Degenerative diseases of the nervous system, such as Alzheimer's, Parkinson's and ALS, are severe, progressive and ultimately fatal. Most existing drugs for these neurodegenerative diseases only... Show moreDegenerative diseases of the nervous system, such as Alzheimer's, Parkinson's and ALS, are severe, progressive and ultimately fatal. Most existing drugs for these neurodegenerative diseases only temporarily relieve symptoms, increase mobility or relieve pain, but do not slow disease progression.This dissertation describes a method to efficiently carry out the development of new drugs that could inhibit disease progression in neurodegenerative diseases. Namely, by using pharmacodynamic biomarkers. These are signaling substances to measure the magnitude of a drug response.These biomarkers can be used in early clinical-pharmacological studies in healthy volunteers or small groups of patients to select the best drug candidates and their expected therapeutic doses as early as possible in the development stage. This helps to make informed choices to advance a potential new drug into large and expensive phase 2 and 3 (registration) studies, or conversely to discontinue development of a non-potential drug as early as possible. This biomarker method was applied in this dissertation to investigate 2 new drugs that could potentially slow disease progression in Alzheimer's and ALS (a RIPK1 inhibitor) or Parkinson's disease (a LRRK2 inhibitor). The research results from multiple early clinical-pharmacological studies in healthy volunteers and patients described in this thesis form the basis for larger phase 2 and 3 follow-up studies that have now been initiated with ALS patients and Parkinson's disease patients. Both with the goal of confirming whether these agents can indeed slow disease progression, which would represent a major breakthrough in the treatment of these conditions. Show less
Sepsis is a life-threatening condition caused by a dysregulated host response to infection, it is associated with significant morbidity, mortality, and with a high financial burden on global... Show moreSepsis is a life-threatening condition caused by a dysregulated host response to infection, it is associated with significant morbidity, mortality, and with a high financial burden on global healthcare systems. Bacterial infections are the primary cause of sepsis, but the growing prevalence of antimicrobial resistance complicates the effectiveness of antimicrobial treatments. Moreover, limited understanding of the host immune response during sepsis hinders the discovery of valuable biomarkers and drug targets. As such, there is an urgent need to improve the treatment of sepsis. To tackle this challenge, we have concentrated our efforts on optimizing current treatment strategies and on facilitating the discovery of novel host inflammatory response directed therapeutics. In this thesis, we have utilized quantitative pharmacological modeling approaches to assess the adequacy of current dose regimens and to evaluate antibiotic pharmacokinetic variability, thereby optimizing antimicrobial therapies for sepsis. Additionally, our researches had aimed to deepen our understanding of the underlying dynamics of sepsis pathology, enabling the identification of promising biomarkers and therapeutic targets for sepsis. Our work demonstrated how quantitative modeling strategies can support the design of optimized treatment strategies, and how systematic model-based integration of disease mechanisms can help to overcome the translational challenges in sepsis drug development. Show less
Colorectal cancer (CRC) is often treated with chemotherapy. However, it is well known that treatment with chemotherapy comes with challenges, such as (severe) adverse events leading to loss of... Show moreColorectal cancer (CRC) is often treated with chemotherapy. However, it is well known that treatment with chemotherapy comes with challenges, such as (severe) adverse events leading to loss of quality of life, treatment discontinuation and sometimes even death. Moreover, chances for curation in the metastatic setting are low. Therefore, a large window of opportunity to improve both safety as well as efficacy of chemotherapeutic treatment for the individual patient exists. A possible approach to improve chemotherapeutic treatment for CRC patients could be the discovery, validation and implementation of new genetic biomarkers. The use of genetic biomarkers allows to identify patients that are at higher risk for severe adverse drug events and to select patients which will benefit the most from chemotherapy. The aim of this thesis was therefore to improve the safety and efficacy of chemotherapeutic drugs in patients with colorectal cancer by individualising drug dosing and choice of drug based on germline genetic biomarkers. The described studies in this thesis brought us a few steps closer to safe and effective use of chemotherapeutic drugs in the individual colorectal cancer patient. Irinotecan should no longer be administered without a UGT1A1 genotype test and a start has been made towards personalised medicine for colorectal cancer patients with peritoneal metastases. Show less
