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Background: Primary ciliary dyskinesia (PCD) is a rare genetic disorder caused by insufficient mucociliary clearance leading to chronic airway infections. The diagnostic guideline of the European Respiratory Society (ERS) primarily recommends the evaluation of the clinical history (e. g. by the PICADAR prediction tool), nasal nitric oxide (nNO) production rate measurements, high-speed videomicroscopy analysis (HSVMA) of ciliary beating, and the assessment of ciliary axonemes via transmission electron microscopy (TEM). Genetic testing can be implemented as a last step., Question: In this study, we aimed to characterise PCD with a defective C1d projection of the ciliary central apparatus (CA) and evaluated the applicability of the ERS diagnostic guideline to this PCD type., Methods: Using a high-throughput sequencing approach of genes encoding C1d components, we identified pathogenic variants in the novel PCD genes CFAP46 and CFAP54 , and the known PCD gene CFAP221 . To fully assess this PCD type, we also analysed individuals with pathogenic variants in CFAP74 ., Results: Careful evaluation revealed that C1d-defective PCD is associated with normal situs composition, normal nNO-production rates, normal ciliary ultrastructure by TEM, and normal ciliary beating by HSVMA. Despite chronic respiratory disease, PICADAR does not reliably detect this PCD type. However, we could show by in-vitro ciliary transport assays that affected individuals exhibit insufficient ciliary clearance., Conclusions: Overall, this study extends the spectrum of PCD genes and highlights that C1d-defective PCD individuals elude diagnosis in the current diagnostic algorithm. To enable diagnosis, genetic testing should be prioritised in future diagnostic guidelines., (Copyright ©The authors 2024. For reproduction rights and permissions contact permissions@ersnet.org.)

Rationale: Primary ciliary dyskinesia (PCD) is a heterogeneous, multisystem disorder characterized by defective ciliary beating. Diagnostic guidelines of the American Thoracic Society and European Respiratory Society recommend measurement of nasal nitric oxide (nNO) for PCD diagnosis. Several studies demonstrated low nNO production rates in PCD individuals, but underlying causes remain elusive. Objectives: To determine nNO production rates in a well-characterized PCD cohort, including subgroup analyses with regard to ultrastructural and ciliary beating phenotypes. Methods: This study included 301 individuals assessed according to European Respiratory Society guidelines. Diagnostic cutoffs for nNO production rates for this study cohort and subgroups with normal and abnormal ultrastructure were determined. Diagnostic accuracy was also tested for the widely used 77 nl/min cutoff in this study cohort. The relationship between nNO production rates and ciliary beat frequencies (CBFs) was evaluated. Results: The study cohort comprised 180 individuals with definite PCD diagnosis, including 160 individuals with genetic diagnosis, 16 individuals with probable PCD diagnosis, and 105 disease controls. The 77 nl/min nNO cutoff showed a test sensitivity of 0.92 and specificity of 0.86. Test sensitivity was lower (0.85) in the subgroup of 47 PCD individuals with normal ultrastructure compared with 133 PCD individuals with abnormal ultrastructure (0.95). The optimal diagnostic cutoff for the nNO production rate for the whole study cohort was 69.8 nl/min (sensitivity, 0.92; specificity, 0.89); however, it was 107.8 nl/min (sensitivity, 0.89; specificity, 0.78) for the subgroup of PCD with normal ultrastructure. PCD individuals with normal ultrastructure compared with abnormal ultrastructure showed higher ciliary motility. Consistently, PCD individuals with higher CBFs showed higher nNO production rates. In addition, laterality defects occurred less frequently in PCD with normal ultrastructure. Conclusions: Measurements of nNO below the widely used 77 nl/min cutoff are less sensitive in detecting PCD individuals with normal ultrastructure. Our findings indicate that higher nNO production in this subgroup with a higher cutoff for the nNO production rate (107.8 nl/min) and higher residual ciliary motility is dependent on the underlying molecular PCD defect. Higher nNO production rates, higher residual CBFs, and the lower prevalence of laterality defects hamper diagnosis of PCD with normal ultrastructure. Adjusting the cutoff of nNO production rate to 107.8 nl/min might promote diagnosing PCD with normal ultrastructure.

The hyperoxia-induced pro-inflammatory response and tissue damage constitute pivotal steps leading to bronchopulmonary dysplasia (BPD) in the immature lung. The pro-inflammatory cytokines are considered attractive candidates for a directed intervention but the complex interplay between inflammatory and developmental signaling pathways requires a comprehensive evaluation before introduction into clinical trials as studied here for the death inducing ligand TRAIL. At birth and during prolonged exposure to oxygen and mechanical ventilation, levels of TRAIL were lower in tracheal aspirates of preterm infants <29 weeks of gestation which developed moderate/severe BPD. These findings were reproduced in the newborn mouse model of hyperoxic injury. The loss of TRAIL was associated with increased inflammation, apoptosis induction and more pronounced lung structural simplification after hyperoxia exposure for 7 days while activation of NFκB signaling during exposure to hyperoxia was abrogated. Pretreatment with recombinant TRAIL rescued the developmental distortions in precision cut lung slices of both wildtype and TRAIL -/- mice exposed to hyperoxia. Of importance, TRAIL preserved alveolar type II cells, mesenchymal progenitor cells and vascular endothelial cells. In the situation of TRAIL depletion, our data ascribe oxygen toxicity a more injurious impact on structural lung development. These data are not surprising taking into account the diverse functions of TRAIL and its stimulatory effects on NFκB signaling as central driver of survival and development. TRAIL exerts a protective role in the immature lung as observed for the death inducing ligand TNF-α before., (© 2022. The Author(s).)

