Research Annemieke Smet

Abstract

Chronic inflammation plays a significant role in both the onset and progression of many diseases, including, but not limited to, cardiovascular disease, chronic infections, cancer, and inflammatory organ diseases such as COPD, NAFLD, and IBD. Furthermore, acute infections may also trigger chronic inflammation and associated long lasting sequelae. As the prevalence of these diseases is increasing in Western societies and also emerging in other regions, research in this area can have a profound societal and scientific impact. Regulated cell death, barrier dysfunction, and immune modulation are key drivers of chronic inflammatory processes (Fig. 1). There is growing evidence for a limited number of common molecular pathways underpinning the regulation of these processes, and hence for a complex interplay in their pathophysiology. In this regard, Infla-Med brings together UAntwerp's leading basic and translational researchers in these three domains to form a bench-to-bedside and back consortium. The collaboration of complementary forces has enabled scientific breakthroughs in inflammation-focused research and has proven crucial in leveraging collaborations and funding in this competitive research field. For instance, Infla-Med's first 'stage' (2016-2019) resulted in more than € 23M in awarded funding with an overall stable 45% success rate since 2016. Moreover, halfway through Infla-Med's second 'stage' (2020-2022), we have already acquired the same amount of competitive grants. In terms of excellence, Infla-Med's principle investigators have achieved remarkable success in securing large, highly competitive grants for interdisciplinary research at local (BOF-GOA/IMPULS), national (FWO-EOS, iBOF), and international (ERA.Net, Innovative Medicines Initiative, coordination of H2020-MSCA-ITN and HE-MSCA-DN projects) levels. This shows that Infla-Med has established a very high-performing synergistic research framework among its principle investigators. The next 'stage' of Infla-Med will focus on discovering additional scientific breakthroughs and increasing our involvement in leading international research networks and acquiring international excellence funding (ERC). Four key strategic decisions support these ambitious aims for Infla-Med's next stage.

Researcher(s)

• Promoter: De Meyer Guido
• Co-promoter: Caljon Guy
• Co-promoter: De Meester Ingrid
• Co-promoter: De Winter Benedicte
• Co-promoter: Francque Sven
• Co-promoter: Segers Vincent
• Co-promoter: Smet Annemieke
• Co-promoter: Vanden Berghe Tom
• Co-promoter: Van Der Veken Pieter
• Co-promoter: Wullaert Andy

Research team(s)

• Physiopharmacology (PHYSPHAR)

Project type(s)

• Research Project

Abstract

A recent Lancet commission highlighted the significant and increasing burden of chronic obstructive pulmonary disease (COPD) and the need for fundamental changes in the way we think about this disease. Currently, the diagnosis of COPD requires spirometric airway limitation (FEV1/FVC <70%). However, significant lung damage may already have occurred before abnormalities in lung function are identified. Recently, the term pre-COPD has therefore been proposed to refer to individuals without airflow obstruction, but who are at increased risk of subsequently developing COPD based on their symptoms, lung function, or structural abnormalities. Importantly, the pathophysiological mechanisms underlying pre-COPD are largely unknown. Recently, our research group demonstrated a >40% reduction of small airways in pre-COPD patients compared to healthy (smoking) controls, which was similar to established GOLD I COPD. In this project, the cellular and molecular mechanisms underlying structural abnormalities in pre-COPD will be extensively investigated, with a focus on inflammation and epithelial remodeling. A comprehensive strategy will be employed, entailing a thorough transcriptomics investigation and validation at the tissue level, complemented by functional experiments utilizing patient-derived bronchial epithelial cell cultures. Understanding early pathological changes in pre-COPD will ultimately enable earlier and improved diagnosis and therapeutic intervention in (pre-)COPD patients.

Researcher(s)

• Promoter: Lapperre Therese
• Co-promoter: Smet Annemieke
• Co-promoter: Verleden Stijn
• Fellow: Voet Hanne

Research team(s)

• Laboratory Experimental Medicine and Pediatrics (LEMP)

Project type(s)

• Research Project

Abstract

Antimicrobial resistance is still one of the most serious global public health threats and is partly due to the inappropriate use of antibiotics. Acute respiratory tract infections (RTIs), affecting the upper respiratory system (e.g. sinusitis, pharyngitis and cold) or the lower part of the respiratory tract (e.g. acute bronchitis and pneumonia), are the main reasons for antibiotic prescription in primary care. Besides causing primary RTIs, bacterial and viral pathogens also play a paramount role in the exacerbations of chronic lung diseases such as chronic obstructive pulmonary disease (COPD). Due to the overlap in presentation of bacterial and viral acute RTIs and the lack of biomarkers that accurately distinguish between viral and bacterial RTIs, clinicians (i.e. pneumologists and intensivists) are forced to prescribe antibiotics to avoid missing a bacterial infection that could progress to a serious infection or sepsis. Furthermore, general practitioners do not always follow the recommended guidelines for antibiotic usage resulting in an overprescribing of antibiotics for self-limiting infections. Diagnostic uncertainty is thus a key contributor to antibiotic overuse that further fuels antimicrobial resistance. Novel biomarker assays discriminating bacterial from viral aetiology at the point-of-care still remain high unmet medical needs. Candidate novel biomarkers are the mucins. These highly glycosylated proteins are the gatekeepers of the mucosal barrier function maintaining epithelial cell homeostasis and act as a primary contact site to entrap microbes and facilitate their removal from the respiratory tract via a process called mucociliary clearance. However, upon bacterial and viral infection, aberrant mucin expression signatures forms a dysfunctional mucus barrier and becomes pathological. Next to this, mucins are highly polymorphic and the presence of genetic differences in the mucin genes can result in several mRNA isoforms of which some can be implicated in disease. Such disease-associated mucin mRNA isoforms can thus act as novel biomarkers to discriminate between bacterial and viral RTIs. Furthermore, given that epithelial cells can enter the bloodstream because of epithelial barrier injury, the peripheral blood provides a unique tool to monitor the levels of shed mucin mRNA isoforms among different patient groups. Indeed, we recently performed a proof-of-concept study in which we identified dynamic blood mucin mRNA isoform signatures that clearly distinguished symptomatic COVID-19 patients from patients with another viral RTI and discriminated between patients with mild and critical COVID-19. In this project, we wish to further unravel the complete peripheral mucin mRNA isoform landscape that associates with RTIs and identify mucin mRNA isoform biomarker panels that differentiate viral from bacterial RTIs using our optimized targeted mucin mRNA isoform sequencing approach in combination with random forest modelling on blood samples from patients with well-defined, well-characterized bacterial and/or viral RTIs.