Mesothelioma is an aggressive tumor originating from the mesothelial cells of the pleural or peritoneal cavity. This thesis focuses on the treatment of pleural mesothelioma. For long, chemotherapy... Show moreMesothelioma is an aggressive tumor originating from the mesothelial cells of the pleural or peritoneal cavity. This thesis focuses on the treatment of pleural mesothelioma. For long, chemotherapy has been the standard of care, leading to an overall survival of about 15 months. In this thesis, a phase 2 trial is performed using two checkpoint inhibitors, nivolumab (anti-PD-1) plus ipilimumab (anti-CTLA-4), to treat recurrent mesothelioma after at least one line of chemotherapy. Of the 34 patients evaluable for response 29% had a partial response and 38% patients had stable disease, so 68% of patients achieved disease control, the primary endpoint. These results exceeded expectations and rejected the alternative hypothesis. In part III translational research was performed on blood samples, pleural biopsies and breath analyses at baseline and after 6 weeks of treatment. In order to find markers of response and try to explain how immunotherapy affects the tumor microenvironment and immune cell subsets of the peripheral blood. Show less
Immunoglobulin G (IgG) antibodies can exert their functions via both Fab-mediated neutralization and Fc-mediated effector functions, both of which are crucial for protective immunity in COVID-19.... Show moreImmunoglobulin G (IgG) antibodies can exert their functions via both Fab-mediated neutralization and Fc-mediated effector functions, both of which are crucial for protective immunity in COVID-19. Importantly, effector functions and resulting inflammatory responses are impacted by the structure of N-glycans linked to the Fc-tail of IgG. Studying antibody glycosylation in emerging infectious diseases such as SARS-CoV-2 allows to gain insight into specific glycan signatures at the early stages of infection, and to investigate whether these reflect how the disease would progress. For example, low fucosylation is a common glyco-phenotypic signature of IgG1 produced against the spike (S) protein of severely ill SARS-CoV-2 infected patients early on in their disease course, but has likewise been described in other disease settings, where the antigen is presented in the context of host-cell membranes (Chapter 2). In this thesis, antibody glycomics signatures of SARS-CoV-2 infection and vaccination have been explored using an established liquid chromatography – mass spectrometry-based method relying on affinity-isolation and proteolytic digestion of both total and anti-S IgG. In Chapter 3, the glycosylation of SARS-CoV-2 anti-S IgG antibodies were found to be vastly skewed relative to total IgG and to change in a highly dynamic fashion. Moreover, IgG glycosylation was shown to be an early severity marker and showed patient stratification potential, with predicting power for intensive care admission within a hospitalized patient population. Early detection of a pro-inflammatory glycosylation pattern may provide a broader intervention window and decrease the number of ICU-admissions. Furthermore, anti-S IgG1 glycosylation levels obtained with LC-MS show promise to supplement clinical parameters and biomarkers of inflammation, that have together been used for the severity score calculation of hospitalized COVID-19 patients. Similarly to SARS-CoV-2 infection, antibodies generated against the spike protein upon BNT162b2 mRNA vaccination also induced a transient afucosylated anti-S IgG1 response in antigen naïve individuals, albeit to a lower extent than in severely ill patients, exemplifying the influence of the type of immunization on antibody glycosylation (Chapter 4). Upon vaccination, the observed initial, mild afucosylated response was additionally accompanied by low fucosyltransferase (FUT8) expression in antigen-specific plasma cells. Furthermore, the observed initial anti-S IgG afucosylation signature may aided mounting a stronger immune response, as indicated by its correlation with antibody amounts following the second vaccination dose. Given the impact of glycosylation on antibody function, deciphering theunderlying regulatory mechanisms influencing IgG glycosylation will be of great importance to better understand the inflammatory potential, vaccine efficacy and protective capacity of vaccine- or pathogen-induced IgG in both body fluids and tissues in the future.In Chapter 5 and 6, the reaction steps of a previously developed linkage-specific sialic acid derivatization workflow were studied in more detail. Key players in such reactions are catalyst, of which novel types with different physico-chemical properties were introduced in Chapter 5. In Chapter 6, prior lactone formation was found to be a prerequisite for subsequent amidation of α2,3-linked sialic acids, which proceeds via direct aminolysis of the C2 lactone. Together, these new insights will be beneficial for the rational optimization of high-throughput (MALDI-)MS-based glycomics and glycoproteomics workflows relying on linkage-specific sialic acid derivatization. Show less
