PCD is a motile ciliopathy associated with persistent airways infections, chronic inflammation and bronchiectasis. Besides a characteristic lack of mucociliary clearance, other intrinsic factors contributing to infection susceptibility are understudied. We investigated differential protein expression to determine dysregulated biological processes in PCD nasal epithelia. Primary human nasal epithelia from PCD (n=6; static cilia, class 1a transmission electron microscopy defects) and healthy (n=3) donors were ALI-cultured for 4-6 weeks (widespread ciliation by tubulin labelling). Mass spectrometry (MS) proteomics quantified relative protein expression between PCD and healthy donors. Differentially quantified proteins were used to determine biological processes by Gene Ontology Protein ANalysis Through Evolutionary Relationships. We identified 151 significant differentially expressed proteins, 98 were up- and 53 down-regulated in PCD, compared with healthy epithelia. Notably, S100 proteins (cytoskeletal organisation) were enriched in PCD, whilst MUC5B (innate immunity critical) and HSP90 (downstream microbicidal activity) were diminished. Dysregulated biological processes were evident including increased oxidative stress, metabolic state, cell death, protein S-nitrosylation and actin-related cytoskeletal dysfunction. Our combination of ALI-culture with MS proteomics captured aberrant cellular functionality in PCD nasal epithelia. Compared with healthy donors, we found differentially expressed proteins indicative of metabolic depletion, cellular damage and structural instability. Findings may explain the diminished capacity of PCD nasal epithelia to respond to microbial challenge.
Primary Ciliary Dyskinesia (PCD) is a rare inherited multi-genic disorder of mucociliary function.Patients with indicative clinical profiles referred to the UK specialist PCD service receive a diagnosis based on multiple factors.These include high-speed video microscopy (HSVM) analysis of ciliary beat pattern (CBP) and ciliary beat frequency (CBF) at 37°C (for in vivo modelling).In PCD, ciliary axonemal defects generate abnormal CBP with/without abnormal CBF.Corresponding and predominant ultrastructural defects are determined by TEM, except in atypical cases.We report an atypical PCD patient (8 months old) with respiratory and nasal symptoms since birth, situs inversus and serous otitis media.HSVM confirmed abnormal and hyperfrequent ciliary function at 37°C on four occasions, but normal ciliary ultrastructure.On two occasions CBP and CBF (mean ±SD) were assessed at 37oC and room temperature (21-24°C).At 37°C CBF was hyperfrequent (34.4 Hz ±13.5 n=11; 26.3 Hz ±3.4 n=6) and CBP consistently abnormal with interrupted, short range, dyskinetic motility.However at room temperature the same cilia reverted to CBF (15.2 Hz ±4.5 n=2; 12.6 Hz ±0.8 n=6) within our normal range (11-20 Hz) with improved ciliary coordination and range of movement, suggesting a PCD variant with temperature sensitive CBP.Recent research suggests that healthy human epithelium maintains a normal CBP at temperatures as low as 2°C, and low temperature ciliary analysis may diagnostically replace HSVM.However, in light of our case study we conclude that temperature sensitive variants of PCD may exist and CBP analysis below 37°C without HSVM may risk PCD misdiagnosis.
