Christopher O'Callaghan

Ciliary beat pattern is associated with specific ultrastructural defects in primary ciliary dyskinesia

Abstract Background The main symptoms of primary ciliary dyskinesia (PCD) are nasal rhinorrhea or blockage and moist-sounding cough. Diagnosis can be difficult and is based on an abnormal ciliary beat frequency, accompanied by specific abnormalities of the ciliary axoneme. It is unknown whether determining ciliary beat pattern related to specific ultrastructural ciliary defects might help in the diagnosis of PCD. Objective We sought to determine ciliary beat pattern and beat frequency (CBF) associated with the 5 common ultrastructural defects responsible for PCD. Methods Nasal brushings were performed on 56 children with PCD. Ciliary movement was recorded using digital high-speed video imaging to assess beat frequency and pattern. Electron microscopy was performed. Results In patients with an isolated outer dynein arm or with an outer and inner dynein arm defect, 55% and 80% of cilia were immotile, respectively. Cilia that moved were only flickering. Mean CBF (± 95% CI) was 2.3 Hz (± 1.2) and 0.8 Hz(± 0.8), respectively. Cilia with an isolated inner dynein arm or a radial spoke defect had similar beat patterns. Cilia appeared stiff, had a reduced amplitude, and failed to bend along their length. Immotile cilia were present in 10% of cilia with an inner dynein arm defect and in 30% of radial spoke defects. Mean CBF was 9.3 Hz (± 2.6) and 6.0 Hz (± 3.1), respectively. The ciliary transposition defect produced a large circular beat pattern (mean CBF, 10.7 Hz [± 1.1]). No cilia were immotile. Conclusions Different ultrastructural defects responsible for PCD result in predictable beat patterns. Recognition of these might help in the diagnostic evaluation of patients suspected of having PCD.

Analysis of ciliary beat pattern and beat frequency using digital high speed imaging: comparison with the photomultiplier and photodiode methods

BACKGROUND The aim of this study was to determine the relationship of the power and recovery stroke of respiratory cilia using digital high speed video imaging. Beat frequency measurements made using digital high speed video were also compared with those obtained using the photomultiplier and modified photodiode techniques. METHOD Ciliated epithelium was obtained by brushing the inferior nasal turbinate of 20 healthy subjects. Ciliated edges were observed by microscopy and the deviation of cilia during their recovery stroke relative to the path travelled during their power stroke was measured. Beat frequency measurements made by digital high speed video analysis were compared with those obtained using the photomultiplier and modified photodiode. RESULTS Cilia were found to beat with a forward power stroke and a backward recovery stroke within the same plane. The mean angular deviation of the cilia during the recovery stroke from the plane of the forward power stroke was only 3.6°(95% CI 3.1 to 4.1). There was a significant difference in beat frequency measurement between the digital high speed video (13.2 Hz (95% CI 11.8 to 14.6)) and both photomultiplier (12.0 Hz (95% CI 10.8 to 13.1), p = 0.01) and photodiode (11.2 Hz (95% CI 9.9 to 12.5), p<0.001) techniques. The Bland-Altman limits of agreement for the digital high speed video were –2.75 to 5.15 Hz with the photomultiplier and –2.30 to 6.06 Hz with the photodiode. CONCLUSION Respiratory cilia beat forwards and backwards within the same plane without a classical sideways recovery sweep. Digital high speed video imaging allows both ciliary beat frequency and beat pattern to be evaluated.

