1,720,988 research outputs found
Precision medicine for Cystic Fibrosis using intestinal organoids
No full textIn this thesis, the use of intestinal organoids for the development and tailoring of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)-modulating treatments is described. Available data indicate that intestinal organoids can be a very helpful tool to preclinically assess novel CFTR-modulating treatments and can serve as a useful model for developing new approaches such as gene editing. Moreover, currently available CFTR-modulator treatment can be repurposed and optimized through testing in intestinal organoids, while pilot studies indicate that we can predict clinical treatment effect in individual subjects using their intestinal organoids as a surrogate.
Although drug screening on organoids is currently possible in 384 wells format, this approach is time-consuming and expertise in organoid culturing is necessary. Moreover, data analysis has not been fully automated, limiting widespread implementation. In the future, screening platforms utilizing robots to culture organoids and artificial intelligence to analyse the data could increase the throughput and effectiveness of drug screening in organoids. In addition, studies directly comparing intestinal organoids with other pre-clinical models such as human bronchial epithelial cells and human nasal epithelial cells should further elucidate how CFTR-function in intestinal organoids relates to respiratory disease severity. Future clinical studies should elucidate thresholds of organoid swelling that can predict whether a patient will or will not respond to a specific drug (combination), and whether these thresholds are identical for drugs with different modes-of-action, or whether drug-specific thresholds are necessary. Moreover, concerted efforts should ensure that widely available organoid biobanks are created that enable access to a diverse range of (intestinal) tissues to further expand our knowledge of CF and CFTR-directed therapeutics
Cystic Fibrosis: a real-world challenge to predict individual outcomes
No full textCystic Fibrosis (CF) is a rare monogenic multisystem disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. More than 2000 different CFTR variants have been identified that differentially affect CFTR protein function. As a result, CF disease manifestations and response to recently developed CFTR-modulating treatments is highly variable among individuals. Without an effective treatment, people with CF can die at a young age.
To develop and select the best personalized therapy for all people with CF, we need more sensitive tests and outcome measures to predict long-term disease progression and response to CFTR-modulating drugs in real-life practice.
This thesis showed that the forskolin-induced swelling (FIS) assay of intestinal organoids is associated with long-term CF disease progression in multiple organs, including long-term lung function decline and the odds of developing CF-related comorbidities such as pancreatic insufficiency, diabetes and liver disease. The sweat chloride test, which is the reference standard biomarker of CF diagnosis, was not associated with long-term CF disease progression. These findings demonstrate the potential value of the FIS assay as a prognostic biomarker of long-term CF disease progression, which is particularly useful to inform individuals with rare CFTR variants with unclear clinical consequences about their long-term prognosis.
On the other hand, this thesis could not identify predictors that were associated with long-term response to CFTR-modulating drugs in people with CF homozygous for the most prevalent F508del mutation. This indicates that prediction of long-term treatment response remains challenging with the currently available tests and outcome measures in this context, emphasizing the need for alternative outcomes and analysis strategies.
In the last part of this thesis, the Q-Life app was developed as a novel personalized electronic patient-reported outcome measure (ePROM) to measure quality of life of people with CF on an individual level. Two observational studies described in this thesis showed that the Q-Life app is a reliable, valid and sensitive ePROM to measure quality of life in a personalized way. The Q-Life app could be a potential new sensitive and relevant outcome measure that can help to optimize personalized treatment of people with CF.
As suggested in this thesis, future research in extensive datasets studying various long-term outcomes in real-life settings should reveal at what precision individual disease states and therapeutic responses can be resolved and predicted through a combination of CFTR and non-CFTR dependent individual assessments
Gut microbiota in early life and its impact on allergic diseases
No full textThis thesis addresses the development of the intestinal microbiota in infancy, investigated by different molecular approaches (all based on rRNA gene analysis), and includes studies describing consequences of early life modulation of microbiota, by supplementation of probiotics, on composition and functionality of the microbiota.
