the inverse approach where a mAb is modified with tetrazine instead of TCO. A range of tetrazines was developed and evaluated regarding their stability and reactivity, and the most promising tetrazine was conjugated to mAb trastuzumab. In a proof of concept study we demonstrated the usefulness of the tetrazine-mAb conjugate for bioorthogonal pretargeted imaging using a live-cell fluorescent imaging experiment, using a TCO-fluorescent probe. Furthermore, we envisaged the translation of this strategy for in vivo pretargeted PET imaging applications. For this, a novel Al18F-NOTA labeled TCO radioligand was developed as a potential counterpart for IEDDA reaction with a tetrazine-tagged antibody.

The radiotracer showed improved in vivo metabolic stability (51.9 ± 5.16% after 1 h) compared to previous reported 18F-labeled TCOs, allowing a clear visualization of tumor tissue in a small-animal pretargeted PET imaging experiment. In an effort to increased absolute tumor uptake, we further developed next-generation TCO derivatives. The novel 18F-labeled dTCO-amide probe showed an extremely fast kinetic rate (10,553 M-1s-1 in 50:50 methanol:water), good stability in saline and plasma up to 4h at 37°C and a favorable biodistribution in healthy mice. Pretargeted µPET imaging experiments in mice bearing LS174T colorectal tumors, previously treated with a tetrazine-modified anti-TAG-72 monoclonal antibody (CC49), showed clear visualisation of tumor tissue with a significant higher uptake when compared to the control.