DNAI2 Mutations Cause Primary Ciliary Dyskinesia with Defects in the Outer Dynein Arm

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder characterized by chronic destructive airway disease and randomization of left/right body asymmetry. Males often have reduced fertility due to impaired sperm tail function. The complex PCD phenotype results from dysfunction of cilia of the airways and the embryonic node and the structurally related motile sperm flagella. This is associated with underlying ultrastructural defects that frequently involve the outer dynein arm (ODA) complexes that generate cilia and flagella movement. Applying a positional and functional candidate-gene approach, we identified homozygous loss-of-function DNAI2 mutations (IVS11+1G > A) in four individuals from a family with PCD and ODA defects. Further mutational screening of 105 unrelated PCD families detected two distinct homozygous mutations, including a nonsense (c.787C > T) and a splicing mutation (IVS3-3T > G) resulting in out-of-frame transcripts. Analysis of protein expression of the ODA intermediate chain DNAI2 showed sublocalization throughout respiratory cilia. Electron microscopy showed that mutant respiratory cells from these patients lacked DNAI2 protein expression and exhibited ODA defects. High-resolution immunofluorescence imaging demonstrated absence of the ODA heavy chains DNAH5 and DNAH9 from all DNAI2 mutant ciliary axonemes. In addition, we demonstrated complete or distal absence of DNAI2 from ciliary axonemes in respiratory cells of patients with mutations in genes encoding the ODA chains DNAH5 and DNAI1, respectively. Thus, DNAI2 and DNAH5 mutations affect assembly of proximal and distal ODA complexes, whereas DNAI1 mutations mainly disrupt assembly of proximal ODA complexes.

Functional analysis of cilia and ciliated epithelial ultrastructure in healthy children and young adults

Background: There are very few data on normal ciliary beat frequency, beat pattern, and ultrastructure in healthy children and adults. A study was undertaken to define ciliary structure, beat frequency and beat pattern in a healthy paediatric and young adult population. Methods: Ciliated epithelial samples were obtained from 76 children and adult volunteers aged 6 months to 43 years by brushing the inferior nasal turbinate. Beating cilia were recorded using a digital high speed video camera which allowed analysis of ciliary beat pattern and beat frequency. Tissue was fixed for transmission electron microscopy. Results: The mean ciliary beat frequency for the paediatric population (12.8 Hz (95% CI 12.3 to 13.3)) was higher than for the adult group (11.5 Hz (95% CI 10.3 to 12.7 Hz), p<0.01, t test); 10% (range 6–24%) of ciliated edges were found to have areas of dyskinetically beating cilia. All samples had evidence of mild epithelial damage. This reflected changes found in all measurements used for assessment of epithelial damage. Ciliary ultrastructural defects were found in less than 5% of cilia. Conclusion: Normal age related reference ranges have been established for ciliary structure and beat frequency. In a healthy population localised epithelial damage may be present causing areas of ciliary dyskinesia.

Streptococcus pneumoniae Deficient in Pneumolysin or Autolysin Has Reduced Virulence in Meningitis

BACKGROUND The role played by pneumolysin and autolysin in pneumococcal meningitis is poorly understood. METHODS A rat model was used to investigate the disease, in which surgical implantation of a cisternal catheter allowed bacterial instillation and cerebrospinal fluid (CSF) sampling. RESULTS CSF infection of rats with wild-type pneumococci caused meningitis within 26 h, whereas isogenic mutants that do not express pneumolysin (DeltaPly) or autolysin (LytA(-)) caused very mild or no disease. Wild-type infections resulted in pneumococci in the CSF and cortical homogenates, but a minority of the rats infected with DeltaPly or LytA(-) had bacteria in these locations at 26 h. Leukocyte numbers in the CSF were similar after infection with all pneumococci; however, neutrophils and monocytes predominated after wild-type infection, whereas lymphocytes and atypical lymphocytes predominated after infection with the mutants. Wild-type pneumococci caused disruption to the ependyma, but this was not observed in rats infected with DeltaPly or LytA(-). Cells surrounding the ventricles in wild type-infected animals expressed caspase 3, and astrocytes had hypertrophy; both findings were absent in rats infected with the mutants. CONCLUSIONS This study provides strong in vivo evidence that pneumolysin and autolysin play crucial roles in the pathogenesis of pneumococcal meningitis.