A bacterial population, weighing more than 1.5 kilogram inhabits the adult human intestinal tract: the gut microbiota, comprising approximately 1014 bacteria. The gut microbiota is the ecological community consisting of commensal, symbiotic and pathogenic microorganisms that resides in a defined environment. After birth, infants are rapidly colonized by microbes from different environments they are exposed to. The classical early colonizers of the infant gut are facultative anaerobes of the phylum Proteobacteria, such as Escherichia coli and other Enterobacteriaceae. These organisms pave the way for strictly anaerobic bacteria by depleting the initial available oxygen in a matter of days. The neonatal period comprises a critical period for microbiota development, also because this is the period during which the (mucosal) immune system matures. The long-term composition and function of the newborn’s gut microbiota is suggested to be programmed during this period, thereby laying the foundation for future health and influencing the risk of developing disease later in life. The development of the gut microbiota continues during the first years of life and its composition is considered to resemble the adult gastrointestinal tract by the age of three. Mode of delivery, gestational age and feeding mode especially affect the infant’s microbiota composition. Besides, administration of antibiotics in early life and supplementation with prebiotics or probiotics (live microorganisms that, when administered in adequate amounts, confer a health benefit on the host) can have a major effect on the intestinal microbiota composition. Studies strongly suggest that dysbiosis (disturbance of the microbial balance in the gut) contributes to the development of various kinds of disease, such as inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), obesity, allergy, asthma and diabetes.
The development of the gut microbiota in early life is a complex process, regulated by endogenous as well as exogenous factors, which are being unraveled at the moment but also need additional in-depth investigations. Ultimately, everyone develops his or her own unique, individual microbiota. The vast majority of those microbiota are associated with maintenance of health. In all those cases it is important not to disturb this developmental process in early life (e.g. with antibiotics). When the microbiota develops in an aberrant way, associated with disease, early intervention (e.g. with pre- or probiotics) seems to be warranted. These interventions must be optimized by personalization and targeted application, an approach which now is within reach: the microbiome has the greatest potential to be translated into the clinic in the very short term. The challenge will be to bring this approach into clinical practice
Azithromycin in cystic fibrosis : pharmacokinetic and therapeutic aspects of maintenance therapy
No full textThis thesis describes the pharmacokinetics of azithromycin in cystic fibrosis (CF) patients during maintenance therapy, azithromycin drug-drug interactions and the effects of long term use of azithromycin on pulmonary function and microbial resistance.
We studied pharmacokinetic parameters of 500 mg azithromycin per day during maintenance (chronic) therapy in adult CF patients. The t1/2 was > 7 d in blood and in isolated PMNs. On average, the concentration in PMNs was 2100 times the concentration in plasma 24 h after dosing azithromycin and 70% of azithromycin in blood was incorporated in or bound to PMNs. Elevated concentrations were found in sputum (12 to 53 mg/L).
The long t ½ indicated that a less frequent dosing scheme could be feasible. We studied pharmacokinetics and sputum penetration of azithromycin in CF patients after a change from daily dosing of azithromycin to a once weekly dosing scheme. The weekly dose was reduced with a factor 3.5 (500 mg daily vs 1000 mg weekly). This lead to a reduction of AZM exposure by a factor (±SD) of 2.5 (±0.8) in plasma, 2.8 (±0.9) in blood and 2.3 (±1.1) in PMNs and 3.0 (±1.5) in sputum concentration. We concluded that reduction in frequency was feasible and resulted in an approximately linear reduction of azithromycin exposure.
In an earlier study, inhibition of the DNA hydrolysing capacity of dornase alfa by azithromycin was reported(Ripoll 1996). We found that in-vitro azithromycin did not inhibit the DNA hydrolysing capacity of dornase alfa at clinically representative sputum concentrations. These data combined with the data found in clinical trials do, in our opinion, not restrict the concurrent use of dornase alfa and azithromycin.
We evaluated the long term effect of azithromycin on pulmonary function by comparing patients infected with P. aeruginosa (using azithromycin) to a non-pseudomonas infected cohort (not using azithromycin). We found an amelioration in pulmonary function in the first year after start of therapy. In the following years a decline in pulmonary function was found. The rate of decline of pulmonary function however, was comparable in both groups.
Comparing 1997 (the last year before introduction of azithromycin maintenance therapy) and 2007 we observed a decrease in incidence of staphylococcal infections and an marked increase, from 10% to 85%, in macrolide resistance of S. aureus. This finding has been documented before in cystic fibrosis patients in Rotterdam (Phaff 2006), and has to be taken into account when azithromicin maintenance therapy is started.
Based on the pharmacokinetic data found in our study and on a literature search of available clinical trials we propose a dosing scheme with 4 weight groups and a dose level between 20 and 33 mg/kg/week in pediatric and adult CF patients.
