Parallel Session 2

Room 2.1: Molecular Biology

Migraine is a common primary headache disorder that is characterised by severe headache episodes lasting 4-72 hours with accompanying sensitivity to light and sound and/or nausea, or vomiting. Patients are generally symptom free in between migraine episodes, which is referred to as the interictal state. Migraine is considered a multi-factorial disease of which the exact pathophysiology is largely unknown. The endocannabinoid system (ES) is thought to play an important role in migraine via its effect on neurotransmitter release in the central nervous system (CNS) and the trigeminovascular system (TGVS). The TGVS has been recognised as one of the factors that mediate migraine headache pain. It has therefore been hypothesised that migraine patients might have an endocannabinoid dysregulation, but direct evidence to support this is limited. Better understanding of the role of endocannabinoids in migraine could lead to the development of new migraine treatments. The aim of this case-control study is therefore to compare endocannabinoid concentrations in cerebrospinal fluid (CSF) between interictal migraine patients and healthy controls. To do this, the concentrations of the endocannabinoids AEA, 2-AG and DHEA were measured in the CSF of 198 interictal migraine patients and 96 healthy controls by using a micro-liquid chromatography-mass spectrometry (LC-MS) system. 

Introduction

In antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, a debilitating systemic inflammatory condition, proteinase 3 (PR3) is one of the two primarily recognized antigens. Anti-PR3 response is crucial for the disease pathophysiology, however, PR3-related research is immensely limited. This is mainly explained by the technical challenges of PR3 purification from human cells as well as recombinant production.

Objective

To set up a methodological platform in order to investigate PR3-reactive B cells in an antigen-specific manner. 

Methods

In order to produce recombinant PR3 (rPR3), a plasmid construct containing PR3 DNA with a His-tag on a C-terminus was designed. The construct was transfected into human embryonic kidney (HEK) cells for transient PR3 expression. PR3 was then purified from the transfection supernatant via His-tag purification and quality checks of all fractions were done with western blotting and silver staining. Next, PR3 was upconcentrated and rebuffered using 10 kDa centrifugal filters. Serum from 10 ANCA-PR3-positive patients was tested for the presence of anti-PR3 antibodies with ELISA, controlled by 8 healthy donors. To establish antigen-specific staining of PR3-reactive B cells, PR3 was directly labelled with two different fluorophores and tested on anti-PR3-expressing mouse hybridoma cells.

Results

Repeated production of rPR3 in HEK cells and its further optimization resulted in progressive increase in rPR3 purity leading to acquisition of outstandingly pure PR3. According to western blot and silver staining, purified PR3 did not contain any major protein contaminants. ELISA showed that PR3 was recognized by 7/10 serum samples of ANCA vasculitis patients, but not by healthy donors. Labelled PR3 allowed to differentiate between anti-PR3 and negative control hybridoma cells on FACS, making it ready for use with human B cells. 

Conclusion

PR3 production and detection were sufficiently set up and optimized. The next steps include testing the fluorescently labelled PR3 with human anti-PR3-expressing B cells.

Room 2.2: Cancer

Non-small cell lung cancer (NSCLC) has a poor outcome, even in curative TNM stages where 5-year survival is 40-75% due to recurrence. Growth-patterns have potential to further subdivide the tumors and improve treatment. Unfortunately, tumor heterogeneity and the inter-observer variability raise problems for reproducible assessment of the growth patterns. Furthermore, the time-consuming nature of this assessment makes large cohort studies unappealing. Digitalization of this process allows for faster, more elaborate and reproduceable analyses. An up-coming method for image analysis is artificial intelligence in the form of neural networks. These networks can be trained to provide a faster and more consistent grading of NSCLC subtypes compared to pathologists (18, 19). 

We aim to determine the predictive value of growth patterns on recurrence in early stage NSCLC.

Our retrospective cohort consists of early stage NSCLC patients between 2000 and 2020. Patients with neo-adjuvant treatment were excluded. Per case, we included all tumor-containing H&E stained surgical slides. After digitizing, the slides were scored by a pathologist and annotated for growth patterns. We used a ResNet50 neural network to create two classification models, one classifying tumor presence, the other classifying the growth pattern. After training, the models were able create heatmaps for both outcomes.

Our cohort consisted of over 800 patients, and over 4500 slides. Our tumor classification model performed with an F1-score (this score balances precision and recall) of more than 90% on the validation set. The preliminary test models for the growth pattern classification model show a F1-scores of up to 70%. 

Currently our models are not able to compete with the latest NSCLC growth pattern models. Transferring a model to other cohorts has proven difficult. Therefore we will continue improving, to reach pathologist-level classification of the growth patterns, approaching our goal to connect quantified growth pattern data and recurrence data.

Wilms’ tumors are pediatric kidney cancers that affect approximately one in 10,000 children, usually before the age of five years. In approximately 12% of these tumors a loss of the tumor suppressor gene Wilms’ tumor 1 (WT1) is found. This gene has multiple essential functions during kidney development and its expression is indispensable for proper nephrogenesis. Since Wilms’ tumors are the result of a disturbed development of the embryonic kidney, they are considered a good model for studying the relationship between normal renal development and tumorigenesis. 

