Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder caused by cilia and sperm dysmotility. About 12% of cases show perturbed 9+2 microtubule cilia structure and inner dynein arm (IDA) loss, historically termed “radial spoke defect.” We sequenced CCDC39 and CCDC40 in 54 “radial spoke defect” families, as these are the two genes identified so far to cause this defect. We discovered biallelic mutations in a remarkable 69% (37/54) of families, including identification of 25 (19 novel) mutant alleles (12 in CCDC39 and 13 in CCDC40). All the mutations were nonsense, splice, and frameshift predicting early protein truncation, which suggests this defect is caused by “null” alleles conferring complete protein loss. Most families (73%; 27/37) had homozygous mutations, including families from outbred populations. A major putative hotspot mutation was identified, CCDC40 c.248delC, as well as several other possible hotspot mutations. Together, these findings highlight the key role of CCDC39 and CCDC40 in PCD with axonemal disorganization and IDA loss, and these genes represent major candidates for genetic testing in families affected by this ciliary phenotype. We show that radial spoke structures are largely intact in these patients and propose this ciliary ultrastructural abnormality be referred to as “IDA and microtubular disorganisation defect,” rather than “radial spoke defect.”

Background and Aims: In addition to its crucial role in dampening tissue-damaging immune responses in the gut, transforming growth factor β (TGFβ) is a potent profibrogenic agent inducing collagen synthesis and regulating the balance between matrix-degrading matrix metalloproteinases (MMPs) and their inhibitors (TIMPs). TGFβ signalling was investigated by analysis of Smad proteins and MMPs/TIMPs in the mucosa overlying strictures in patients with Crohn’s disease (CD). Methods: Specimens were collected from macroscopically normal mucosa overlying strictured and non-strictured gut of patients with fibrostenosing CD. Isolated myofibroblasts were cultured with anti-TGFβ blocking antibody or TGFβ1. TGFβ transcripts were analysed by quantitative reverse transcription-PCR (RT-PCR). Smad proteins and MMPs were determined by immunoblotting. MMP-12 activity was measured by a real-time MMP-12 activity assay. An in vitro wound-healing scratch assay was used to assess myofibroblast migration. Results: TGFβ transcripts, phosphorylated Smad2–Smad3 (pSmad2–3) and TIMP-1 proteins were higher in mucosa overlying strictures than in mucosa overlying non-strictured areas. In contrast, mucosa overlying strictured gut had lower expression of Smad7, MMP-12 and MMP-3. Myofibroblasts from mucosa overlying strictured gut showed higher TGFβ transcripts, a greater pSmad2–3 response to TGFβ, increased TIMP-1, lower Smad7, increased collagen production and reduced migration ability compared with myofibroblasts from mucosa overlying non-strictured gut. TGFβ blockade increased myofibroblast MMP-12 production and migration, more obviously in myofibroblasts isolated from mucosa overlying non-strictured compared with strictured gut. Conclusions: Changes in TGF-β signalling and MMP production were identified in the mucosa overlying strictures in CD which may give a window into the process of fibrosis.

Nitric oxide is continually synthesised in the respiratory epithelium and is upregulated in response to infection or inflammation. Primary ciliary dyskinesia (PCD) is characterised by recurrent sinopulmonary infections due to impaired mucociliary clearance. Despite chronic infections, nasal nitric oxide in such patients is markedly reduced and is used as a screening test for this condition. These low levels were first described >15 yrs ago but the underlying mechanisms have yet to be fully elucidated. We review epithelial nitric oxide synthesis, release and measurement in the upper airways with particular reference to PCD. The key hypotheses that have been proposed to explain the low nitric oxide levels in this condition are explored and the potential benefits of augmenting airway nitric oxide levels are considered. Further work in these patients clarifying both whether the respiratory epithelium is able to biosynthesise normal levels of nitric oxide and the role played by abnormalities in the anatomy of the paranasal sinuses is essential. While nitric oxide augmentation is unlikely to be beneficial in common PCD phenotypes, it has potential in the treatment of secondary dyskinesias and may also improve treatment of bacterial infections, particularly where biofilms are implicated.

The diagnosis of primary ciliary dyskinesia (PCD) requires the analysis of ciliary function and ultrastructure. Diagnosis can be complicated by secondary effects on cilia such as damage during sampling, local inflammation or recent infection. To differentiate primary from secondary abnormalities, re-analysis of cilia following culture and re-differentiation of epithelial cells at an air-liquid interface (ALI) aids the diagnosis of PCD. However changes in ciliary beat pattern of cilia following epithelial cell culture has previously been described, which has brought the robustness of this method into question. This is the first systematic study to evaluate ALI culture as an aid to diagnosis of PCD in the light of these concerns. We retrospectively studied changes associated with ALI-culture in 158 subjects referred for diagnostic testing at two PCD centres. Ciliated nasal epithelium (PCD n = 54; non-PCD n = 111) was analysed by high-speed digital video microscopy and transmission electron microscopy before and after culture. Ciliary function was abnormal before and after culture in all subjects with PCD; 21 PCD subjects had a combination of static and uncoordinated twitching cilia, which became completely static following culture, a further 9 demonstrated a decreased ciliary beat frequency after culture. In subjects without PCD, secondary ciliary dyskinesia was reduced. The change to ciliary phenotype in PCD samples following cell culture does not affect the diagnosis, and in certain cases can assist the ability to identify PCD cilia.