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1.  CCDC39 is required for assembly of inner dynein arms and the dynein regulatory complex and for normal ciliary motility in humans and dogs 
Nature genetics  2010;43(1):72-78.
Primary ciliary dyskinesia (PCD) is an inherited disorder characterized by recurrent infections of the upper and lower respiratory tract, reduced fertility in males and situs inversus in about 50% of affected individuals (Kartagener syndrome). It is caused by motility defects in the respiratory cilia that are responsible for airway clearance, the flagella that propel sperm cells and the nodal monocilia that determine left-right asymmetry1. Recessive mutations that cause PCD have been identified in genes encoding components of the outer dynein arms, radial spokes and cytoplasmic pre-assembly factors of axonemal dyneins, but these mutations account for only about 50% of cases of PCD. We exploited the unique properties of dog populations to positionally clone a new PCD gene, CCDC39. We found that loss-of-function mutations in the human ortholog underlie a substantial fraction of PCD cases with axonemal disorganization and abnormal ciliary beating. Functional analyses indicated that CCDC39 localizes to ciliary axonemes and is essential for assembly of inner dynein arms and the dynein regulatory complex.
doi:10.1038/ng.726
PMCID: PMC3509786  PMID: 21131972
2.  Quantitative analysis of ciliary beating in primary ciliary dyskinesia: a pilot study 
Background
Primary ciliary dyskinesia (PCD) is a rare congenital respiratory disorder characterized by abnormal ciliary motility leading to chronic airway infections. Qualitative evaluation of ciliary beat pattern based on digital high-speed videomicroscopy analysis has been proposed in the diagnosis process of PCD. Although this evaluation is easy in typical cases, it becomes difficult when ciliary beating is partially maintained. We postulated that a quantitative analysis of beat pattern would improve PCD diagnosis. We compared quantitative parameters with the qualitative evaluation of ciliary beat pattern in patients in whom the diagnosis of PCD was confirmed or excluded.
Methods
Nasal nitric oxide measurement, nasal brushings and biopsies were performed prospectively in 34 patients with suspected PCD. In combination with qualitative analysis, 12 quantitative parameters of ciliary beat pattern were determined on high-speed videomicroscopy recordings of beating ciliated edges. The combination of ciliary ultrastructural abnormalities on transmission electron microscopy analysis with low nasal nitric oxide levels was the “gold standard” used to establish the diagnosis of PCD.
Results
This “gold standard” excluded PCD in 15 patients (non-PCD patients), confirmed PCD in 10 patients (PCD patients) and was inconclusive in 9 patients. Among the 12 parameters, the distance traveled by the cilium tip weighted by the percentage of beating ciliated edges presented 96% sensitivity and 95% specificity. Qualitative evaluation and quantitative analysis were concordant in non-PCD patients. In 9/10 PCD patients, quantitative analysis was concordant with the “gold standard”, while the qualitative evaluation was discordant with the “gold standard” in 3/10 cases. Among the patients with an inconclusive “gold standard”, the use of quantitative parameters supported PCD diagnosis in 4/9 patients (confirmed by the identification of disease-causing mutations in one patient) and PCD exclusion in 2/9 patients.
Conclusions
When the beat pattern is normal or virtually immotile, the qualitative evaluation is adequate to study ciliary beating in patients suspected for PCD. However, when cilia are still beating but with moderate alterations (more than 40% of patients suspected for PCD), quantitative analysis is required to precise the diagnosis and can be proposed to select patients eligible for TEM.
doi:10.1186/1750-1172-7-78
PMCID: PMC3562218  PMID: 23057704
Cilia; Electron microscopy; High-speed videomicroscopy; Kartagener syndrome; Nitric oxide
3.  Otologic features in children with primary ciliary dyskinesia 
OBJECTIVE
To evaluate otologic features in children with primary ciliary dyskinesia (PCD).
DESIGN
retrospective study
SETTING
pediatric referral center
PATIENTS
58 PCD patients, distributed in four age-groups (I: 0–5 years; II: 6–11 years; III: 12–17 years and IV: above 18 years with 47, 50, 34 and 10 cases, respectively). Follow-up: 2 to 6 years in each age-group. Ultrastructural defects of outer or inner dynein arms, and central complex (CC): 33, 13 and 11 cases, respectively.