In 2013 the putative Wilms’ tumor cancer stem cell was identified as ALDH and NCAM1 double positive. The aim of our study was to investigate how these NCAM1+/ALDH1+ cancer stem cells relate to the nephron progenitor cells in the normal embryonic kidney. We used flowcytometry to study the effect of a Foxd1 driven WT1 knock-out on the ALDH activity and NCAM1 expression in mouse embryonic kidneys of embryonic day 12.5 and 18.5. An AldeRed assay was performed to assess the ALDH activity and NCAM1 expression was analyzed with an antibody staining. 

Since the initial protocols did not give satisfactory results, multiple adaptations were made. We optimized the AldeRed assay conditions by varying the incubation times and DEAB reagent concentrations. In addition, we compared two NCAM1 antibodies and examined the most optimal order of the assay and antibody staining. We found that the most optimal AldeRed assay incubation time for mouse embryonic kidney cells was 15 minutes. In addition, better results were obtained when the NCAM antibody staining preceded the AldeRed assay, contrary to what was advised by the manufacturer of the assay. Currently we are using these optimized protocols to analyze mouse embryonic kidney cells with a Foxd1 driven WT1 knock-out.

Room 2.3: Immunology

Adoptive T cell transfer (ACT) is a promising immunotherapeutic strategy in the treatment of cancer, but suffers from poor clinical efficacy. Low T cell survival upon reinfusion is a major drawback and results from the long and cumbersome ex vivo expansion protocol. Recent advancements in the field of nanomedicine allow for the in vivo activation of tumour-specific T cells with so-called artificial antigen-presenting cells (aAPCs). Optimal nanoparticle size, shape and surface ligand density are required for robust T cell activation. Polymersomes, polymeric nanovesicles, emerge as an exciting aAPC platform because their size, shape and ligand density can be easily tuned. To investigate the effect of these design parameters on T cell activation in vitro, we developed a small aAPC library comprised of differently sized (100 nm and 300 nm) and morphologically distinct polymersomes (spheres and tubes) functionalised with three different densities of anti-CD3 and anti-CD28 antibodies. Our findings propose that T cell activation by polymersome aAPCs is dependent on a complex interplay between particle size, shape and ligand density, in which large size, tubular shape and high ligand density enhance T cell activation. Interestingly, the flexibility of the tubular shape reduces the effect of ligand density, most likely due to dynamic clustering of surface ligands at the aAPC-T cell interface. In conclusion, this study emphasises the high potential of polymersomes as a platform for aAPCs and warrants further research into the in vivo application of this system to enhance ACT in cancer immunotherapy.

Inflammatory Bowel Disease (IBD) is a chronic inflammatory disease of the gastrointestinal (GI) tract, including Crohn’s disease (CD) and ulcerative colitis (UC). IBD is a complex, immune-mediated inflammatory disease, characterized by marked dysregulation of mucosal immune responses against intestinal microbiota. By growing T cell clones from biopsies, the interaction between CD4+ T-cells, other immune cells and bacteria can be studied. This will provide clues on the relationship between CD4+ T cells with the tissue microenvironment in IBD-related chronic inflammatory processes. The hypothesis of this study is that microbiota-specific T cells from IBD patients have been rewired by the inflammatory environment and have an altered functional response to commensal bacterial antigens than cells of the same specificity in healthy intestinal mucosa. We generated T-cell clones specific to C. Aldenese, C. Perfrigens and B. ovatus. These T-cell clones were tested for their bacterial specify with proliferation assays and will be functionally characterised with the use of imaging mass cytometry and Luminex assays.

Room 2.4: Neonatal

High rates of aneuploidy in human pre-implantation embryos are estimated to be an important  cause of pregnancy loss after in vitro fertilization (IVF). During spermatogenesis most histones,  constituting chromatin, are replaced by protamines to facilitate the dense packaging required. Aberrations are associated with infertility and affect IVF outcome. The Baart group recently  demonstrated that retained histones are transmitted to the human embryo. Aberrant expression  of retained Histone H3 at lysine residue 9 (H3K9me3) at the pericentromeric region, crucial for  genome integrity, may result in aneuploidy due to chromosome malsegregation and improper  DNA repair. Therefore, it is hypothesised that the transmission of a compromised  heterochromatic signature by the sperm, contributes to the high rates of aneuploidy reported in  the human embryo and thereby impacts on early embryo development and IVF outcome. In this  study, our aim is to establish a method to quantify existing variation in the epigenetic signature of  cHC in human sperm by performing Western blot analysis on surplus sperm of patients  undergoing IVF treatment. To establish a solid method, we first needed to optimize the Western blot protocol. First, the efficiency of the protein transfer on a nitrocellulose or PVDF membrane was compared. Furthermore, dot blots were used to investigate the most suitable antibodies for  H3 and H3K9me3 and optimize the total protein stain (TPS) for protein normalization between  blots. Our results showed that the PVDF membrane gave a higher background signal than the  nitrocellulose membrane. Additionally, dot blot results showed that the antibodies tested are  suitable for detection of H3 and H3K9me3. However, the TPS remains present on the blot even  after destaining maximally according to the manufacturers use. Future perspective is utilising the optimized protocol to determine levels of H3, H3K9me3 and the H3K9me3/H3 ratios and  investigate the correlation with preimplantation embryo development and IVF outcomes. 

