A range of 30%-62% of functioning multiciliated airway cells is sufficient to maintain ciliary airway clearance.

BACKGROUND: Primary ciliary dyskinesia (PCD) is a genetic disorder caused by aberrant motile cilia function that results in defective ciliary airway clearance and subsequently to recurrent airway infections and bronchiectasis. QUESTION: How many functional multiciliated airway cells are sufficient to maintain ciliary airway clearance? METHODS: To answer this question we exploited the molecular defects of the X-linked recessive PCD variant caused by pathogenic variants in DNAAF6 (PIH1D3), characterized by immotile cilia in the affected males. We carefully analyzed the clinical phenotype, molecular defect (immunofluorescence and transmission-electron microscopy) and performed in vitro (particle tracking in air-liquid interface cultures) and in vivo (radiolabeled tracer studies) studies to assess ciliary clearance of respiratory cells from females with heterozygous and males with hemizygous pathogenic DNAAF6 variants. RESULTS: PCD males with hemizygous pathogenic DNAAF6 variants displayed exclusively immotile cilia, absence of ciliary clearance and severe PCD symptoms. Due to random or skewed X-chromosome inactivation in six females with heterozygous pathogenic DNAAF6 variants, 54.3%±10 (range 38%-70%) of multiciliated cells were defective. Nevertheless, in vitro and in vivo assessment of the ciliary airway clearance was normal or slightly abnormal. Consistently, heterozygous female individuals showed no or only mild respiratory symptoms. CONCLUSIONS: Our findings indicate that 30%-62% of functioning multiciliated respiratory cells are able to generate either normal or slightly reduced ciliary clearance. Because heterozygous females displayed either no or subtle respiratory symptoms, complete correction of 30% of cells by precision medicine might be able to improve ciliary airway clearance in PCD individuals as well as clinical symptoms.

CCDC151 mutations cause primary ciliary dyskinesia by disruption of the outer dynein arm docking complex formation.

A diverse family of cytoskeletal dynein motors powers various cellular transport systems, including axonemal dyneins generating the force for ciliary and flagellar beating essential to movement of extracellular fluids and of cells through fluid. Multisubunit outer dynein arm (ODA) motor complexes, produced and preassembled in the cytosol, are transported to the ciliary or flagellar compartment and anchored into the axonemal microtubular scaffold via the ODA docking complex (ODA-DC) system. In humans, defects in ODA assembly are the major cause of primary ciliary dyskinesia (PCD), an inherited disorder of ciliary and flagellar dysmotility characterized by chronic upper and lower respiratory infections and defects in laterality. Here, by combined high-throughput mapping and sequencing, we identified CCDC151 loss-of-function mutations in five affected individuals from three independent families whose cilia showed a complete loss of ODAs and severely impaired ciliary beating. Consistent with the laterality defects observed in these individuals, we found Ccdc151 expressed in vertebrate left-right organizers. Homozygous zebrafish ccdc151(ts272a) and mouse Ccdc151(Snbl) mutants display a spectrum of situs defects associated with complex heart defects. We demonstrate that CCDC151 encodes an axonemal coiled coil protein, mutations in which abolish assembly of CCDC151 into respiratory cilia and cause a failure in axonemal assembly of the ODA component DNAH5 and the ODA-DC-associated components CCDC114 and ARMC4. CCDC151-deficient zebrafish, planaria, and mice also display ciliary dysmotility accompanied by ODA loss. Furthermore, CCDC151 coimmunoprecipitates CCDC114 and thus appears to be a highly evolutionarily conserved ODA-DC-related protein involved in mediating assembly of both ODAs and their axonemal docking machinery onto ciliary microtubules.

Ventilation inhomogeneity in children with primary ciliary dyskinesia.

BACKGROUND: The lung clearance index (LCI) derived from the multiple breath inert gas washout (MBW) test reflects global ventilation distribution inhomogeneity. It is more sensitive than forced expiratory volume in 1 s (FEV(1)) for detecting abnormal airway function and correlates closely with structural lung damage in children with cystic fibrosis, which shares features with primary ciliary dyskinesia (PCD). Normalised phase III slope indices S(cond) and S(acin) reflect function of the small conducting and acinar airways, respectively. The involvement of the peripheral airways assessed by MBW tests has not been previously described in PCD. METHODS: A cross-sectional MBW study was performed in 27 children and adolescents with verified PCD, all clinically stable and able to perform lung function tests. LCI, S(cond) (n=23) and S(acin) (n=23) were derived from MBW using a mass spectrometer and sulfur hexafluoride as inert marker gas. MBW indices were compared with present age, age at diagnosis and spirometry findings, and were related to published normative values. RESULTS: LCI, S(cond) and S(acin) were abnormal in 85%, 96% and 78% of patients with PCD and in 81%, 93% and 79%, respectively, of 13/27 subjects with normal FEV(1). LCI and S(acin) correlated significantly while S(cond) did not correlate with any other lung function parameters. None of the lung function measurements correlated with age or age at diagnosis. CONCLUSIONS: PCD is characterised by marked peripheral airway dysfunction. MBW seems promising in the early detection of lung damage, even in young patients with PCD. The relationship of MBW indices to the outcome of long-term disease and their role in the management of PCD need to be assessed.