Alzheimer’s disease (AD) is the most common cause of dementia and quickly becoming one of the most burdening diseases of the century. Effective treatments are still missing, partially because its... Show moreAlzheimer’s disease (AD) is the most common cause of dementia and quickly becoming one of the most burdening diseases of the century. Effective treatments are still missing, partially because its pathogenesis is still incompletely understood. This thesis explores the role of iron in AD, how it interacts with the immune system to influence disease pathogenesis and whether it could serve as potential biomarker. The first part of this thesis describes the importance of translational MRI, and how it can be used to increase our understanding of neurological diseases and help identify biomarkers. Subsequently, we used translational MRI to characterize the differences in iron accumulation in the brain between patients with AD and healthy elderly. The second part of this thesis investigated how the immune cells of the brain, microglia, interact with the accumulated iron. Using a combination of advanced multispectral immunofluorescence on brain tissue from AD patients and a human stem-cell derived microglia model, we studied the activation pattern of iron-accumulating microglia in human brains and emulated microglial iron accumulation in vitro. This enabled us to study the effect of iron on the gene expression patterns and function of the brain’s immune cells. Show less
This thesis highlights, firstly, the importance of early CRC detection by presenting results of a CRC diagnostic proteomic biomarker signature with high discriminative power. Secondly, a strong... Show moreThis thesis highlights, firstly, the importance of early CRC detection by presenting results of a CRC diagnostic proteomic biomarker signature with high discriminative power. Secondly, a strong robust, independent prognostic tumor stroma ratio (TSR) biomarker, which confirms to be of important clinical value. The TSR has the ability to stratify colon cancer patients according to their prognostic outcome in a highly reproducible and low-cost manner. It has shown to link patients with a high intra tumor stromal content and a worse prognosis. Literature shows a wealth of evidence that supports this prognostic value in CRC as well as in other cancers. This PhD research therefore concludes that it should be implemented in the official guidelines of the TNM classification to improve stratification for CRC patients in daily routine pathological evaluation. The prospective, international, multicentre UNITED study will hopefully overcome the last hurdle for this clinical implementation. Lastly, this thesis offers more insight in the elusiveness of the tumor microenvironment and stromatogenesis that contributes to the aggressiveness of some CRC tumors. The biological differences, interconnections and changes in the microenvironment presented give multiple leads for further research and new personalized treatment possibilities. Show less
Stalenhoef voerde een een gerandomiseerd onderzoek uit op de Spoedeisende Hulp-afdelingen van het LUMC en geaffilieerde ziekenhuizen onder leiding van prof. Jaap van Dissel (Infectieziekten), over... Show moreStalenhoef voerde een een gerandomiseerd onderzoek uit op de Spoedeisende Hulp-afdelingen van het LUMC en geaffilieerde ziekenhuizen onder leiding van prof. Jaap van Dissel (Infectieziekten), over de toepassing van een klinische beslisregel om het risico in te schatten bij patiënten die zich presenteren met pyelonefritis, acute prostatitis of urosepsis.Het onderzoek waaraan 370 patiënten deelnamen, toonde aan dat het aantal ziekenhuisopnames met de toepassing van deze beslisregel significant werd verlaagd. Het aantal thuis behandelde patiënten dat op een later moment toch werd opgenomen, was hierbij hoger dan verwacht. Dit kwam mede doordat er bij een aantal patiënten toch een andere diagnose werd gesteld op het moment dat de kweekuitslagen bekend werden. Verder onderzoek richt zich op verbetering van de beslisregel door deze te combineren met een biomarker die de acute immuunrespons reflecteert. Show less