Primary ciliary dyskinesia (PCD) is a genetically heterogeneous condition characterised by progressive lung disease arising from abnormal motile ciliary function. Approximately half of patients have situs inversus. The estimated prevalence of PCD in the UK South Asian population is 1:2,265. Early, accurate diagnosis is key to implementing appropriate management but clinical diagnostic tests can be equivocal. The aim of this study was to determine the importance of genetic screening for primary ciliary dyskinesia in a UK South Asian population with a typical clinical phenotype, where standard testing is inconclusive. Next-generation sequencing was used to screen 86 South Asian patients who had a clinical history consistent with PCD. The effect of a CCDC103 c.461A>C p.His154Pro missense variant on phenotypic variability was tested and variant pathogenicity was assessed by oligomerisation assay. Sixteen of 86(19%) patients carried a homozygous CCDC103 p.His154Pro mutation which was found to disrupt protein oligomerisation. Variable diagnostic test results were obtained including normal nasal nitric oxide levels (7/15 patients tested), normal ciliary beat pattern or frequency (9/16 cases) and a spectrum of partial and normal dynein arm retention (9/16 cases). Fifteen (94%) patients or their sibling (s) had situs inversus suggesting in CCDC103 p.His154Pro patients without situs inversus the diagnosis is missed. In conclusion, the CCDC103 p.His154Pro mutation is more prevalent than previously thought in the South Asian community and causes PCD that can be difficult to diagnose using pathology-based clinical tests. Genetic testing is critical when there is a strong clinical phenotype with inconclusive or normal diagnostic tests
Background: Primary ciliary dyskinesia (PCD) is a rare genetic disease that impedes ciliary function, leading to recurrent upper and lower airway infections. The Southampton PCD Diagnostic Centre has been commissioned by NHS England since 2006. We aimed to describe the characteristics of patients diagnosed at the centre. Methods: We conducted a cross-sectional study using data from local PCD registry, electronic health records and medical notes for all patients diagnosed with PCD in Southampton from 2006 to 2016. Results: Of 1,720 referrals, we diagnosed 142 patients with PCD (8%). Diagnosis was always based on at least one diagnostic test. The number of tests has increased in the last 5 years particularly in genotyping (65% vs 56% before 2011). Nasal nitric oxide was measured in 82% of patients over 5 years of age. Overall, 84% had high-speed video analysis, of which 70% showed predominantly static cilia, with the remainder exhibiting a variety of beat patterns; 131 patients had transmission electron microscopy, which showed 'hallmark' defects in 70% of cases; and 59% were genotyped. Median age at initial assessment was 10 years, younger for the 43% with situs abnormalities (7 vs 12 years, p=0.062). 11% had congenital heart disease, considerably higher than previously reported (3-6%). 72% reported neonatal respiratory symptoms, of which 58% needed respiratory support. Mean FEV1% was low in both children and adults (79% vs 71% respectively, p=0.26) at the time of diagnosis. Conclusion: We found a high prevalence of congenital heart disease and neonatal respiratory symptoms. Lung function was impaired in both children and adults. These findings highlight the need for early care for PCD in specialised centres.
Background: There is no gold standard to diagnose PCD so combination testing is used. High-speed video microscopy analysis (HSV) of ciliary function is highly sensitive but requires expensive equipment and specialists. Ability of non-specialists to diagnose PCD using simple equipment would improve diagnosis in resource-limited countries. Aim: To compare PCD diagnostic decisions of a non-specialist using clinical data and observational light microscopy (OLM), with a specialist multidisciplinary (MDT) service. Methods: 24 nasal brushings were analysed blind by OLM at room temperature (RT) by an ERS Fellow, and by HSV at 37oC by specialists (normal, abnormal, equivocal, insufficient). Final diagnostic decisions (PCD negative, PCD positive, equivocal) by the Fellow based on OLM and clinical data were compared to MDT decisions using clinical data, nitric oxide, HSV and electron microscopy. Results: Comparing tests: OLM was 42.3% sensitive and 50% specific compared to specialist HSV. Most brushings were equivocal using either tests (13 by OLM at RT and 15 by HSV at 37oC). When clinical data were added, there were significant differences between final diagnosis by Fellow and MDT (p<0.05). TheMDT9s outcomes were: 14 PCD negative, 7 equivocal and 3 PCD positive. The Fellow identified all 3 positives (all static cilia) but could not exclude PCD in 12/14 negatives as subtle changes were difficult to visualise; and 1 case was miss-diagnosed as positive. Conclusion: PCD diagnosis cannot be reliably made using OLM and clinical data but requires state-of-the-art diagnostics and specialist multidisciplinary assessment.
