Ciliogenesis In Culture Research Articles

Mannose-binding lectin 2 gene polymorphism and lung damage in primary ciliary dyskinesia.

Mannose-binding lectin (MBL) plays an important role in innate immunity and has been reported to be associated with the age-related decline in lung function in cystic fibrosis. MBL polymorphisms are associated with lung function decline in Primary Ciliary Dyskinesia (PCD). We performed sputum microbiology, spirometry pre- and post-administration of salbutamol, ciliary motion analysis, ultrastructural assessment of cilia, ciliogenesis in culture, and chest high resolution computed tomography in children with a clinical history of respiratory tract infections and/or presence of bronchiectasis suggestive of PCD or secondary ciliary dyskinesia (SCD). All subjects were evaluated for single nucleotide polymorphisms in the gene encoding MBL-2. The diagnosis of PCD was established in 45 subjects, while in the remaining 53 the diagnosis was SCD. A significant bronchodilator response was observed only in PCD associated with the MBL2-3 genotype, which is known to be associated with low/undetectable MBL serum levels. Also, bronchiectasis severity was significantly greater in subjects with MBL2-3 in both PCD and SCD. No other association was found between MBL genotypes and clinical findings. MBL plays a relatively minor role as a disease modifier in PCD. A similar finding in SCD supports the likely significance of this result.

Primary ciliary dyskinesia: critical evaluation of clinical symptoms and diagnosis in patients with normal and abnormal ultrastructure

BackgroundPrimary ciliary dyskinesia (PCD) is a rare disorder with variable disease progression. To date, mutations in more than 20 different genes have been found. At present, PCD subtypes are described according to the ultrastructural defect on transmission electron microscopy (TEM) of the motile cilia. PCD with normal ultrastructure (NU) is rarely reported because it requires additional testing. Biallelic mutations in DNAH11 have been described as one cause of PCD with NU.The aim of our study was to describe the clinical characteristics of a large population of patients with PCD, in relation to the ultrastructural defect. Additionally, we aimed to demonstrate the need for biopsy and cell culture to reliably diagnose PCD, especially the NU subtype.MethodsWe retrospectively analyzed data from 206 patients with PCD. We compared the clinical characteristics, lung function, microbiology and imaging results of 68 patients with PCD and NU to those of 90 patients with dynein deficiencies and 41 patients with central pair abnormalities. In addition, we aimed to demonstrate the robustness of the diagnosis of the NU subtype in cell culture by data from genetic analysis.ResultsPCD with NU comprised 33% (68/206) of all patients with PCD. Compared to other subtypes, patients with PCD and NU had a similar frequency of upper and lower respiratory tract problems, as well as similar lung function and imaging. With the currently widely applied approach, without cell culture, the diagnosis would have been missed in 16% (11/68) of patients with NU. Genetic analysis was performed in 29/68 patients with PCD and NU, and biallelic mutations were found in 79% (23/29) of tested patients.ConclusionsWe reported on the clinical characteristics of a large population of patients with PCD and NU. We have shown that systematic performance of biopsy and cell culture increases sensitivity to detect PCD, especially the subtype with NU.PCD with NU has similar clinical characteristics as other PCD types and requires biopsy plus ciliogenesis in culture for optimal diagnostic yield.

Rapid diagnosis of primary ciliary dyskinesia: cell culture and soft computing analysis

Diagnosis of primary ciliary dyskinesia (PCD) sometimes requires repeated nasal brushing to exclude secondary ciliary alterations. Our aim was to evaluate whether the use of a new method of nasal epithelial cell culture can speed PCD diagnosis in doubtful cases and to identify which are the most informative parameters by means of a multilayer artificial neural network (ANN). A cross-sectional study was performed in patients with suspected PCD. All patients underwent nasal brushing for ciliary motion analysis, ultrastructural assessment and evaluation of ciliary function after ciliogenesis in culture by ANN. 151 subjects were studied. A diagnostic suspension cell culture was obtained in 117 nasal brushings. A diagnosis of PCD was made in 36 subjects (29 of whom were children). In nine out of the 36 patients the diagnosis was made only after a second brushing, because of equivocal results of both tests at first examination. In each of these subjects diagnosis of PCD was confirmed by cell culture results. Cell culture in suspension evaluated by means of ANN allows the separation of PCD from secondary ciliary dyskinesia patients after only 5 days of culture and allows diagnosis to be reached in doubtful cases, thus avoiding the necessity of a second sample.

Simplified cell culture method for the diagnosis of atypical primary ciliary dyskinesia

Background:The diagnosis of primary ciliary dyskinesia (PCD) can be challenging, and it may be particularly difficult to distinguish primary ciliary disease from the secondary changes after infections.Objectives:The purpose of the...