In this thesis, methods were developed, and antibody glycosylation was characterized in order to further the clinical application of antibody glycosylation analysis. New mass spectrometric... Show moreIn this thesis, methods were developed, and antibody glycosylation was characterized in order to further the clinical application of antibody glycosylation analysis. New mass spectrometric workflows were introduced in Chapters 2 and 8. Chapter 7 showed the differential characteristics of murine IgG glycosylation of different strains and highlighted the profound differences between humans and mice, with regard to IgG glycosylation. Chapters 4 and 8 showed the use of controlled human situations to study the regulatory mechanisms of IgG and IgA glycosylation. Finally, Chapters 3, 5 and 6, identified glycosidic differences with specified (patho)physiological conditions, which might be exploited for patient stratification in the future. Show less
Ewing sarcoma is an aggressive primary malignant bone tumor with high degree of tumor vascularization and is the second most common sarcoma of bone in children and young adults. Patients... Show more Ewing sarcoma is an aggressive primary malignant bone tumor with high degree of tumor vascularization and is the second most common sarcoma of bone in children and young adults. Patients with disseminated disease at diagnosis or early relapse have a poor prognosis. To identify novel therapies and biomarkers for these patients we focused on the chemokines and chemokine receptors in Ewing sarcoma cells and their role its tumor microenvironment. CCL21 CXCL14, CXCR7 and the ratio between CXCR4-1 and CXCR4-2 have been identified as candidate prognostic markers, CCL21 immunotherapy as potential therapy and CXCR4 as potential therapeutic target in EWS. In addition, the presented peptide-based life cell imaging methods improve the ability to study CXCR4 cell membrane expression and dynamics qualitatively and quantitatively. This approach might be helpful for the measurement of anti-CXCR4 therapy efficacy. This work identified specific the chemokine signaling pathways that can be used to target Ewing sarcoma and its tumor microenvironment. Show less
Part I describes the prognostic effect and interactions of the immune system in breast cancer patients. Part II of the thesis describes the prognostic effect of these prognostic immune parameters... Show morePart I describes the prognostic effect and interactions of the immune system in breast cancer patients. Part II of the thesis describes the prognostic effect of these prognostic immune parameters and biomarkers molecular subtypes and stem cell marker ALDH-1, which are known to be strong breast cancer prognostic factors, in elderly breast cancer patients compared to their younger counterparts. Show less
The aim of this thesis was to identify novel lifespan regulating loci that influence human longevity and population mortality. To this end, we performed two genome-wide association studies, one of... Show moreThe aim of this thesis was to identify novel lifespan regulating loci that influence human longevity and population mortality. To this end, we performed two genome-wide association studies, one of long-lived individuals from the family-based Leiden Longevity Study (LLS) and an extended one of long-lived individuals from multiple cohorts of European descent. Using the latter, we identified two genome-wide significant loci, the TOMM40/APOE/APOC1 locus and an intergenic locus on chromosome 5q33.3. In addition, our gene set analysis with the LLS data showed that genetic variation in genes involved in the insulin/IGF-1 signaling and telomere maintenance pathways is associated with human longevity. Since our genetic studies identified a limited number of longevity loci, we additionally examined whether leukocyte telomere length (LTL) could be used as a biomarker of healthy aging. We showed that LTL meets three of the four criteria for a biomarker of healthy aging in the LLS, i.e., LTL changes with chronological age and is associated with health, in this case immune-related parameters, and prospective mortality. To identify novel longevity loci, future research may benefit from a better definition of the healthy aging phenotype, combining study designs, and the use of novel methods and technologies, such as next-generation sequencing. Show less
The aims of this thesis were to gain insight into specific disease processes in Huntington__s Disease (HD) and to identify biomarkers. To achieve these aims, cognitive functioning, structural brain... Show moreThe aims of this thesis were to gain insight into specific disease processes in Huntington__s Disease (HD) and to identify biomarkers. To achieve these aims, cognitive functioning, structural brain characteristics and intrinstic functional brain connectivity of premanifest and early HD subjects were examined. Cortical, subcortical and the intermediate white matter brain tissue shows evidence of structural and functional decline. We found evidence that disease processes, such as altered metabolism, excessive iron accumulation and cell loss, play a role in the changes. We conclude that changes occur