The comparable rate of decline in P. aeruginosa infected and uninfected patients is a valuable achievement of current CF treatment, including azithromycin maintenance therapy, but cannot be contributed to azithromycin alone, since, in the timeframe we analyzed, also other anti Pseudomonas therapies have been introduce
Intestinal organoids as model for cystic fibrosis
No full textRecent advances in adult stem cell culture technology have enabled long-term in vitro expansion of intestinal organoids or ‘mini-guts’. In this thesis, we used the organoid model to develop a novel assay to measure function of CFTR, the protein mutated in subjects with cystic fibrosis (CF). This methodology enables accurate quantitation of the individual's residual CFTR function and response to CFTR-restoring drugs. It was used to (i) establish relations between the CFTR genotype, residual CFTR function, and response to therapy, (ii) discover novel approaches for optimal repair of mutant CFTR and (iii) guide the development of personalized/precision medicine for CF patients. The potential of the model has been provided, validation of its predictive capacity is next. This assay will play an important role in the development of future therapeutic options that aim to bring curative treatments to all CF subjects
Effective and sustainable patient participation: The patient’s perspective in CF care and CF research
No full textThis thesis focuses on how the Dutch CF Foundation defined the patients’ perspective as a starting point for the improvement of quality of care and the making of a patient prioritized research agenda. Quality of care and can be improved if we take into account the patients’ perspective in a structured way. Professional and systematical involvement of patient representatives in the development of standards of care for example, paves the way for useful insights, that contribute to a golden standard if merged with the expertise of medical experts. Quality of care also improves when criteria are defined that are meaningful to the people with CF. CF centers of expertise were assessed systematically based on criteria defined by patients themselves. The program is feasible and manageable for a professional patient organization to run, and showed a high efficacy. The results of the assessment show that quality of care improves when the using the patients’ perspective in the procedure. The patient organization is also able to address specific issues with regard to people with CF, offering them early intervention if a certain problem occurs (coaching project). This program is highly effective.
The process to close the gap between the patients’ unmet needs and outcomes of relevant research is described. A wide range of conditions has been laid bare, in order to set up a patient prioritized research agenda. Acting in the best interest of the patient is no longer the ultimate task of clinicians, but also of researchers
Molecular and functional characterisation of rare CFTR variants
No full textCystic fibrosis (CF) is caused by mutations in the CFTR gene. The CFTR gene encodes for the CFTR protein, which is an ion channel and indirectly responsible for fluid transport in various organs. People with CF suffer from various symptoms, including recurrent airway infections, pancreatic insufficiency and intestinal malabsorption. There are over 2000 different variants of the CFTR gene known, these variants all differentially affect CFTR quantity or function. Additionally, response to CFTR modulator treatment also differs per mutation.
In this thesis we use PDIOs to study rare CFTR variants and aim to predict the effect of the CFTR intragenic profile and CFTR modulator response on the in vivo disease expression. First, we use PDIOs to identify pwCF with CFTR mutations that are currently not eligible for CFTR modulator therapy that could benefit from this treatment. Next, we gain insight in the complexity of the CFTR gene and investigate the use of PDIOs for the linking of rare, complex genotypes to CFTR function and disease phenotype. Finally, we developed new assay models to increase the dynamic range of functional CFTR measurements in PDIOs, and potentially improve automation and accessibility of CFTR function measurements
The role of physical activity and fitness in peope with Cystic Fibrosis compared to healthy people
No full textThis thesis describes the physical activity behaviors and factors and settings which influence this behavior in Dutch youth with and without a chronic disease and disability. In addition, the (possible) changes in cardiorespiratory fitness as a result of improved Cystic Fibrosis (CF) care, possible limiting factors and new promising medication were described. The prevalence of severe fatigue and the relation between psychosocial and clinical measured factors were assessed. Higher levels of cardiorespiratory fitness are related to survival and higher quality of life scores in people with CF. Therefore, increasing our knowledge regarding cardiorespiratory fitness in this period subject to change, is clinically relevant.
In 2010 up to 2014, only 29% of the Dutch youth met the physical activity guidelines (to be at least moderately active for at least 60 minutes per day) and 43% had at least two hours of screen time. A lot of daily physical activity comes from cycling to school. Unfortunately, physical activity of especially scholars from cluster schools is lower. The Dutch youth with a chronic disease is not able to participate unlimited in sports and exercise.
More than half (55%) of our pediatric population (n=60) with CF, with no ventilatory limitation, has still low cardiorespiratory fitness levels, despite improved CF care. Children and adolescents performed a cardiopulmonary exercise test (CPET) to establish cardiorespiratory fitness levels (peak oxygen uptake- VO2peak). Patients were categorized as having low cardiorespiratory fitness when VO2peak relative to body weight < 82% of predicted. Logistic regression analysis showed that glucose intolerance and colonization with P. Aeruginosa were most strongly related to lower levels of fitness.
The maximal cardiac output during maximal exercise, determined by stroke volume and heart rate, is lower in both children and adults with CF (n=19) compared to healthy people (n=22). A blunted heart rate response, also named chronotropic incompetence, might be a cause for this lower cardiac output. Training intensities based on predicted heart rates seem inadequate for people with CF.