Relative Roles of Pneumolysin and Hydrogen Peroxide from Streptococcus pneumoniae in Inhibition of Ependymal Ciliary Beat Frequency

ABSTRACT Ciliated ependymal cells line the ventricular system of the brain and the cerebral aqueducts. This study characterizes the relative roles of pneumolysin and hydrogen peroxide (H2O2) in pneumococcal meningitis, using the in vitro ependymal ciliary beat frequency (CBF) as an indicator of toxicity. We have developed an ex vivo model to examine the ependymal surface of the brain slices cut from the fourth ventricle. The ependymal cells had cilia beating at a frequency of between 38 and 44Hz. D39 (wild-type) and PLN-A (pneumolysin-negative) pneumococci at 108 CFU/ml both caused ciliary slowing. Catalase protected against PLN-A-induced ciliary slowing but afforded little protection from D39. Lysed PLN-A did not reduce CBF, whereas lysed D39 caused rapid ciliary stasis. There was no effect of catalase, penicillin, or catalase plus penicillin on the CBF. H2O2 at a concentration as low as 100 μM caused ciliary stasis, and this effect was abolished by coincubation with catalase. An additive inhibition of CBF was demonstrated using a combination of both toxins. A significant inhibition of CBF at between 30 and 120 min was demonstrated with both toxins compared with either H2O2 (10 μM) or pneumolysin (1 HU/ml) alone. D39 released equivalent levels of H2O2 to those released by PLN-A, and these concentrations were sufficient to cause ciliary stasis. The brain slices did not produce H2O2, and in the presence of 108 CFU of D39 or PLN-A per ml there was no detectable bacterially induced increase of H2O2release from the brain slice. Coincubation with catalase converted the H2O2 produced by the pneumococci to H2O. Penicillin-induced lysis of bacteria dramatically reduced H2O2 production. The hemolytic activity released from D39 was sufficient to cause rapid ciliary stasis, and there was no detectable release of hemolytic activity from the pneumolysin-negative PLN-A. These data demonstrate that D39 bacteria released pneumolysin, which caused rapid ciliary stasis. D39 also released H2O2, which contributed to the toxicity, but this was masked by the more severe effects of pneumolysin. H2O2 released from intact PLN-A was sufficient to cause rapid ciliary stasis, and catalase protected against H2O2-induced cell toxicity, indicating a role for H2O2 in the response. There is also a slight additive effect of pneumolysin and H2O2 on ependymal toxicity; however, the precise mechanism of action and the role of these toxins in pathogenesis remain unclear.

Diagnosing primary ciliary dyskinesia

A nationally funded diagnostic service should lead to improved outcome The National Specialist Commissioning Advisory Group (NSCAG) has funded three centres to establish and provide a national diagnostic service for England for children and adults suspected of suffering from primary ciliary dyskinesia (PCD). This is welcomed, as state of the art diagnostic testing will be available nationally which will increase the numbers of patients diagnosed with a condition in which early diagnosis has a very significant effect on both short-term and long-term morbidity. Inheritance is autosomal recessive with an incidence of around 1:15 000 in the Caucasian population and, as expected, we have found a much higher incidence in ethnic groups where consanguineous marriages are common. Accurate diagnosis will allow appropriate genetic counselling of families. PCD is caused by one of a number of different ciliary defects that result in ineffective mucociliary clearance. Although most patients with PCD have symptoms from birth or early infancy,1 the diagnosis is frequently delayed2 and it is likely that a significant number of patients are never diagnosed.3 Failure to diagnose PCD leads to progressive and permanent lung destruction owing to obstruction of the airways with secretions and subsequent infection, leading to bronchiectasis. …

Ciliated air-liquid cultures as an aid to diagnostic testing of primary ciliary dyskinesia.