Researcher(s)

• Promoter: Smet Annemieke
• Co-promoter: De Winter Benedicte

Research team(s)

• Laboratory Experimental Medicine and Pediatrics (LEMP)

Project type(s)

• Research Project

Abstract

Inflammatory bowel diseases (IBD) are very heterogenous diseases that, due to their disabling and chronic nature and the high costs associated with their disease management, entail a great burden both for patients and for society. Currently, there are no available biomarkers to help clinicians choose the most appropriate treatment for each patient, forcing them to do so empirically. One way to improve treatment, is focussing on personalized medicine where efforts must be directed towards the identification of molecular markers to assess mucosal healing as the main therapeutic endpoint. According to the recent guidelines, mucosal healing is defined as a composite term of endoscopic improvement and histologic remission, but its evaluation still remains challenging as objective measurements at the molecular level to monitor mucosal healing and select the appropriate therapy are currently lacking. Novel molecular markers that could fit these criteria are the mucins. These highly glycosylated proteins are the gatekeepers maintaining mucosal barrier function. However, aberrant mucin expression, as characterized by a depletion of secreted mucin expression and overexpression of transmembrane mucins, has been described in IBD. Own data also highlighted that increased expression of MUC1, MUC3, MUC4 and MUC13 associates with IBD presentation and activity and is involved in barrier dysfunction by affecting expression of junctional proteins and cell polarity. Furthermore, mucins are highly polymorphic and the presence of genetic differences in the mucin genes can result in several mRNA isoforms of which some can be implicated in disease. In the context of the IOF-SBO (ID: 42601) and IOF-POC DEVELOP (ID: 48068) projects, we unraveled a mucin mRNA isoform landscape of 400 unique mucin mRNA isoforms that is 1) expressed in the intestinal tract of IBD and control patients and 2) includes mucin mRNA isoform panels that associate with disease/inflammatory status (IBD or control), IBD subtype (CD, UC) and location (ileum, colon). These panels were identified using our optimized targeted isoform nextgeneration sequencing (NGS) pipeline. In this project, we wish to further standardize our biomarker platform based on these mucin mRNA isoform panels. To do so, we will first introduce automation into our targeted isoform NGS workflow to minimize variability at library preparation level, eliminate pipetting errors and improve the throughput time, reproducibility, consistency and data quality. Subsequently, we will validate our mucin mRNA isoform panels on external clinical samples of different origin, such as biopsies, blood and stool, to evaluate their clinical efficacy as (minimally) invasive biomarkers predicting the probability of treatment success in IBD patients and thus to offer personalised medicine to the IBD population.

Researcher(s)

• Promoter: Smet Annemieke
• Co-promoter: De Winter Benedicte

Research team(s)

• Laboratory Experimental Medicine and Pediatrics (LEMP)

Project type(s)

• Research Project

Abstract

Gastric cancer (GC) is the fifth most common cancer and the fourth leading cause of cancer-related death worldwide. Although Helicobacter pylori is the primary cause of GC, the disease is complex and involves multiple genetic, molecular, environmental, and microbiological factors. One of the hallmark features of gastric adenocarcinomas is aberrant mucin expression which drives tumorigenesis by influencing cellular growth and survival. Mucins are the gatekeepers of the mucus barrier covering the epithelium underneath and are heavily glycosylated. They are expressed at the apical surfaces of epithelial cells either as secretory or transmembrane mucins and play a crucial role in the maintenance of mucosal barrier homeostasis by communicating between the microbial flora and the mucosal immune system. Furthermore, these aberrantly expressed glycoproteins are also linked to the initiation, progression, and poor prognosis of GC. Of particular interest is the transmembrane MUC13 mucin. In healthy human beings, MUC13 is predominantly expressed in the intestines with only a very low level of expression in the stomach. In the diseased stomach, however, MUC13 expression is significantly upregulated by IL-1β (a key mediator in Helicobacter-related GC) and more specifically in adenocarcinoma and during the early events of the carcinogenesis process. Furthermore, MUC13 contains serine and tyrosine residues for potential phosphorylation and a protein kinase C consensus phosphorylation motif in its cytoplasmic domain that could play a critical role in tumorigenesis via cell signalling pathways that protect tumour cells from death. Currently, the exact role of MUC13 in the gastric carcinogenesis process remains poorly understood. From a certain point on, however, development of gastric adenocarcinoma may be H. pylori-independent, since colonization decreases in later steps of carcinogenesis, particularly in patients who develop intestinal metaplasia and dysplasia, and is finally lost in adenocarcinoma. Nowadays, the gastric microbiome is believed to contribute to cancer progression as well. The GC microbiome seems to be enriched with intestinal or oral taxa which can be assigned to an increase in pH, caused by H. pylori, and to specific interactions of the microbiota with the gastric mucosa. More specifically, the carbohydrate structures present on mucins, like MUC13, can act as binding sites or metabolic substrates for bacteria and the abundancy of MUC13 plays thus an important determinant in the site-specific colonization of bacteria in the gastric mucosa. Nevertheless, which specific tumour-enriched bacterial species can act as potential drivers in MUC13-mediated gastric carcinogenesis remains largely unknown. Therefore, this study aims to 1) identify the tumour-enriched bacterial taxa, other than H. pylori, involved in MUC13-driven gastric carcinogenesis and 2) unravel the MUC13-mediated mechanisms affecting tumour cell death.