Background:CEP164 encodes a centrosomal protein required for assembly of primary cilia. More recently it has been suggested it may also have a role in formation of multiple motile cilia. Pathogenic variants in CEP164 are known to cause nephronophthisis-related ciliopathies but a causative link to the motile ciliopathy primary ciliary dyskinesia (PCD) has not been proven. Aim: To assess airway cilia in a patient with a clinical history consistent with PCD, and bi-allelic variants in CEP164. Method: A patient with bronchiectasis in the UK 100,000 Genomes Project was found to have compound heterozygous stop gain variants in CEP164, and no other relevant variants. The patient underwent PCD diagnostic functional testing including high-speed video microscopy and transmission electron microscopy (TEM) of nasal epithelial cells. In addition, localisation of CEP164 protein was assessed by immunofluorescence (IF). Results: Cilia displayed a dyskinetic ciliary beat pattern, with long cilia and cilia with bulbous tips. Air liquid interface culture partially resolved dyskinesia, however an abnormal 'staggered' beat pattern was evident and the presence of long cilia persisted. Ciliary ultrastructure was normal by TEM. IF analysis demonstrated an absence of CEP164 labelling at the centriolar region. Conclusion: Supported by BEAT-PCD, we provide evidence that presence of CEP164 is vital to correct formation and function of respiratory cilia in addition to primary cilia, and that pathogenic variants in CEP164 are responsible for the PCD phenotype in this patient. We suggest CEP164 should be considered a candidate gene for PCD.
PYRRO-C3D is a cephalosporin-3-diazeniumdiolate nitric oxide (NO) donor prodrug designed to selectively deliver NO to bacterial infection sites. The objective of this study was to assess the activity of PYRRO-C3D against nontypeable Haemophilus influenzae (NTHi) biofilms and examine the role of NO in reducing biofilm-associated antibiotic tolerance. The activity of PYRRO-C3D on in vitro NTHi biofilms was assessed through CFU enumeration and confocal microscopy. NO release measurements were performed using an ISO-NO probe. NTHi biofilms grown on primary ciliated respiratory epithelia at an air-liquid interface were used to investigate the effects of PYRRO-C3D in the presence of host tissue. Label-free liquid chromatography-mass spectrometry (LC/MS) proteomic analyses were performed to identify differentially expressed proteins following NO treatment. PYRRO-C3D specifically released NO in the presence of NTHi, while no evidence of spontaneous NO release was observed when the compound was exposed to primary epithelial cells. NTHi lacking β-lactamase activity failed to trigger NO release. Treatment significantly increased the susceptibility of in vitro NTHi biofilms to azithromycin, causing a log fold reduction (10-fold reduction or 1-log-unit reduction) in viability (P < 0.05) relative to azithromycin alone. The response was more pronounced for biofilms grown on primary respiratory epithelia, where a 2-log-unit reduction was observed (P < 0.01). Label-free proteomics showed that NO increased expression of 16 proteins involved in metabolic and transcriptional/translational functions. NO release from PYRRO-C3D enhances the efficacy of azithromycin against NTHi biofilms, putatively via modulation of NTHi metabolic activity. Adjunctive therapy with NO mediated through PYRRO-C3D represents a promising approach for reducing biofilm-associated antibiotic tolerance.
'%3e%3cpath fill='%23012169' d='M0 0v30h60V0z'/%3e%3cpath stroke='%23fff' stroke-width='6' d='m0 0 60 30m0-30L0 30'/%3e%3cpath stroke='%23C8102E' stroke-width='4' d='m0 0 60 30m0-30L0 30' clip-path='url(%23b)'/%3e%3cpath stroke='%23fff' stroke-width='10' d='M30 0v30M0 15h60'/%3e%3cpath stroke='%23C8102E' stroke-width='6' d='M30 0v30M0 15h60'/%3e%3c/g%3e%3c/svg%3e)