In our adult CF population (n=77), 26% reported to experience severe fatigue. Patients could be categorized as experiencing ‘severe fatigue’ when the score on the Checklist Individual Strength-20 questionnaire was at least 35. Linear regression analysis showed a relation between fatigue and pulmonary function and functional capacity, measured with the Modified Shuttle Test. Stronger relations were however found for fatigue and patient-reported outcome measures such as physical and emotional functioning.
In a small group of people (n=7) with CF and the S1251N mutation, we found an increase in pulmonary function and body mass index after a median of 15 [IQR 13-16] months of treatment with ivacaftor, a CFTR modulator drug. Remarkable was a decrease in cardiorespiratory fitness, which might be explained by a lower work of breathing.
Maintaining and improving cardiorespiratory fitness remains an important treatment aim for CF physiotherapy care
Nasal epithelial cells as model for cystic fibrosis
No full textCystic fibrosis (CF) is an inheritable disease in which the chloride channel CFTR does not function properly. This leads to the formation of thick, sticky mucus, causing problems in multiple organs, particularly in the lungs, and can result in premature death. CF is caused by a DNA mutation in the CFTR gene. Approximately 80% of individuals with CF are eligible for CFTR-repairing drugs, depending on their mutation type. However, due to the exorbitant cost of these drugs, only 12% of people with CF receive this therapy. The effectiveness of (new) drugs for CF can be tested in the lab by modeling water and salt transport in cultured cells. In this thesis, we developed protocols for culturing nasal cells and nasal organoids (3D mini-organs), obtained from a simple nose brush, as model for CF. We utilized these nasal organoids in the forskolin-induced swelling (FIS) assay to predict individual responses to the currently available drugs. Additionally, we used the nasal organoids to identify drugs that activate alternative chloride channels other than CFTR. This could serve as an alternative therapy to restore salt and water transport, irrespective of the type of mutation. We discovered drugs that induce fluid transport in the nasal organoids, but it remains unknown by which ion channel they operate. Furthermore, we investigated the differences between cultured nasal and bronchial cells to understand the representativeness of nasal cells for the lower airways, where most symptoms in people with CF occur
Pseudomonas aeruginosa colonization in patients with cystic fibrosis; population structure, the Dutch clone and effects of segregation
No full textMore than half of the patients with cystic fibrosis (CF) are infected with Pseudomonas aeruginosa in their lungs. It was thought that patients acquired unique strains of this bacterium from the environment, however in the last decades epidemic clones have been described in other countries that are most likely transmitted between CF patients. Infection with these epidemic clones has been associated with worse clinical outcome. To prevent transmission of such clones a strict segregation policy was implemented in the Netherlands without extensive knowledge of the local epidemiology. CF patients are advised not to meet, are isolated from other patients when visiting the out-patient clinic or when hospitalized and events like summer camps were discontinued. This thesis describes the population structure of P. aeruginosa as highly diverse and highly specific for different patient populations. In CF patients two dominant genotypes are described, ST406 and ST497, that are associated with specific age groups and are different from the epidemic genotypes found abroad. The epidemic strains described abroad are not found in Dutch CF patients. ST406 is harbored by 50% of CF patients between 15 and 25 years of age in 2007 and ST497 was detected mainly in older CF patients. ST406 is not associated with worse clinical outcome, but appears resistant to more antibiotic classes than unique strains and is associated with more inhaled antibiotic therapy. High persistence and possibly ease of transmission seem to have contributed to its spread. Investigating the genome, transcriptome and phenotypic features of two ST406 strains cultured from the same patient with three years in between demonstrated that ST406 is a slow growing, low virulent strain and adaptation to the CF lung niche was driven mostly by point mutations that coincided with differences in transcriptome levels and phenotypic changes, most notably with differences in biofilm formation. Some of the adaptations found in ST406 were also described for other chronic CF related strains, indicating parallel evolution of different P. aeruginosa strains to the CF lung niche. These affected genes might provide targets for future therapies.
Evaluating the effect of five years of segregation in the Netherlands yielded little difference in P. aeruginosa population structure and no statistically significant difference in acquisition of chronic infection. However, in post-hoc analysis there was a significant difference in chronic acquisition in young patients (under 15 years of age). Also there were no new acquisitions with ST406 in the newly infected patients during segregation. These findings suggest that continuing with strict segregation reduces the acquisition of chronic infection at least in young CF patients as well as the spread of shared genotypes that are associated with resistance to more antibiotic classes
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