Researcher(s)

• Promoter: Smet Annemieke
• Co-promoter: De Winter Benedicte
• Fellow: Oosterlinck Baptiste

Research team(s)

• Laboratory Experimental Medicine and Pediatrics (LEMP)

Project type(s)

• Research Project

Abstract

Loss of mucosal barrier integrity is a significant contributor in the pathophysiology of inflammatory bowel diseases (IBD) and colorectal cancer (CRC), but the role of transmembrane mucins, as epithelial signalling receptors mediating barrier dysfunction, is poorly understood. Upon inflammation, aberrantly expressed transmembrane mucins are likely to be the first point of contact between host tissue and the gut microbiota that penetrate the defective secreted mucus layer and co-induce barrier dysfunction. Furthermore, the presence of genetic differences in mucin genes can give rise to a large repertoire of structurally diverse mucin mRNA isoforms via alternative splicing. While most isoforms encode similar biological functions, other have the potential to alter protein function resulting in progression towards disease. Currently, the mucin mRNA isoforms implicated in mucosal barrier dysfunction, is a scientific field to discover. My recent work showed that the transmembrane MUC1 and MUC13 mucins are involved in GI barrier dysfunction upon inflammation. Using a novel long-read RNA sequencing approach, we also discovered a higher number of novel and unique MUC1 and MUC13 mRNA isoforms in inflamed colonic tissue compared to healthy tissue which explains the increased MUC1 and MUC13 expression seen in IBD patients. Therefore, the core aim of this project is to unravel the transmembrane mucin isoform landscape, with a dedicated but open focus on MUC1 and MUC13, mediating mucosal barrier dysfunction in IBD and CRC as well as the different levels of regulation (i.e. genetic alterations, microbial and inflammatory triggers) mediating mucin alternative splicing. I propose an unconventional approach encompassing a broadly applicable framework with next-generation sequencing tools, human sample collections and translational models. This work could lead to a new paradigm shift greatly advancing novel applications for disease follow-up and pathology-specific targeting.

Researcher(s)

• Promoter: Smet Annemieke
• Fellow: Smet Annemieke

Research team(s)

• Laboratory Experimental Medicine and Pediatrics (LEMP)

Project type(s)

• Research Project

Abstract

Upon gastrointestinal and reproductive homeostasis, there is an intimate crosstalk between mucins (MUCs) and the microbiome at the mucosal surface to maintain barrier integrity but a disbalance between both actors could dictate disease development, such as inflammatory bowel diseases (IBD) and aerobic vaginitis (AV). Indeed, thinning of the epithelium and aberrant MUC1 and MUC13 expression have been associated with barrier dysfunction in IBD. Also MUC1 seems to be increasingly expressed upon vaginal infection. Such defective mucus layer will thus allow microbiota to come in close contact with these mucins and co-elicit inflammation and mucosal damage. Nevertheless, which microbes interact with MUC1 and MUC13 in response to inflammation and dysbiosis and mediate barrier dysfunction in IBD and AV remains poorly understood. Therefore, we will first characterize microbiome diversity and function in IBD and AV using shotgun metagenomic sequencing. In parallel, we will also unravel the MUC1 and MUC13 mRNA isoform landscape using targeted isoform sequencing. Combining both sequencing data will allow to identify microbial-mucin isoform associations in IBD and AV. Finally, we will investigate the mechanism(s) by which abundant microbial species in IBD and AV interact with the aberrantly expressed MUC1 and MUC13 isoforms resulting in barrier dysfunction. To do so, bacterial mutant approaches and human epithelial organoid cultures established from IBD and AV samples will be used.

Researcher(s)

• Promoter: Smet Annemieke
• Co-promoter: Ceuleers Hannah
• Co-promoter: Lebeer Sarah

Research team(s)

• Laboratory Experimental Medicine and Pediatrics (LEMP)

Project type(s)

• Research Project

Abstract

The aim of this network initiative is to exploit our collective interest and complementary expertise to enhance insight in the pathophysiological mechanisms of disorders, including Helicobacter-related gastric diseases, inflammatory bowel disease (IBD), coeliac disease, intestinal ischemia, functional gastrointestinal disorders, non-alcoholic fatty liver disease (NAFLD), cirrhosis and chronic kidney disease (CKD), which we hypothesize to have common ground in the GI tract. We will investigate key areas of uncertainty regarding the exact role of intestinal barrier function (the microbiome, the epithelial barrier and the mucosal immune system) in the different gut-organ-axes and associated pathologies. Specifically, in the next 5 years we aim to gain in depth understanding in: 1) the cellular and molecular mechanisms that act within the gut wall (mucosal mediators, mucosal immune system, enteric nervous system) and signal from the gut lumen (microbiome and metabolites) to regulate intestinal barrier function. 2) how changes in the intestinal barrier affect communication from the gut to other organs via neuronal, metabolic, endocrine and immunological pathways. 3) how (patho-)physiological mechanisms at the level of the organs (e.g. psychobiological processes in the brain) interfere with intestinal barrier function through organ-gut (i.e. efferent) signaling mechanisms.