MAIN OUTCOME MEASURES
Frequency of: acute otitis media (AOM), otitis media with effusion (OME), otorrhea, chronic otitis media, hearing loss, middle ear surgery and type of antibiotic regimen according to age and type of defect.
RESULTS
Recurrent AOM decreased from group I (68%) to group IV (0%), p<0.00001. OME was more severe in groups I to III than in group IV, p=0.02. Otorrhea decreased in group IV: 30% versus 80% in other groups, p<0.001. One half of patients with tubes eventually had tympanic perforation. Hearing loss was moderate in groups I to III and mild in group IV. Continuous antibiotics could be slightly reduced only in group IV. CC defect was a significant marker of severity for all of these criteria.
CONCLUSIONS
Despite continuous antibiotics, the middle ear condition in PCD remained severe throughout childhood with improvement only after the age of 18 years. Grommet placement failed to improve the middle ear condition. CC defect is a marker of severity.
doi:10.1001/archoto.2010.183
PMCID: PMC3307375  PMID: 21079168
Adolescent; Anti-Bacterial Agents; administration & dosage; Audiometry; Bronchiectasis; etiology; Chi-Square Distribution; Child; Child, Preschool; Female; Hearing Loss; etiology; Humans; Infant; Kartagener Syndrome; complications; Male; Otitis Media; etiology; Retrospective Studies; cilia; Kartagener syndrome; serous otitis media; conductive hearing loss
4.  CC2D2A mutations in Meckel and Joubert syndromes indicate a genotype-phenotype correlation 
Human mutation  2009;30(11):1574-1582.
The Meckel syndrome (MKS) is a lethal fetal disorder characterized by diffuse renal cystic dysplasia, polydactyly, a brain malformation that is usually occipital encephalocele and/or vermian agenesis, with intrahepatic biliary duct proliferation. Joubert syndrome (JBS) is a viable neurological disorder with a characteristic “molar tooth sign” (MTS) on axial images reflecting cerebellar vermian hypoplasia/dysplasia. Both conditions are classified as ciliopathies with an autosomal recessive mode of inheritance. Allelism of MS and JBS has been reported for TMEM67/MKS3, CEP290/MKS4, and RPGRIP1L/MKS5. Recently, one homozygous splice mutation with a founder effect was reported in the CC2D2A gene in Finnish fetuses with MKS, defining the 6th locus for MKS. Shortly thereafter, CC2D2A mutations were reported in JBS also. The analysis of the CC2D2A gene in our series of MKS fetuses, identified 14 novel truncating mutations in 11 cases. These results confirm the involvement of CC2D2A in MKS and reveal a major contribution of CC2D2A to the disease. We also identified three missense CC2D2A mutations in two JBS cases. Therefore and in accordance with the data reported regarding RPGRIP1L, our results indicate phenotype-genotype correlations, as missense and presumably hypomorphic mutations lead to JBS while all null alleles lead to MKS.
doi:10.1002/humu.21116
PMCID: PMC2783384  PMID: 19777577
Meckel-Gruber syndrome; MKS; Joubert syndrome; JBS; CC2D2A; ciliopathy
5.  Effect of neutrophil elastase and its inhibitor EPI-hNE4 on transepithelial sodium transport across normal and cystic fibrosis human nasal epithelial cells 
Respiratory Research  2010;11(1):141.
Background
Hyperactivity of the epithelial sodium (Na+) channel (ENaC) and increased Na+ absorption by airway epithelial cells leading to airway surface liquid dehydration and impaired mucociliary clearance are thought to play an important role in the pathogenesis of cystic fibrosis (CF) pulmonary disease. In airway epithelial cells, ENaC is constitutively activated by endogenous trypsin-like serine proteases such as Channel-Activating Proteases (CAPs). It was recently reported that ENaC activity could also be stimulated by apical treatment with human neutrophil elastase (hNE) in a human airway epithelial cell line, suggesting that hNE inhibition could represent a novel therapeutic approach for CF lung disease. However, whether hNE can also activate Na+ reabsorption in primary human nasal epithelial cells (HNEC) from control or CF patients is currently unknown.
Methods
We evaluated by short-circuit current (Isc) measurements the effects of hNE and EPI-hNE4, a specific hNE inhibitor, on ENaC activity in primary cultures of HNEC obtained from control (9) and CF (4) patients.