throughout the brain from the earliest disease phase onwards. Hence, both premanifest and manifest HD should not be regarded as a disorder of the basal ganglia, but as a disease affecting the whole brain. Candidate biomarkers that have the potential to objectively reflect the early changes and the progressive nature of the disease are measures of subcortical atrophy, integrity of white matter pathways and of intrinsic functional brain connectivity. Iron, creatine, and N-acetylaspartate concentrations in the caudate nucleus and putamen may prove to be useful as markers of disease state for objectifying transitional disease processes from premanifest to manifest HD. Visuospatial working memory could be applied as a state marker for stage two HD. Show less
The aim of this thesis was to find potential MRI biomarkers for Huntington__s disease (HD). Therefore, after an overview of the current literature on MRI biomarkers, followed by examinations of... Show moreThe aim of this thesis was to find potential MRI biomarkers for Huntington__s disease (HD). Therefore, after an overview of the current literature on MRI biomarkers, followed by examinations of volumetric MRI, magnetization transfer imaging (MTI), diffusion tensor imaging (DTI) and magnetic resonance spectroscopy (MRS) were applied in patients in different disease stages of HD. The main conclusions demonstrate that choosing the optimal biomarker for evaluating therapeutic effects is dependent on the disease stage and therapeutic compound. To evaluate the premanifest stages of the disease volumetric MRI and DTI are most suitable. When the transition period is the desired timeframe for evaluation, also MRS can be very useful, especially if the compound in question has a direct potential influence on certain pathogenic pathways which in turn have an impact on specific metabolites. Future research should focus on combining multiple imaging techniques; __multimodal imaging__. A composite MRI biomarker has the potential to distinguish between disease groups more accurately than a single biomarker and in this way improve the evaluation of therapeutic compounds. Show less
This thesis describes the development of a novel alcohol clamp, a new method to obtain stable plasma levels of alcohol and its application in CNS-research. The method might have several advantages... Show moreThis thesis describes the development of a novel alcohol clamp, a new method to obtain stable plasma levels of alcohol and its application in CNS-research. The method might have several advantages that were explored in subsequent studies described in this thesis. The stability of the alcohol clamp was used to examine functional effect profiles and time-dependence of different CNS-effects. The tests to examine these effects were chosen based upon a prior review of the literature, during which the most sensitive CNS-tests were selected. Hereafter, we studied the alcohol clamping method as a tool to compare alcohol disposition capacities between different (ethnic) populations and as a tool to compare their different CNS-responses to multiple stable alcohol levels. We also investigated whether the clamping method could be useful as a future benchmarking entity in CNS-research, based on its fMRI effects on the brain at rest and its efficacy on tremor symptoms. Finally, we employed the method in an interaction study with a compound that is in development for addictive disorders including alcoholism. This thesis has examined several examples of situations where the alcohol clamp has been a useful research instrument during alcohol research and in early drug development. Show less
Survival rates after surgical treatment of gastric, colon and rectal cancer can improve with preoperative and/or postoperative adjuvant treatment with chemo- and/or radiotherapy. The role of... Show moreSurvival rates after surgical treatment of gastric, colon and rectal cancer can improve with preoperative and/or postoperative adjuvant treatment with chemo- and/or radiotherapy. The role of epigenetic aberrancies such as DNA methylation is established to play a pivotal role in gastrointestinal carcinogenesis and malignant progression. This thesis describes novel epigenetic biomarkers that could tailor (neo)adjuvant treatment regimens to the patients who would benefit from them. The same markers could further be used to exclude those patients who would not gain more survival-years and would be unnecessarily exposed to the morbidity/mortality of additional chemo- and/or radiation therapy. Currently, nodal status is the single most important prognostic indicator in gastrointestinal cancers however this can only be reliably assessed by the pathologist after surgery. Currently, neoadjuvant regimens are more and more being studied and shown to be of benefit. This development necessitates biomarkers that are available before the operation. An important derived conclusion from this thesis is that the preoperative availability of primary tumor DNA methylation biomarkers holds great potential for tailoring neoadjuvant therapy. The thesis also describes technical advancements that will enable primary tumor DNA methylation assessment in small amounts of tissue as preoperative diagnostic tumor biopsies harvested during gastroscopy or colonoscopy Show less