Researcher(s)

• Promoter: Smet Annemieke

Research team(s)

• Laboratory Experimental Medicine and Pediatrics (LEMP)

Project type(s)

• Research Project

Abstract

The Laboratory of Experimental Medicine and Pediatrics - within the Faculty of Medicine and Health Sciences and closely linked to the Antwerp University Hospital - focusses its research on the study of inflammation in a clinically relevant context built on interdisciplinary methodologies and collaborations. To remain in the forefront of research we perform ground-breaking experimental, as well as clinical and translational research from bench to bedside and vice versa, using innovative and high-end methodologies including organoids, rodent models, cell cultures, different next-generation omics approaches and clinical trials. We challenge you to write down a project that will have an added value to one of the research lines currently explored at LEMP (www.uantwerpen.be/en/research-groups/lemp) and briefly described below. Loss of mucosal barrier integrity is a significant contributor in the pathophysiology of mucosal inflammatory/infectious diseases (e.g. IBD, gastrointestinal cancers, RSV, COVID-19). The role of transmembrane mucins, as epithelial signalling receptors mediating barrier dysfunction, is poorly understood. Furthermore, the presence of genetic differences in mucin genes can give rise via alternative splicing to a large repertoire of structurally diverse mucin mRNA isoforms encoding similar biological functions or altering protein function resulting in progression towards disease. Currently, the mucin mRNA isoform landscape implicated in mucosal barrier dysfunction is a field to discover. Volatile organic compounds (VOCs) are compounds that are by-products of cell metabolism and induced by inflammation. The human body houses thousands of VOCs which are exhaled and can serve as non-invasive markers for disease. Hence, breathomics is applied to search for clinically relevant diagnostic, prognostic and predictive biomarkers for inflammation-related diseases in adults and children (thoracic cancers, COVID-19, asthma, COPD, BPD in neonates, gastrointestinal diseases) and to monitor the effect of air pollution on human health. However, there is a need for further identification and data mining of volatiles, linking VOCs to metabolic processes. Chronic low-grade inflammation is a key factor in obesity. As its treatment remains challenging over all age groups, research focusses on new treatment strategies for obesity, that minimize dropout and weight regain. Pathophysiological processes (hypoxia) that lead to comorbidities like cardiovascular and metabolic morbidity and obstructive sleep apnoea are also of interest. Kidney transplantation is the best treatment for patients with end-stage renal disease. As diagnosis requires invasive procedures, there is a need of sensitive, non-invasive markers of an early-stage acute rejection and the early diagnosis of glomerular damage in children and adults with various underlying diseases (diabetes, obesity or sickle cell anaemia). Visceral pain is a key feature of the gastrointestinal disorders IBD and IBS. The management of visceral hypersensitivity is challenging and requires further research towards new treatment targets. Unravelling the immunopathogenesis of chronic Hepatitis B infections is essential in the quest for novel treatment approaches. While the ineffective T-cell responses are well-known, B cells have been left largely understudied, urging a deeper understanding of the role of the humoral immune response in chronic HBV at the level of HBV-specific antibody production and of the phenotypic/functional level of B cells. Non-Alcoholic Fatty Liver Disease (NAFLD) is the global leading cause of chronic liver disease but pharmacological treatment remains poorly successful. Changes in liver hemodynamics and in parenchymal oxygenation contribute to the steatohepatitis and progressive disease worsening and are a potential drugable target. Furthermore, the role of NAFLD on extrahepatic vascular alterations contributing to cardiovascular disease warrants further study.

Researcher(s)

• Promoter: De Winter Benedicte
• Co-promoter: Lamote Kevin
• Co-promoter: Ledeganck Kristien
• Co-promoter: Smet Annemieke
• Co-promoter: Van Eyck Annelies
• Fellow: Oosterlinck Baptiste

Research team(s)

• Laboratory Experimental Medicine and Pediatrics (LEMP)

Project type(s)