Results
Neither hNE nor EPI-hNE4 treatments did modify Isc in control and CF HNEC. Incubation with aprotinin, a Kunitz-type serine protease inhibitor that blocks the activity of endogenous CAPs, decreased Isc by 27.6% and 54% in control and CF HNEC, respectively. In control and CF HNEC pretreated with aprotinin, hNE did significantly stimulate Isc, an effect which was blocked by EPI-hNE4.
Conclusions
These results indicate that hNE does activate ENaC and transepithelial Na+ transport in both normal and CF HNEC, on condition that the activity of endogenous CAPs is first inhibited. The potent inhibitory effect of EPI-hNE4 on hNE-mediated ENaC activation observed in our experiments highlights that the use of EPI-hNE4 could be of interest to reduce ENaC hyperactivity in CF airways.
doi:10.1186/1465-9921-11-141
PMCID: PMC2959028  PMID: 20932306
6.  Muco-ciliary differentiation of nasal epithelial cells is decreased after wound healing in vitro 
Allergy  2009;64(8):1136-1143.
Epithelial damage and modifications of cell differentiation are frequent in airway diseases with chronic inflammation, in which Transforming Growth Factor-β1 (TGF-β1) plays an important role. The aim of this study was to evaluate the differentiation of human nasal epithelial cells (HNEC) after wound healing and the potential effects of TGF-β1.
Basal, mucous and ciliated cells were characterized by cytokeratin-14, MUC5AC and βIVtubulin immunodetection, respectively. Their expression was evaluated in situ in nasal polyps and in an in vitro model of wound healing in primary cultures of HNEC after wound closure, under basal conditions and after TGF-β1 supplementation. Using RT-PCR, the effects of TGF-β1 on MUC5AC and DNAI1 genes, specifically transcribed in mucous and ciliated cells, were evaluated.
In situ, high TGF-β1 expression was associated with low MUC5AC and βIVtubulin expression. In vitro, under basal conditions, MUC5AC expression remained stable, cytokeratin-14 expression was strong and decreased with time, while βIV tubulin expression increased. TGF-β1 supplementation down-regulated MUC5AC and βIV tubulin expression as well as MUC5AC and DNAI1 transcripts.
After a wound, differentiation into mucous and ciliated cells was possible and partially inhibited in vitro by TGF-β1, a cytokine that may be involved in epithelial remodeling observed in chronic airway diseases.
doi:10.1111/j.1398-9995.2009.02003.x
PMCID: PMC2846321  PMID: 19245428
Axonemal Dyneins; Cell Differentiation; drug effects; Cells, Cultured; Cilia; metabolism; Down-Regulation; Dyneins; metabolism; Epithelial Cells; cytology; metabolism; Humans; Keratin-14; metabolism; Mucin 5AC; genetics; metabolism; Mucins; metabolism; Nasal Mucosa; cytology; metabolism; Nasal Polyps; metabolism; pathology; Transforming Growth Factor beta1; metabolism; pharmacology; Tubulin; metabolism; Wound Healing; ciliated cells; human nasal epithelial differentiation; mucous cells; TGF-beta 1; wound healing
7.  Phagocytosis of Aspergillus fumigatus conidia by primary nasal epithelial cells in vitro 
BMC Microbiology  2008;8:97.
Background
Invasive aspergillosis, which is mainly caused by the fungus Aspergillus fumigatus, is an increasing problem in immunocompromised patients. Infection occurs by inhalation of airborne conidia, which are first encountered by airway epithelial cells. Internalization of these conidia into the epithelial cells could serve as a portal of entry for this pathogenic fungus.
Results
We used an in vitro model of primary cultures of human nasal epithelial cells (HNEC) at an air-liquid interface. A. fumigatus conidia were compared to Penicillium chrysogenum conidia, a mould that is rarely responsible for invasive disease. Confocal microscopy, transmission electron microscopy, and anti-LAMP1 antibody labeling studies showed that conidia of both species were phagocytosed and trafficked into a late endosomal-lysosomal compartment as early as 4 h post-infection. In double immunolabeling experiments, the mean percentage of A. fumigatus conidia undergoing phagocytosis 4 h post-infection was 21.8 ± 4.5%. Using combined staining with a fluorescence brightener and propidium iodide, the mean rate of phagocytosis was 18.7 ± 9.3% and the killing rate 16.7 ± 7.5% for A. fumigatus after 8 h. The phagocytosis rate did not differ between the two fungal species for a given primary culture. No germination of the conidia was observed until 20 h of observation.