BACKGROUND The diagnosis of primary ciliary dyskinesia (PCD) can prove difficult because of secondary damage of ciliated tissue. METHODS Here we audit culturing cells, obtained by nasal brushing, to a ciliated phenotype using an air-liquid interface method to determine if the effects of secondary damage on cilia were reduced following culture. RESULTS Of 231 patients consecutively referred for diagnostic testing, culture was attempted in 187, with 101 (54%) becoming ciliated. Of the 90 brush biopsy samples with a low dyskinesia score (< 40%), 71 grew cilia after culture (79% success). Significant secondary damage (> 40% dyskinesia) was present in 69 (43%) of the initial brush biopsy samples, and of these, 18 (26%) became ciliated after culture. In these samples, ciliary dyskinesia was significantly (P < .001) reduced (64% ± 6.8% before culture, 31% ± 4.5% after culture). Ciliary beat frequency (CBF) after cell culture was similar to CBF before culture. Cell culture helped to exclude PCD in eight patients for whom ciliary dyskinesia was present in > 70% of the initial brush biopsy sample, a level at which a rebiopsy would normally be requested. In six patients in whom no cilia were found in the initial brush biopsy samples, ciliated cell culture was successful and excluded the diagnosis. PCD was diagnosed in 28 patients and ciliated cell culture was successful in 12 (43%) showing identical ciliary beat pattern and electron microscopy findings. CONCLUSIONS Ciliary dyskinesia was reduced following cell culture to a ciliated phenotype compared with the initial brush biopsy sample. The specific PCD phenotype was maintained after culture.

Sensitivities of Human Monocytes and Epithelial Cells to Pneumolysin Are Different

ABSTRACT The Streptococcus pneumoniae pore-forming toxin, pneumolysin, is an important virulence factor in pneumococcal pneumonia. The effect of pneumolysin on human lung epithelial and monocyte cell viability was compared. Pneumolysin caused a dose-dependent loss of viability of human lung epithelial (A549 and L132) and monocyte (U937 and THP-1) cell lines. Analysis of the dose-response curves revealed similar log 50% inhibitory concentration (pIC50) values for A549, L132, and THP-1 of 0.12± 0.1, 0.02± 0.04, and 0.12± 0.13 hemolytic units (HU), respectively, but U937 cells showed a significantly greater pIC50 of 0.42± 0.12 HU. Differentiation of A549 and L132 with phorbol ester or THP-1 with gamma interferon had no effect on their sensitivity to pneumolysin. However, a significant decrease in the potency of pneumolysin against U937 cells followed gamma interferon treatment. The Hill slopes of the inhibition curves were greater than unity, indicating that pneumolysin may act with positive cooperativity. Analysis of pneumolysin-treated THP-1 cells by electron microscopy revealed membrane lesions of between 100 and 200 nm in diameter.

A Receptor-targeted Nanocomplex Vector System Optimized for Respiratory Gene Transfer

Synthetic vectors for cystic fibrosis (CF) gene therapy are required that efficiently and safely transfect airway epithelial cells, rather than alveolar epithelial cells or macrophages, and that are nonimmunogenic, thus allowing for repeated delivery. We have compared several vector systems against these criteria including GL67, polyethylenimine (PEI) 22 and 25 kd and two new, synthetic vector formulations, comprising a cationic, receptor-targeting peptide K(16)GACSERSMNFCG (E), and the cationic liposomes (L) DHDTMA/DOPE or DOSEP3/DOPE. The lipid and peptide formulations self assemble into receptor-targeted nanocomplexes (RTNs) LED-1 and LED-2, respectively, on mixing with plasmid (D). LED-1 transfected airway epithelium efficiently, while LED-2 and GL67 preferentially transfected alveolar cells. PEI transfected airway epithelial cells with high efficiency, but was more toxic to the mice than the other formulations. On repeat dosing, LED-1 was equally as effective as the single dose, while GL67 was 30% less effective and PEI 22 kd displayed a 90% reduction of efficiency on repeated delivery. LED-1 thus was the only formulation that fulfilled the criteria for a CF gene therapy vector while GL67 and LED-2 may be appropriate for other respiratory diseases. Opportunities for PEI depend on a solution to its toxicity problems. LED-1 formulations were stable to nebulization, the most appropriate delivery method for CF.