• Research Project

Abstract

Inflammatory bowel diseases (IBD) are very heterogenous diseases that, due to their disabling and chronic nature and the high costs associated with their disease management, entail a great burden both for patients and for society. Currently, there are no available biomarkers to help clinicians choose the most appropriate treatment for each patient, forcing them to do so empirically. One way to improve treatment, is focussing on personalized medicine where efforts must be directed towards the identification of molecular markers to assess mucosal healing as the main therapeutic endpoint. Novel markers that could fit these criteria are the mucins. These highly glycosylated proteins are the gatekeepers maintaining mucosal barrier function. However, aberrant mucin expression, as characterized by a depletion of secreted mucin expression and overexpression of transmembrane mucins, has been described in IBD and own data highlighted that increased expression of MUC1, MUC3 and MUC13 is involved in barrier dysfunction by affecting expression of junctional proteins and cell polarity. Furthermore, mucins are highly polymorphic and the presence of genetic differences in the mucin genes can result in several mRNA isoforms of which some can be implicated in disease. The mucin mRNA isoform landscape associated with IBD still remains an unexplored conundrum. We have recently performed a proof of concept study in which we identified novel and known mRNA isoforms of MUC1, MUC3A, MUC5AC, MUC12, MUC13 and MUC17 associated with IBD. We wish to further validate our mucin mRNA isoform sequencing data to unravel the mucin mRNA isoforms as biomarkers to improve follow-up and treatment in IBD. Therefore, in this project we will design a mucin mRNA isoform-based biomarker assay to improve treatment management in IBD. To do so, we will first validate and complement our PacBio proof-of-concept isoform sequencing data and unravel which of the identified mucin mRNA isoforms are aberrantly expressed in IBD, using long-read ONT nanopore sequencing and short-read Illumina sequencing, respectively. These additional sequencing technologies will allow to select the optimal set of IBD-associated mucin mRNA isoforms for further implementation in a novel multiplex RT-qPCR-based biomarker assay. This biomarker assay will then be used to unravel which of the available and novel therapeutics impacts on aberrant mucin mRNA isoform expression and subsequent barrier function/integrity (as marker for mucosal healing) using patient-derived organoid cultures and to evaluate the efficacy of the IBD-associated mucin mRNA isoforms as minimally invasive biomarkers predicting the probability of treatment success at baseline in IBD patients.

Researcher(s)

• Promoter: Smet Annemieke
• Co-promoter: De Winter Benedicte

Research team(s)

• Laboratory Experimental Medicine and Pediatrics (LEMP)

Project type(s)

• Research Project

Abstract

Next to inflammation, intestinal barrier dysfunction is an important mechanism related to the pathogenesis of inflammatory bowel diseases (IBD). The mechanisms underlying an altered barrier function in IBD, in particular the role of mucins (MUC), remain largely unexplored. Our own pilot data show an increased expression of MUC1 and MUC13 in inflamed biopsies from IBD patients. These MUCs are thought to disturb cell polarity complexes and tight junctions eventually resulting in an intestinal barrier dysfunction. Nevertheless, the exact role of MUC1 and MUC13 in the epithelial response to acute or chronic inflammation remains poorly understood. Therefore, we will first unravel the mechanisms by which these mucins affect intestinal barrier permeability upon inflammation using 3D-mini guts (organoids), the Ussing chamber technique and IBD mouse models. In parallel, the obtained results will be translated to IBD patients in order to identify MUC1 and MUC13 as novel targets for therapy and/or biomarkers, as still a large number of patients fail to respond or obtain full remission with the current therapies. To do so, the mechanisms of action of MUC1 and MUC13 affecting barrier integrity will be verified. Thereafter, the expression levels of MUC1, MUC13 and their barrier mediators will be investigated by single-cell RNA sequencing and subsequently correlated to the mucosal permeability and clinical outcome parameters.

Researcher(s)

• Promoter: Smet Annemieke
• Co-promoter: De Winter Benedicte
• Fellow: Ceuleers Hannah

Research team(s)

• Laboratory Experimental Medicine and Pediatrics (LEMP)

Project type(s)

• Research Project

Abstract

Infection with SARS-CoV-2 mostly leads to a mild self-limiting respiratory tract illness, however, some patients develop severe progressive pneumonia, multiorgan failure, and death. This project aims to determine factors that dictate the course of COVID19 beyond cytokines. We have prior data that specific aberrantly expressed mucins, triggered by SARS-CoV-2, regulate ACE2 expression and affect lung barrier integrity. Such mucin alterations are clinically relevant as excessive mucin production is seen in severe COVID-19 illness obstructing the respiratory tract and complicating recovery. Here, we will first identify differentially expressed mucin isoforms in COVID-19 patients exhibiting the entire spectrum of disease severity. Thereafter, therapeutics currently used for COVID-19 will be screened for their ability to reduce mucin abundance. As mucin expression is also a critical factor in microbiome homeostasis and dysbiosis might modulate COVID-19 severity, this project secondly aims to map the microbiome associated with different degrees of disease severity. Unravelling mucin isoform-microbiome interactions that shape the course of SARS-CoV-2 infection will lead to the future identification of those patients who are in danger of progressing to severe disease. This project will also improve the choice for an appropriate treatment as well as the time frame of treatment options once infection occurs.

Researcher(s)

• Promoter: De Winter Benedicte
• Co-promoter: Malhotra Surbhi
• Co-promoter: Smet Annemieke
• Co-promoter: Verstraeten Aline

Research team(s)

• Laboratory Experimental Medicine and Pediatrics (LEMP)

Project type(s)