Axonemal protein complexes, such as outer (ODA) and inner (IDA) dynein arms, are responsible for the generation and regulation of flagellar and ciliary beating. Studies in various ciliated model organisms have shown that axonemal dynein arms are first assembled in the cell cytoplasm and then delivered into axonemes during ciliogenesis. In humans, mutations in genes encoding for factors involved in this process cause structural and functional defects of motile cilia in various organs such as the airways and result in the hereditary disorder primary ciliary dyskinesia (PCD). Despite extensive knowledge about the cytoplasmic assembly of axonemal dynein arms in respiratory cilia, this process is still poorly understood in sperm flagella. To better define its clinical relevance on sperm structure and function, and thus male fertility, further investigations are required. Here we report the fertility status in different axonemal dynein preassembly mutant males (DNAAF2/ KTU, DNAAF4/ DYX1C1, DNAAF6/ PIH1D3, DNAAF7/ZMYND10, CFAP300/C11orf70 and LRRC6). Besides andrological examinations, we functionally and structurally analyzed sperm flagella of affected individuals by high-speed video- and transmission electron microscopy as well as systematically compared the composition of dynein arms in sperm flagella and respiratory cilia by immunofluorescence microscopy. Furthermore, we analyzed the flagellar length in dynein preassembly mutant sperm. We found that the process of axonemal dynein preassembly is also critical in sperm, by identifying defects of ODAs and IDAs in dysmotile sperm of these individuals. Interestingly, these mutant sperm consistently show a complete loss of ODAs, while some respiratory cilia from the same individual can retain ODAs in the proximal ciliary compartment. This agrees with reports of solely one distinct ODA type in sperm, compared to two different ODA types in proximal and distal respiratory ciliary axonemes. Consistent with observations in model organisms, we also determined a significant reduction of sperm flagellar length in these individuals. These findings are relevant to subsequent studies on the function and composition of sperm flagella in PCD patients and non-syndromic infertile males. Our study contributes to a better understanding of the fertility status in PCD-affected males and should help guide genetic and andrological counselling for affected males and their families., Competing Interests: The authors have declared that no competing interests exist.

Axonemal dynein ATPases direct ciliary and flagellar beating via adenosine triphosphate (ATP) hydrolysis. The modulatory effect of adenosine monophosphate (AMP) and adenosine diphosphate (ADP) on flagellar beating is not fully understood. Here, we describe a deficiency of cilia and flagella associated protein 45 (CFAP45) in humans and mice that presents a motile ciliopathy featuring situs inversus totalis and asthenospermia. CFAP45-deficient cilia and flagella show normal morphology and axonemal ultrastructure. Proteomic profiling links CFAP45 to an axonemal module including dynein ATPases and adenylate kinase as well as CFAP52, whose mutations cause a similar ciliopathy. CFAP45 binds AMP in vitro, consistent with structural modelling that identifies an AMP-binding interface between CFAP45 and AK8. Microtubule sliding of dyskinetic sperm from Cfap45 -/- mice is rescued with the addition of either AMP or ADP with ATP, compared to ATP alone. We propose that CFAP45 supports mammalian ciliary and flagellar beating via an adenine nucleotide homeostasis module.

Bronchopulmonary dysplasia (BPD), characterized by impaired alveolarization and vascularization in association with lung inflammation and apoptosis, often occurs after mechanical ventilation with oxygen-rich gas (MV-O2). As heightened expression of the proinflammatory cytokine TNF-α has been described in infants with BPD, we hypothesized that absence of TNF-α would reduce pulmonary inflammation, and attenuate structural changes in newborn mice undergoing MV-O2 Neonatal TNF-α null (TNF-α(-/-)) and wild type (TNF-α(+/+)) mice received MV-O2 for 8 h; controls spontaneously breathed 40% O2 Histologic, mRNA, and protein analysis in vivo were complemented by in vitro studies subjecting primary pulmonary myofibroblasts to mechanical stretch. Finally, TNF-α level in tracheal aspirates from preterm infants were determined by ELISA. Although MV-O2 induced larger and fewer alveoli in both, TNF-α(-/-) and TNF-α(+/+) mice, it caused enhanced lung apoptosis (TUNEL, caspase-3/-6/-8), infiltration of macrophages and neutrophils, and proinflammatory mediator expression (IL-1β, CXCL-1, MCP-1) in TNF-α(-/-) mice. These differences were associated with increased pulmonary transforming growth factor-β (TGF-β) signaling, decreased TGF-β inhibitor SMAD-7 expression, and reduced pulmonary NF-κB activity in ventilated TNF-α(-/-) mice. Preterm infants who went on to develop BPD showed significantly lower TNF-α levels at birth. Our results suggest a critical balance between TNF-α and TGF-β signaling in the developing lung, and underscore the critical importance of these key pathways in the pathogenesis of BPD. Future treatment strategies need to weigh the potential benefits of inhibiting pathologic cytokine expression against the potential of altering key developmental pathways., (Copyright © 2016 the American Physiological Society.)