Nasal nitric oxide and pulmonary radioaerosol mucociliary clearance as supplementary tools in diagnosis of primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) is a rare, usually autosomal recessive inherited disorder, characterized by abnormalities in ciliary structure and/or function. Frequent, intermittent or chronic airway infections precipitated by impaired airway mucociliary clearance may cause permanent lung damage and reduced lung function. Early diagnosis is considered important for the prevention of lung damage, but diagnosis is probably often delayed or even missed since diagnosis of PCD is both complex and time consuming, and yet not always exact. The aims of this PhD thesis were to evaluate the discriminative capacity and "real-life" clinical application of two candidates for supplemental diagnostic testing for PCD: Nasal nitric oxide (nNO) measurement placed as a first line test to point out probable PCD patients for further investigation or exclude patients, regardless of age, Pulmonary radioaerosol mucociliary clearance (PRMC) as a second line test for PCD investigation in children from 5 years of age. And additionally, Proposing an algorithm for the pathway of diagnosing PCD based on these two studies and recommendations from the literature. Nasal NO and PRMC demonstrated to be two highly valid supplementary diagnostic tools to be placed in each end of the diagnostic pathway when investigating selected patients referred for PCD work up. Nasal NO measurement demonstrated to have an obvious place as a first line test in the pathway of PCD investigation and PRMC as second line test as a supplement to ciliary function test and EM-test in cases of difficult diagnoses. Neither of these tests can stand alone in diagnosis or excluding of PCD. PCD remains to be a diagnosis that should be made at a tertiary PCD centre, as clinical evaluation of referred patients is crucial before excluding the disease.

Pulmonary radioaerosol mucociliary clearance in diagnosis of primary ciliary dyskinesia.

BACKGROUND: Methods relying on nasal ciliary motility for the diagnosis of primary ciliary dyskinesia (PCD) are often hampered by secondary ciliary dyskinesia. A functional test for pulmonary mucociliary clearance, which is not influenced by secondary nasal ciliary defects, would be a valuable tool in a PCD workup. METHODS: The diagnostic validity and repeatability of a pulmonary radioaerosol mucociliary clearance (PRMC) test for the diagnosis of PCD was assessed in the following three sequentially performed substudies: (1) a preliminary cross-sectional study of PRMC in patients with known PCD; (2) a prospective blinded trial of patients referred for suspicion of PCD; and (3) an implementation study of PRMC as a routine method used in a PCD workup. PRMC was studied after (99m)Tc-albumin colloid aerosol inhalation, and the results were compared to (1) the results of nasal ciliary motility studies, (2) ciliary ultrastructure, and (3) the final clinical diagnosis. The repeatability of PRMC was assessed in 14 patients. RESULTS: A total of 95 patients, 5 to 74 years of age, were included in the study (57 patients in whom PCD was diagnosed, 26 non-PCD patients, and 12 patients referred for PCD workup without a conclusive workup result). In substudy 1, 14 of 15 patients with known PCD showed impaired PRMC; the results were inconclusive in 1 patient. In substudy 2, among 59 patients referred for PCD workup PRMC test results, compared to nasal ciliary motility, showed a sensitivity of 88% and a specificity of 100%. In substudy 3, among 21 patients referred for PCD investigation who were included in a routine PCD workup after PRMC implementation, 71% of PRMC test results were in alignment with nasal ciliary motility. Repeatability of interpretation was seen in 13 of 14 cases. A conclusive PRMC after only one test was found in 81 of 95 patients (85%). CONCLUSION: PRMC is a noninvasive functional test for total tracheobronchial mucociliary clearance with a high sensitivity and specificity for PCD, a high rate of conclusive results after only one test and a further ability to separate PCD from focal pulmonary mucociliary defects.

Patient-specific three-dimensional explant spheroids derived from human nasal airway epithelium: a simple methodological approach for ex vivo studies of primary ciliary dyskinesia.