• Research Project

Abstract

Inflammatory bowel diseases (IBD), colorectal cancer (CRC) and gastric cancer (GC), still remain disease entities with a high morbidity burden and are major contributors to health problems worldwide. The burden of IBD is rising globally with substantial variation in levels and trends of disease. Most available therapies with biologicals are directed against the inflammatory responses, but still a substantial number of patients fail to respond or to obtain full remission. In a later stage, some patients will even need surgery and are facing an increased risk of developing colon cancer. GC and CRC are respectively the third and fourth leading cause of cancer deaths worldwide, as prognosis in the advanced tumour stage still remains poor. While chemotherapy is the cornerstone of cancer therapy, limited efficacy and development of resistance to chemotherapeutic drugs are major challenges in the treatment of these epithelial cancers. Next to this, immunotherapy does not provide major advances in metastatic CRC and GC as the response rate still remains low. One way to improve treatment, is focussing on personalized medicine where efforts must be directed towards the identification of patients who are likely to respond to a specific treatment regimen and optimize its efficacy. This personalization can focus on molecular biomarkers to maximize efficacy and minimize adverse events. Ideally, such biomarkers should have both a prognostic and predictive potential. Assessment of mucosal healing upon therapy is the golden standard to validate treatment efficacy in IBD, but validated markers for disease monitoring have still their limitations. Furthermore, knowledge of the molecular basis of CRC and GC has advanced at a rapid pace in recent years, which led to the identification of potential biomarkers. However, due to the large heterogeneity of these cancer types, such markers are not present in the majority of cancer patients, resulting in restricted efficiency for clinical application. Novel markers that could fit these criteria are the transmembrane MUC1 and MUC13 mucins. Both mucins are highly overexpressed in IBD, CRC and GC patients, making them valuable new markers for clinical implementation. Furthermore, mucins are highly polymorphic and the presence of genetic differences in the MUC1 and MUC13 genes can result in several mRNA isoforms of which some can be implicated in disease. The MUC1 and MUC13 mRNA isoforms associated with IBD, GC and CRC still remain an unexplored conundrum. We have recently performed a proof of concept study in which we identified the MUC1 and MUC13 mRNA isoforms associated with inflammation in IBD. We wish to build upon these data and extend our search to unravel the MUC1 and MUC13 mRNA isoforms as biomarkers to improve follow-up and treatment in IBD, CRC and GC. Therefore, in this project, we will develop and validate a novel biomarker assay, based on MUC1 and MUC13 mRNA isoforms with prognostic and predictive potential, to predict disease outcome and therapy response in IBD, CRC and GC. To do so, we will unravel the MUC1 and MUC13 mRNA isoforms aberrantly expressed in IBD, CRC and GC patients and correlate their expression levels with clinical patient data. Thereafter, we will generate a multiplex RT-qPCR based on a selected isoform panel and validate our assay on tissue and liquid biopsies to evaluate the efficacy of the isoforms as non-invasive prognostic and predictive biomarkers.

Researcher(s)

• Promoter: Smet Annemieke
• Co-promoter: De Winter Benedicte

Research team(s)

• Laboratory Experimental Medicine and Pediatrics (LEMP)

Project type(s)

• Research Project

Abstract

Infection with SARS-CoV-2 mostly leads to a mild self-limiting respiratory tract illness, however, some patients progress to develop severe progressive pneumonia, multiorgan failure, and death. The project aims to determine factors that dictate the severity of COVID-19. Firstly, guided by our prior data of interaction of certain mucins with the ACE2 receptor and the clinical evidence of excessive mucin production in severe COVID-19 illness, we intend to characterize different mucins for their role in both the initiation and progression of COVID-19. Secondly, based on a severe degree of edematous interstitial lung tissue pathology observed in COVID-19 autopsies and its hypothesized link to abnormally low PaO2 observed clinically, the project intends to characterize aquaporin (AQP) water channels that are responsible for fluid transport across cells. This has important therapeutic relevance for COVID-19 as specific AQP inhibitors have been shown to attenuate inflammation and lung injury and to block mucin hypersecretion. Lastly, mucin expression is also a critical factor in microbiome homeostasis and based on, so far, scarce data that co-infection with other respiratory pathogens and other microbial interactions might modulate COVID-19 severity, the project aims to characterize the microbiome associated with different degrees of disease severity. Identifying factors that shape the course of SARS-CoV-2 infection will lead to identification of plausible targets to treat COVID-19.

Researcher(s)

• Promoter: De Winter Benedicte
• Co-promoter: Jorens Philippe
• Co-promoter: Kumar-Singh Samir
• Co-promoter: Malhotra Surbhi
• Co-promoter: Smet Annemieke
• Co-promoter: Verstraeten Aline

Research team(s)

• Laboratory Experimental Medicine and Pediatrics (LEMP)

Project type(s)

• Research Project

Abstract

Respiratory Syncytial Virus (RSV) is worldwide the leading cause of serious airway infections. It's so common that most children will be infected by age 2. In adults and older children, RSV symptoms are mostly mild and typically mimic a common cold, but younger children can develop very severe disease. Preventive and therapeutic options are limited and currently it is not clear why some children develop severe disease while others do not. We therefore aim to investigate host- and virus-related factors that influence disease severity. To reach this objective, we will isolate RSV from children with respiratory disease, characterize the isolates and objectify differences between them. Next, we will investigate the effect of different virus isolates on the immunological response induced by a human respiratory epithelial cell line, since epithelial cells are the primary target cells and are implicated in the pathogenic reaction upon RSV infection. Lastly we want to evaluate whether the same clinical isolate induces a different immunological response in respiratory epithelial cells isolated from different patients. We will thus not only gain fundamental insights in the causes of RSV induced disease, but we will finally also correlate the virus- and host-related risk factors identified in the lab with clinical symptoms observed in patients. With this project we aim to identify patients prone to severe disease in an early stage, thus improving therapeutic options and disease outcome.