The secondary nature of ciliary (dis)orientation in secondary and primary ciliary dyskinesia

ObjectiveCiliary orientation (COR) is an important parameter of mucociliary clearance and ciliary disorientation has been reported in cases of acquired abnormalities [secondary ciliary dyskinesia (SCD)] and in a very few cases as the single abnormality in primary ciliary dyskinesia (PCD). The etiology, pathogenesis, consequences and relevance of ciliary (dis)orientation are still unclear. Material and MethodsTo elucidate the primary or secondary nature of ciliary (dis)orientation, COR was measured in 179 non-PCD and 59 PCD patients. COR was measured in biopsies and after ciliogenesis in culture and was correlated with a number of functional and ultrastructural parameters. COR was defined as the SD of the angles of lines through the central pair of microtubules using transmission electron microscopy. Internationally accepted normal values for COR are ≤20°; COR values of 20–35° indicate increased disorientation; and COR values >35° represent a random orientation. ResultsFor non-PCD biopsies, COR increased with increasing SCD, from 15±7° (n=54) for normal (<5%) SCD to 28±8° (n=16) for severe (>25%) SCD. No correlation was found between COR and ciliary beat frequency. However, increased COR values (28±8°) were found for immotility (n=8), compared to (coordinated) ciliary activity (19±9°) (n=121). After ciliogenesis no ultrastructural abnormalities were found and COR was normal (13±5°; n=308). COR can therefore be considered to be secondary in non-PCD and correlates with SCD percentage and ciliary motility. In biopsies from PCD patients with dynein deficiency and with normal ultrastructure, COR was increased, to 28±11° (n=32) and 21±7° (n=15), respectively, and in cases with central pair abnormalities COR was random (38±11°; n=12). After ciliogenesis COR remained random in the PCD group with central pair abnormalities (38±9°; n=15), and was increased in the PCD groups with dynein deficiency (24±10°; n=35) and normal ultrastructure (25±8°; n=17). Ciliary disorientation was never found as the single abnormality. Conclusion COR can be considered to be secondary in PCD. Both ciliary (im)motility and SCD percentage contribute to COR.

Diagnostic evaluation of mucociliary transport: from symptoms to coordinated ciliary activity after ciliogenesis in culture.

Mucociliary transport is one of the most important local defense mechanisms of the airways, but it is prone to many and frequent acquired abnormalities and to inherited abnormalities. These abnormalities result in basic physiologic disturbances leading to a number of respiratory symptoms and signs. In order to critically evaluate the diagnostic value of parameters of this mucociliary cascade, the results of ciliary investigations in over 500 individuals (controls, acquired, and inherited abnormalities) were reviewed. Ciliary beat frequency, ciliary coordination and ultrastructural abnormalities were measured and evaluated in biopsies and after ciliogenesis in culture. There is a considerable overlap for all investigated parameters in biopsy material of controls, secondary, and primary ciliary dyskinesia. There is not one parameter that is diagnostic for primary ciliary dyskinesia or can be used as an exclusion criterion. After ciliogenesis in culture, cilia are always completely normal except for the inherited abnormalities. Absence of coordinated ciliary activity after ciliogenesis in culture is 100% sensitive and specific for the diagnosis of primary ciliary dyskinesia.

Ciliary Function Analysis for the Diagnosis of Primary Ciliary Dyskinesia: Advantages of Ciliogenesis in Culture

The gold standard for the diagnosis of primary ciliary dyskinesia (PCD) is a dynein deficiency shown with transmission electron microscopy. However, there are many cases of PCD without dynein deficiency. When considering ciliary function, there are similar problems of sensitivity in diagnosis and there is also a major lack of specificity. Based on the normal ciliary function and ultrastructure and the absence of secondary abnormalities after ciliogenesis in sequential monolayer-suspension culture, the diagnostic value of ciliary function analysis after ciliogenesis was investigated in more than 70 PCD and 640 non-PCD cases. In biopsies, ciliary immotility was found in 66% of PCD cases but was also found in 8% of non-PCD cases. PCD was later confirmed in 61% of the biopsies with ciliary immotility. Normal ciliary beat frequency (CBF) was found in 20% of PCD biopsies. Coordinated ciliary activity was observed in 10% of PCD cases. After ciliogenesis in culture, ciliary immotility was present in 78% of the PCD cases but never in non-PCD cases. CBF was normal after ciliogenesis in 7% of the PCD cases and was always found in non-PCD cases. Absence of coordinated ciliary activity was found in 100% of PCD cases and 0% of non-PCD cases. In conclusion, while ciliary function analysis in a biopsy never proves, nor excludes the diagnosis of PCD, after ciliogenesis in culture CBF measurement can be diagnostic for PCD and reaches 100% specificity and sensitivity when considering coordinated ciliary activity, making it the single 100% diagnostic parameter for PCD.

Nasal ciliary beat frequency is age independent

Evaluate the age-dependency of ciliary beat frequency (CBF) in biopsies after ciliogenesis in culture. Retrospective analysis of CBF and ciliary ultrastructure in biopsies and after ciliogenesis from 203 individuals, aged 3 months to 74 years. All patients with successful culture were included. Ciliogenesis was obtained using the sequential monolayer-suspension culture technique for dissociated nasal epithelial cells. CBF was measured using computerized microscope photometry. Secondary ultrastructural abnormalities were evaluated in transmission electron microscopy. There was no correlation between age and CBF, in either the biopsies (7.0 +/- 2.6 Hz; n = 113) or after ciliogenesis in culture (8.1 +/- 1.3 Hz; n = 203). Even in individuals older than 70 years, CBF was normal in bioptic material (6.7 +/- 1.7 Hz) and after ciliogenesis in culture (7.9 +/- 1.0 Hz). Also, in individuals less than 1 year of age CBF was normal in biopsies as well as after ciliogenesis. CBF correlated inversely with the percentage of secondary ultrastructural abnormalities in the biopsies as seen with transmission electron microscopy: 8.1 +/- 1.8 Hz when ciliary ultrastructure was normal and 3.5 +/- 3.3 Hz in cases of severe secondary ciliary dyskinesia. After ciliogenesis in culture, ciliary ultrastructure was always normal, as was CBF. CBF is age independent but correlates with secondary ultrastructural abnormalities. CBF after ciliogenesis in culture is the intrinsic one.