Replicative ageing of fibroblasts has often been used as a model for organismal ageing. The general assumption that the ageing process is mirrored by cellular senescence in vitro is based on lower... Show moreReplicative ageing of fibroblasts has often been used as a model for organismal ageing. The general assumption that the ageing process is mirrored by cellular senescence in vitro is based on lower replicative capacity of human fibroblasts from donors of higher chronological age, but these inverse relations have not been reported unequivocally. The relation between chronological age and fibroblast growth characteristics was assessed in nonagenarian subjects of the Leiden 85+ Study. A high remaining replicative capacity impressively showed that even in the very elderly a crucial number of cells with high mitotic capacity are left to give rise to fibroblast strains with the capacity for more than 100 population doublings. During the course of fibroblast growth in vitro, beta-galactosidase activity has been shown to be a reliable biomarker for replicative senescence. In myoblast cultures the relation between mixed cultures and clonal cultures was studied, showing marked heterogeneity between clonal cultures that all had a significantly lower replicative capacity when compared to mixed cultures, indicating heterogeneity of cells within one tissue compartment in their in vivo history. In a formal review on the replicative capacity of fibroblasts from patients suffering from accelerated ageing syndromes, age related diseases and donor age it was found that except for premature ageing syndromes, the replicative capacity of fibroblasts in vitro does not mirror key characteristics of human life histories. Show less
Although cannabis is especially known for its recreational use as a __soft drug__, its potential therapeutic properties have been recognized for hundreds of years. Since the isolation of THC from... Show moreAlthough cannabis is especially known for its recreational use as a __soft drug__, its potential therapeutic properties have been recognized for hundreds of years. Since the isolation of THC from Cannabis sativa L, the discovery of cannabinoid receptors and their natural ligands (endocannabinoids) the interest in the development of novel cannabinoids as medicine is accelerating. This thesis describes useful cannabis-biomarkers and the clinical pharmacology of some cannabinoid agonists and antagonists in early phase drug development. This includes a novel mode of pure intrapulmonary THC administration that can be used as a benchmark for novel CB1/CB2-agonists, or to demonstrate inhibitory activity of CB1-antagonists. In addition, the pharmacodynamics and pharmacokinetics of two novel CB1/CB2 agonists are evaluated and compared with the pharmacodynamic effect profile of THC. The clinical trials carried out for this research were performed at the Centre for Human Drug Research, Leiden, The Netherlands. Show less
The muscular dystrophies are a group of neuromuscular disorders characterized by progres_sive muscle weakness and wasting. Although the underlying genetic defects of a large number of muscular... Show moreThe muscular dystrophies are a group of neuromuscular disorders characterized by progres_sive muscle weakness and wasting. Although the underlying genetic defects of a large number of muscular dystrophies are now know, the molecular mechanisms resulting in the devastating effects of the disease are not yet clear. Furthermore, the muscular dystrophies differ in clinical presentation and severity. The processes responsible for this di_vergence are largely unknown as well. In this thesis, gene expression profiling has been applied to study the molecular and cellular mechanisms and subsequent biological processes that play a role in muscular dystrophy. To characterize the processes associated with regeneration, we have compared gene expression levels in hindlimb muscle tissue of mdx and control mice in a temporal study. Additionally, we have determined the gene expression profiles of dif_ferentiating human myoblasts in vitro, since regeneration processes recapitulate myogenesis. We also set out to compare gene expression levels of different mouse models for muscular dystrophy to find common and distinct molecular mechanisms that underlie different forms of muscular dystrophy. Accordingly, we first had to determine the effects of genetic background variation between inbred mouse strains, and to study the feasibility of alternative experimental designs. Show less