Researcher(s)

• Promoter: Verhulst Stijn
• Co-promoter: Delputte Peter
• Co-promoter: Smet Annemieke
• Fellow: Stobbelaar Kim

Research team(s)

• Laboratory Experimental Medicine and Pediatrics (LEMP)

Project type(s)

• Research Project

Abstract

Chronic infection with the human pathogen Helicobacter pylori plays a crucial role in the initial steps of gastric cancer (GC) development by causing enhanced inflammation and progressive changes in the gastric mucosa, like alterations in mucin expression and distribution. Aberrant expression of transmembrane mucins (MUCs) has been linked to the initiation, progression and poor prognosis of GC. Of particular interest is the MUC13 mucin. In healthy human beings, MUC13 is predominantly expressed in the intestines with only a very low level of expression in the stomach. In the diseased stomach, however, MUC13 expression is significantly upregulated by IL-1β (a key mediator in Helicobacter-related GC) and more specifically in adenocarcinoma and during the early events of the carcinogenesis process. Furthermore, MUC13 contains serine and tyrosine residues for potential phosphorylation and a protein kinase C consensus phosphorylation motif in its cytoplasmic domain that could play a critical role in tumorigenesis via cell signalling pathways that protect tumour cells from death. Currently, the exact role of MUC13 in the gastric carcinogenesis process remains poorly understood. Therefore, in the first part of this project (work package 1 (WP1)), we will investigate the role of IL-1β-induced MUC13 expression in different modes of programmed cell death (including apoptosis, necroptosis, pyroptosis and ferroptosis) and autophagy (as a form of cell survival) in GC cells and identify its downstream intracellular mechanism involved using in vitro, in vivo and translational approaches. In addition, we will also verify whether cell death resistance mediated by MUC13 already occurs in gastric pre-neoplastic lesions (i.e. atrophy and intestinal metaplasia). From a certain point on, however, development of gastric adenocarcinoma may be H. pylori independent, since colonization decreases in later steps of carcinogenesis, particularly in patients who develop intestinal metaplasia and dysplasia, and is finally lost in adenocarcinoma. Nowadays, the gastric microbiome is believed to contribute to cancer progression as well. The GC microbiome seems to be enriched with intestinal or oral taxa which can be assigned to an increase in pH, caused by H. pylori, and to specific interactions of the microbiota with the gastric mucosa. More specifically, the carbohydrate structures present on mucins, like MUC13, can act as binding sites or metabolic substrates for bacteria and the abundancy of MUC13 plays thus an important determinant in the site-specific colonization of bacteria along the gastrointestinal tract. Nevertheless, which specific tumour-enriched bacterial species can act as potential drivers in MUC13-mediated gastric carcinogenesis remains largely unknown. Here (WP2), we will identify which tumour-enriched bacterial taxa other than H. pylori are associated with aberrant MUC13 expression during the gastric disease process and are thus involved in MUC13-mediated gastric cancer development. To approach this, 16s rRNA sequencing will be used to identify potential candidate species which will be further investigated in a germ free mouse model.

Researcher(s)

• Promoter: Smet Annemieke
• Fellow: Oosterlinck Baptiste

Research team(s)

• Laboratory Experimental Medicine and Pediatrics (LEMP)

Project type(s)

• Research Project

Abstract

Irritable bowel syndrome (IBS) is a disease without a clear cause which is associated with abdominal pain and altered bowel habits. A disturbed intestinal permeability and an increased sensitivity of internal organs are causal factors of disease. An important pathological role lies in the 'gut-brain interaction' in which signals from the intestine are conveyed to the brain and vice versa. This interaction is essential for a healthy bowel function and involves signaling molecules such as histamine, proteases, bile and fatty acids. Also the microbial content (the 'microbiome') of the intestine plays an important role in the gut-brain interaction. In this respect, we are particularly interested in the intestinal barrier function. The mucous membranes in the intestine provide an active barrier that allows passage of nutrients but impedes non-nutrients and toxins. It is well known that certain chronic diseases are associated with a disturbed intestinal barrier, also called 'leaky gut'. Possibly, a 'leaky gut' is also involved in IBS and that is what we will investigate in this project. We will study the role of different mediators such as vasoactive intestinal polypeptide, intestinal alkaline posphatase, serine protease inhibitors and mucoprotectants in a preclinical rat model and directly translate the importance of these mediators to the human situation validating their occurence and activity in human colonic samples. -

Researcher(s)

• Promoter: De Winter Benedicte
• Co-promoter: De Schepper Heiko
• Co-promoter: Smet Annemieke
• Fellow: Hanning Nikita

Research team(s)

• Laboratory Experimental Medicine and Pediatrics (LEMP)

Project type(s)

• Research Project

Abstract

This project aims to advance the currently available sequencing technologies at the University of Antwerp (UA) by acquiring a third generation sequencing (3GS) platform. The flagship of the third generation, single-molecule longread sequencers, PacBio Sequel, harnesses the natural process of DNA replication and enables real-time observation of DNA synthesis. 3GS promises to open new avenues for sequencing-based research beyond the current state-of-the-art for this consortium, which consists of more than 14 UA research groups in various disciplines of medicine, biology and bioinformatics. Furthermore, several third parties have also committed to utilize this technology for their ongoing and future research studies. 3GS will be utilized by this consortium to (i) sequence prokaryotic and eukaryotic genomes, and difficult-to-sequence genome regions, (ii) identify new genes and mutations in various rare Mendelian disorders, (iii) identify epigenetic modifications to better understand biological processes like gene expression and host-pathogen interactions, (iv) precisely profile the human, murine, and environmental microbiome in disease and under various environmental stressors, and (v) develop novel preventive therapies for infection-prone disorders for better drug targeting. The analysis of the large amount of genomic and transcriptomic data generated by the various research groups will be coordinated by the UZA/UA bioinformatics group Biomina.