BACKGROUND: Three-dimensional explant spheroid formation is an ex vivo technique previously used in studies of airway epithelial ion and water transport. Explanted cells and sheets of nasal epithelium form fully differentiated spheroids enclosing a partly fluid-filled lumen with the ciliated apical surface facing the outside and accessible for analysis of ciliary function. METHODS: We performed a two-group comparison study of ciliary beat pattern and ciliary beat frequency in spheroids derived from nasal airway epithelium in patients with primary ciliary dyskinesia (PCD) and in healthy controls. Nasal ciliary cells and sheets were removed on day 1 by nasal brush biopsy and analyzed with regard to ciliary beat pattern-and frequency using high-speed video imaging for standard reference values. Three-dimensional explant spheroid formation was initiated in the same individual on the same day by incubation of cells and sheets from a separate brush biopsy. Harvested spheroids were analyzed earliest possible and values of spheroid ciliary beat pattern and frequency were compared to the corresponding reference values from day 1. RESULTS: Spheroids formed fast in serum-free culture medium. Formation was successful in 15 out of 18 (82%) sampled individuals. Thus, formation was successful in seven healthy controls and eight PCD patients, while unsuccessful in 3 with PCD due to infection. Median (range) number of days in culture before harvesting of spheroids was 4 (1-5) in healthy versus 2 (1-5) in PCD. Spheroid ciliary beat pattern and frequency were unchanged compared to their corresponding day 1 standard reference values. Spheroid ciliary beat frequency discriminated highly significant between healthy controls (9.3 Hz) and PCD patients (2.4 Hz) (P < 0.0001). Survival of spheroids was 16 days in a single healthy person. CONCLUSION: Patient-specific three-dimensional explant spheroid formation from a minimal invasive nasal brush biopsy is a feasible, fast and valid ex vivo method to assess ciliary function with potential of aiding the diagnosis of PCD. In addition, it may be a useful model in the investigation of pathophysiological aspects and drug effects in human nasal airway epithelium.

Hand-held tidal breathing nasal nitric oxide measurement--a promising targeted case-finding tool for the diagnosis of primary ciliary dyskinesia.

BACKGROUND: Nasal nitric oxide (nNO) measurement is an established first line test in the work-up for primary ciliary dyskinesia (PCD). Tidal breathing nNO (TB-nNO) measurements require minimal cooperation and are potentially useful even in young children. Hand-held NO devices are becoming increasingly widespread for asthma management. Therefore, we chose to assess whether hand-held TB-nNO measurements reliably discriminate between PCD, and Healthy Subjects (HS) and included Cystic Fibrosis (CF) patients as a disease control group known to have intermediate nNO levels. METHODS: In this cross sectional, single centre, single occasion, proof-of-concept study in children and adults with PCD and CF, and in HS we compared feasibility, success rates, discriminatory capacity, repeatability and agreement between a hand-held electrochemical device equipped with a nNO software application sampling at flow rates 2 ml/s or 5 ml/s, and two stationary chemiluminescence devices, applying both tidal breathing and velum closure techniques. RESULTS: Measurements were done in 16 PCD patients, 21 patients with CF and 20 HS aged between 3.8 and 60.9 years. Hand-held TB-nNO showed high success rate (96.5-100%) vs. velum closure nNO techniques (70.2-89.5%). Hand-held TB-nNO sampling at flow rate 5 ml/s showed equally high discriminative power (PCD vs. HS [p<0.0001] and PCD vs. CF [p<0.0001]) and reaching close to 100% sensitivity and specificity, superior repeatability (CV% = 10%) and equal limits of agreement compared to TB-nNO by stationary devices and even compared to velum closure sampling. CONCLUSION: Hand-held TB-nNO discriminates significantly between PCD, CF and HS and shows promising potential as a widespread targeted case-finding tool for PCD, although further studies are warranted before implementation.

Lesions of the lacrimal drainage system: a clinicopathological study of 643 biopsy specimens of the lacrimal drainage system in Denmark 1910-1999.

PURPOSE: To determine the frequency of histologically verified lesions of the lacrimal drainage system in Denmark between the years 1910 and 1999. Furthermore, to correlate the clinical diagnosis with the pathology diagnosis. METHODS: Retrospective review of all pathology reports from 1910 to 1999 in the files of the Eye Pathology Institute, University of Copenhagen, describing a lesion of the lacrimal drainage system. In addition, a retrospective review of all reports describing a lesion of the lacrimal drainage system from the Danish Pathology Database. All specimens were re-evaluated, except in cases with a primary diagnosis of dacryocystitis. In these cases a sample of 25% was re-evaluated. RESULTS: A total of 643 lesions were collected. Dacryocystitis was the most frequent lesion, constituting 508 cases (79%). The remaining cases were diagnosed as dacryolithiasis (62 cases; 7.9%), tumour (29 cases; 4.5%), trauma (19 cases; 3.0%), congenital malformation (nine cases; 1.4%), canaliculitis (eight cases; 1.2%) and granulomatous inflammation (eight cases; 1.2%). Seventeen tumours were malignant, of which B-cell lymphoma was the most common (six cases). In 0.6% of cases with a clinical diagnosis of dacryocystitis/lithiasis a non-suspected malignant tumour was diagnosed. Micro-organisms were uncommon in dacryocystitis (9%) but frequent in cases of dacryolithiasis (87%). CONCLUSION: Dacryocystitis was by far the most frequent lesion of the lacrimal drainage system referred for histopathological evaluation. Dacryolithiasis was often associated with micro-organisms, especially Gram positive rods. Histopathology is necessary to confirm suspected tumours, more than half of which were inflammatory lesions, and to detect tumours that sometimes masquerade as inflammation.