Scientists are much like children. The lab is our playground, we still love the ‘why question’ and at the Embryology department we continue to get to the (biomedical) bottom of embryo development to find answers to the question we all asked as children: ‘where do babies come from?’. In my JRP2, I am focusing on a sub-sub-aspect of this question by studying how the placenta and uterus closely interact to maintain early pregnancy. A successful interplay between the maternal uterus and the trophoblasts cells from the fetal placenta is crucial to provide nutrients to the embryo and develop the placenta. With immunofluorescence and 3D culture of placenta, we aim to better understand the molecular mechanisms driving this interplay. Insights from the first trimester of pregnancy are still sparse due to the tissue being mostly inaccessible for practical and ethical reasons. In the early 20th century embryologists started collecting human embryonic tissues and doing histology to identify the tissue structures. The historical collections closely describe embryo and placenta development and are still a valuable source of knowledge. In our group we are in the special position to have access to first trimester pregnancy tissue, which allows us to further visualize, with present-day techniques, early pregnancy interactions between placenta and uterus. All in all, a talk about early pregnancy, microscopes, embryo tissue collection in the past and present, to hopefully inspire your childlike curiosity. 

During pregnancy, the maternal immune system must fight off pathogens, while remaining tolerant towards the semi-allogeneic foetus. After the first trimester, maternal blood enters the placenta and surrounds the foetal villi, where nutrients, gases and waste are exchanged between mother and foetus. Here, the maternal immune system is in direct contact with foetal tissue. Surprisingly little is known about the immune cell composition of this intervillous blood (IVB). Previously, the immune cell composition of IVB in healthy term pregnancies was unveiled, which differed in many ways from peripheral blood (PB), but until today the immune cell composition of IVB in earlier stages of the placenta remained to be elucidated. 

Here we analysed the immune profile of IVB and PB of women undergoing second trimester abortions (gestational week 13-22). We used flow cytometry to compare second trimester IVB with paired PB samples, as well as with earlier acquired data from term pregnancies, in order to see changes in immune profile over time. Furthermore, migration assays were used to evaluate the chemotactic capacity of placenta-conditioned medium. 

We found an enrichment of monocytes and CD56bright NK cells in second trimester IVB. The migration assay confirmed this by showing that second trimester conditioned medium attracted high numbers of classical, but not intermediate and non-classical monocytes. T cells showed a more activated and more exhausted phenotype in IVB compared to PB. Additionally, a shift was observed from monocytes being the dominant subset in second trimester IVB, to CD3+ T cells being dominant in term IVB. 

This characterisation of the immune profile of IVB is the first step in understanding foetal-maternal tolerance, and the dynamic changes of immune composition over the course of pregnancy. 

Workshops

The lab is a treacherous place. At first sight, the biomedical lab is fun, exciting, and challenging.  But underneath a layer of pipettes, 96 well plates and R code, lies a much more important and often overlooked aspect of the life at work: Lab Politics. Lab politics can be described as all diplomatic relationships between the stakeholders of a laboratory. As a student in the lab, you will encounter tricky situations, and everyone will have a different approach to solve these issues. Often, a solution can be found by following guidelines and recommendations from colleagues and your supervisors. But how do you deal when situations get out of hand, or when you have a problem with your supervisor? Understanding and reacting to motives and intentions is an invaluable tool to be successful in any environment. Managing your way through conflicts is learned mainly through experience. During the workshop, you may be assigned a role as a stakeholder in a scenario. In the end, the goal is to get acquainted with the power of Lab Politics and to be more confident in representing your ideals and motives in the lab!

Imagine, you have worked in the lab for months and you finally get your most anticipated results. They are great and you can’t wait to share them with the world. But how? Research articles are great to convey your findings to fellow researchers, yet they are certainly not your average family member’s cup of tea. Letting the world (and your curious friends) know what you have done all that time behind a lab bench or a computer screen, and why your discoveries are worthwhile, is a key aspect of being a scientist. However, contrary to what you might think, it is not the easiest aspect. As all things in life, it takes practice to communicate science in the best way possible. But, once done well, science communication is a great tool to reach a broad audience and spark their enthusiasm in research. During this workshop we will discuss the further significance of science communication, look at tips on how to translate science to friends, family (and perhaps complete strangers) and put these tips into practice. At the end you’ll be ready to spread some science awesomeness!

How to make science fun for children? I think we all remember science classes that were so boring that you rather would have done something else. Or, maybe, you do not remember them at all! However, as we all know, science is not boring but very interesting and exciting. At Technolab Leiden, where I am currently doing my internship, we want to engage children in science and teach them about science in an interactive way. At Technolab it is all about learning together, by DOING. In this workshop, we will create an interactive lesson for children about a scientific topic, following Technolab’s basic principles. What will be your role as a teacher, what is the goal of your lesson, how to engage children and what kind of methods are suitable for your topic? We will discover this and more during this workshop.