Conclusion
HNEC can phagocytose fungal conidia but killing of phagocytosed conidia is low, although the spores do not germinate. This phagocytosis does not seem to be specific to A. fumigatus. Other immune cells or mechanisms are required to kill A. fumigatus conidia and to avoid further invasion.
doi:10.1186/1471-2180-8-97
PMCID: PMC2440385  PMID: 18564423
8.  Mutations of DNAI1 in Primary Ciliary Dyskinesia 
Rationale: Primary ciliary dyskinesia (PCD) is a rare, usually autosomal recessive, genetic disorder characterized by ciliary dysfunction, sino-pulmonary disease, and situs inversus. Disease-causing mutations have been reported in DNAI1 and DNAH5 encoding outer dynein arm (ODA) proteins of cilia.
Objectives: We analyzed DNAI1 to identify disease-causing mutations in PCD and to determine if the previously reported IVS1+2_3insT (219+3insT) mutation represents a “founder” or “hot spot” mutation.
Methods: Patients with PCD from 179 unrelated families were studied. Exclusion mapping showed no linkage to DNAI1 for 13 families; the entire coding region was sequenced in a patient from the remaining 166 families. Reverse transcriptase–polymerase chain reaction (RT-PCR) was performed on nasal epithelial RNA in 14 families.
Results: Mutations in DNAI1 including 12 novel mutations were identified in 16 of 179 (9%) families; 14 harbored biallelic mutations. Deep intronic splice mutations were not identified by reverse transcriptase–polymerase chain reaction. The prevalence of mutations in families with defined ODA defect was 13%; no mutations were found in patients without a defined ODA defect. The previously reported IVS1+2_3insT mutation accounted for 57% (17/30) of mutant alleles, and marker analysis indicates a common founder for this mutation. Seven mutations occurred in three exons (13, 16, and 17); taken together with previous reports, these three exons are emerging as mutation clusters harboring 29% (12/42) of mutant alleles.
Conclusions: A total of 10% of patients with PCD are estimated to harbor mutations in DNAI1; most occur as a common founder IVS1+2_3insT or in exons 13, 16, and 17. This information is useful for establishing a clinical molecular genetic test for PCD.
doi:10.1164/rccm.200603-370OC
PMCID: PMC2648054  PMID: 16858015
cilia; dynein; dextrocardia; Kartagener syndrome; mutation
9.  Verruculogen associated with Aspergillus fumigatus hyphae and conidia modifies the electrophysiological properties of human nasal epithelial cells 
BMC Microbiology  2007;7:5.
Background
The role of Aspergillus fumigatus mycotoxins in the colonization of the respiratory tract by conidia has not been studied extensively, even though patients at risk from invasive aspergillosis frequently exhibit respiratory epithelium damage. In a previous study, we found that filtrates of A. fumigatus cultures can specifically alter the electrophysiological properties of human nasal epithelial cells (HNEC) compared to those of non pathogenic moulds.
Results
We fractionated the organic phase of filtrate from 3-day old A. fumigatus cultures using high-performance liquid chromatography. The different fractions were tested for their ability to modify the electrophysiological properties of HNEC in an in vitro primary culture model.
The fraction collected between 20 and 30 min mimicked the effects of the whole filtrate, i.e. decrease of transepithelial resistance and increase of potential differences, and contained secondary metabolites such as helvolic acid, fumagillin, and verruculogen. Only verruculogen (10-8 M) had effects similar to the whole filtrate. We verified that verruculogen was produced by a collection of 67 human, animal, plant and environmental A. fumigatus isolates. Using MS-MS analysis, we found that verruculogen was associated with both mycelium and conidia extracts.
Conclusion
Verruculogen is a secondary metabolite that modifies the electrophysiological properties of HNEC. The role of these modifications in the colonization and invasion of the respiratory epithelium by A. fumigatus on first contact with the epithelium remains to be determined.
doi:10.1186/1471-2180-7-5
PMCID: PMC1797047  PMID: 17244350

Results 1-9 (9)