Een bijdrage tot de kennis over trilhaarbeweging en trilhaarziekten

Cilia are tiny cell organelles (6-7 µm length) that protrude from the cell surface of almost all cell types of the human body. They are equipped with an extensive protein machinery and have a significant role in several human tissues. The motile cilia are important for extracellular fluid movement in the entire respiratory tract, in the cerebrospinal canal and in the female reproductive tract. Moreover, motile cilia govern the left-right patterning of the organs during embryogenesis. The tail of a spermatozoon also has the same structure as a motile cilium. Primary ciliary dyskinesia (PCD) is a rare congenital disorder of the motile respiratory cilia. Patients with PCD have chronic upper and lower respiratory tract symptoms, situs inversus in almost half of the cases and an increased incidence of male infertility. The diagnosis of the disorder is difficult, and until now, there is no consensus on a diagnostic algorithm. Evaluation of the efficacy of ciliary motion, counting and describing ciliary beat pattern by light microscopy, evaluation of the ciliary structure by electron microscopy, measurement of nasal nitric oxide, which is often low in patients with PCD and eventually confirmation of genetic mutations in known PCD genes, form the cornerstones for the diagnosis. The genetic origin of the disorder is very heterogeneous and not completely unravelled. Ciliogenesis is a specialized technique that induces de novo regrowth of cilia on the respiratory epithelium, after dedifferentiation. Secondary changes can be excluded in this way. In this PhD thesis, we studied motile respiratory cilia in health and disease. In the first part of this thesis, we explore the effect of nebulized drugs on ciliary activity in vitro. This is of high clinical relevance. Although several drugs have a proven positive benefit/risk ratio in patients with CF, this has not been extensively studied in other diseases. Prudence is warranted when extrapolating therapies from CF care to other diseases. When efficacy is not proven, absence of side effects becomes even more important. We therefore advise to evaluate the influence of new inhaled compounds on ciliary activity. We devised an in vitro cell system that allows testing the influence of nebulized drugs on ciliary function: during nebulization and also after nebulization, to assess the duration of any effect. We confirm that salbutamol has a cilio-stimulatory effect, and that hypertonic saline has a transient inhibitory effect. All the tested drugs, manufactured for inhalation, did not influence ciliary activity, but we identified Obracin , a tobramycin containing solution manufactured for intravenous application, as a cilio-inhibitory drug when used for nebulization. In the second part, we have contributed to determine the validity of diagnostic tests for PCD. We show that PCD with normal ultrastructure is present in a large subset of patients with PCD and that these patients have similar symptoms compared to those with ultrastructural changes. We unequivocally prove that ciliogenesis in culture is the only technique capable to detect this difficult diagnosis in all cases with normal ultrastructure. Nasal NO is used as a screening test for the diagnosis of PCD. We demonstrate that, on its own, it is an insufficiently sensitive and specific test. Additional tests are needed when the clinical suspicion for PCD is high. Genetic testing can contribute to the diagnosis of PCD. Genetic analysis in our patients with PCD and normal ultrastructure confirmed the diagnosis by the presence of biallelic disease causing mutations in a PCD gene in 79% of the tested patients. This unequivocally proves the correct diagnosis in this PCD subtype. In collaboration with the group of H. Omran in Münster, we unraveled a part of the complex cascade of events during ciliogenesis: CCNO and MCIDAS were identified as important inducers of formation of multiple motile cilia at the cell surface. Mutations in these genes cause a Reduced Generation of Multiple Motile Cilia, formerly known as ciliary aplasia. Patients with PCD not only need a diagnosis but need follow-up and optimal treatment. In the last part of the thesis, the value of LCI as outcome measure in patients with PCD was studied. In our cross-sectional study, LCI was more frequently abnormal than FEV1. Compared to FEV1 , LCI was a better predictor of structural abnormalities on chest CT scan. In patients with PCD, LCI holds promise as parameter for clinical follow-up and possibly as outcome parameter in clinical trials.LIST OF ABBREVIATIONS 9 GENERAL INTRODUCTION AND OBJECTIVES 11 Introduction 13 Cilia 13 Primary Ciliary Dyskinesia 21 Objectives 39 RESULTS 41 Chapter 1 The influence of nebulized drugs on ciliary activity 43 Chapter 2 Diagnostic tests for primary ciliary dyskinesia 61 In vitro ciliogenesis is needed not to miss PCD with normal ultrastructure 63 The diagnostic accuracy of nasal nitric oxide 87 The genetic origin of PCD 99 Chapter 3 LCI as outcome measure for use in clinical trials 133 GENERAL DISCUSSION 145 FUTURE PERSPECTIVES 155 REFERENCES 159 SUMMARY/SAMENVATTING 167 CURRICULUM VITAE 173nrpages: 178status: publishe