Researcher(s)

• Promoter: Malhotra Surbhi
• Co-promoter: Kumar-Singh Samir
• Co-promoter: Laukens Kris
• Co-promoter: Mortier Geert
• Co-promoter: Smet Annemieke
• Co-promoter: Vanden Berghe Wim
• Co-promoter: Van Rie Annelies

Research team(s)

• Laboratory of Medical Microbiology (LMM)

Project type(s)

• Research Project

Abstract

Inflammation-induced gastrointestinal diseases, such as inflammatory bowel diseases (IBD) and Helicobacter-induced gastric pathologies (in particular cancer), are major contributors to health problems worldwide. There is emerging evidence that a loss of the gastrointestinal mucosal barrier integrity is a significant contributor to the pathophysiology of these disorders. Inappropriate overexpression of transmembrane mucins can affect the gastrointestinal mucosal barrier. Of particular interest is the transmembrane MUC13. This mucin, which is predominantly expressed in the intestines and only at a very low level in the stomach, has been shown to be significantly upregulated in the inflamed stomach and more specifically in adenocarcinoma and early events of the carcinogenesis process. Aberrant expression of MUC13 has also been seen in inflamed colonic mucosal biopsies from IBD patients. These findings clearly show the importance of the pro-inflammatory activity of MUC13 signalling during the gastric disease process and in IBD and necessitates further research. Therefore, this project firstly (work package 1(WP1)) aims to determine the role of gastrointestinal MUC13 signaling during inflammation. In particular, the role of MUC13 in epithelial cell apoptosis as well as the signal transducers regulating its expression will be identified using cell and mice models of gastric disease and IBD. The inappropriate overexpression of transmembrane mucins can affect the mucosal barrier integrity by disrupting cell polarity and cell-cell interactions resulting in loss of tight junctions. Tight junctions regulate cell polarity and their formation is dependent on the PAR3-PAR6-aPKC (PAR) complex. Dysfunction of the PAR complex can be mediated by a receptor tyrosine kinase, belonging to the ErbB family, which can be activated by transmembrane mucins. Additionally, the vasointestinal peptide (VIP) has been implicated in tight junction regulation and mucin expression and seems to be affected upon inflammation. The second aim of this project (WP2) is thus to investigate whether aberrant MUC13 expression has an effect on the integrity of the gastrointestinal mucosal barrier by participating in receptor tyrosine kinase activation and thereby contributing to the disruption of the PAR complex and VIP signaling. For this purpose, immunoprecipitation, microscopic and transfection assays and in vivo models will be used. Finally (WP3), MUC13 expression and the key molecules in MUC13-induced gastrointestinal mucosal barrier disruption will be validated in a translational set-up using biopsies from patients with gastric disease and IBD. This might result in new strategies to treat these disorders and/or in the identification of new diagnostic biomarkers.

Researcher(s)

• Promoter: Smet Annemieke
• Fellow: Breugelmans Tom

Research team(s)

• Laboratory Experimental Medicine and Pediatrics (LEMP)

Project type(s)

• Research Project

Abstract

Inflammation-induced gastrointestinal diseases, such as inflammatory bowel diseases (IBD) and Helicobacter-induced gastric pathologies (in particular cancer), are major contributors to health problems worldwide. There is emerging evidence that a loss of the gastrointestinal mucosal barrier integrity is a significant contributor to the pathophysiology of these disorders. Inappropriate overexpression of transmembrane mucins can affect the gastrointestinal mucosal barrier. Of particular interest is the transmembrane MUC13. This mucin, which is predominantly expressed in the intestines and only at a very low level in the stomach, has been shown to be significantly upregulated in the inflamed stomach and more specifically in adenocarcinoma and early events of the carcinogenesis process. Aberrant expression of MUC13 has also been seen in inflamed colonic mucosal biopsies from IBD patients. These findings clearly show the importance of the pro-inflammatory activity of MUC13 signalling during the gastric disease process and in IBD and necessitates further research. Therefore, this project firstly aims to determine the role of gastrointestinal MUC13 signaling during inflammation. The inappropriate overexpression of transmembrane mucins, such as MUC13, can affect the mucosal barrier integrity by disrupting cell polarity and cell-cell interactions resulting in loss of tight junctions. Tight junctions regulate cell polarity and their formation is dependent on the PAR3-PAR6-aPKC (PAR) complex. Dysfunction of the PAR complex can be mediated by a receptor tyrosine kinase, belonging to the ErbB family, which can be activated by transmembrane mucins. Additionally, the vasointestinal peptide (VIP) has been implicated in tight junction regulation and mucin expression and seems to affected upon inflammation. The second aim of this project is thus to investigate whether aberrant mucin expression has an effect on the integrity of the gastrointestinal mucosal barrier by participating in receptor tyrosine kinase activation and thereby contributing to the disruption of the PAR complex and VIP signaling. Finally, the key molecules in gastrointestinal mucosal barrier disruption will be validated in a translational set-up using biopsies from patients with gastric disease (including cancer) and IBD. This might result in new strategies to treat these disorders and/or in the identification of new diagnostic biomarkers.

Researcher(s)

• Promoter: Smet Annemieke
• Fellow: Smet Annemieke

Research team(s)

• Laboratory Experimental Medicine and Pediatrics (LEMP)

Project type(s)

• Research Project