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.

In the African continent, the sensitization pattern and clinical profile are unknown in patients with rhinitis/rhinosinusitis attending the outpatient ear, nose, and throat (ENT) clinics. We therefore aimed to analyze the clinical characteristics of rhinitis/rhinosinusitis patients in Democratic Republic of Congo (DRC), classify allergic rhinitis (AR) according to the Allergic Rhinitis and Its Impact on Asthma criteria, and evaluate the sensitization profile and its associated factors. From January to May 2009, 423 patients with rhinitis symptoms attending the Outpatient ENT clinic of the University Hospital and Saint Joseph Hospital of Kinshasa were evaluated for allergy symptoms, severity, and duration of symptoms and underwent skin-prick tests (SPTs) for a panel of 15 allergens. Of 423 patients 35.2% had positive SPT results, with 40.9% showing polysensitization. Dermatophagoides pteronyssinus (DPT) (68.5%) and cockroach (36.2%) were the most common allergens among sensitized patients. Patients with rhinitis/rhinosinusitis mainly presented in decreasing order with sneezing, facial pain/pressure, nasal obstruction, postnasal discharge, nose itching, clear nasal discharge, and eye itching. Persistent and moderate/severe AR represented 61.4 and 69.3%, respectively. Sensitization was independently associated with younger age, rhinoconjunctivitis, and reaction to nonspecific trigger factors. In conclusion, 35.2% of patients attending the ENT Outpatient Clinic in DRC for rhinitis problems had a positive SPT to at least one allergen, with mainly DPT and cockroach allergens being involved; and a substantial portion showed persistent and moderate/severe AR. Therefore, allergy should not be neglected as an etiologic factor in rhinologic disease in the African continent.

Primary Ciliary Dyskinesia is a heterogeneous genetic disease that is characterized by cilia dysfunction of the epithelial cells lining the respiratory tracts, resulting in recurrent respiratory tract infections. Despite lifelong physiological therapy and antibiotics, the lungs of affected patients are progressively destroyed, leading to respiratory insufficiency. Recessive mutations in Dynein Axonemal Intermediate chain type 1 (DNAI1) gene have been described in 10% of cases of Primary Ciliary Dyskinesia. Our goal was to restore normal ciliary beating in DNAI1–deficient human airway epithelial cells. A lentiviral vector based on Simian Immunodeficiency Virus pseudotyped with Vesicular Stomatitis Virus Glycoprotein was used to transduce cultured human airway epithelial cells with a cDNA of DNAI1 driven by the Elongation Factor 1 promoter. Transcription and translation of the transduced gene were tested by RT–PCR and western blot, respectively. Human airway epithelial cells that were DNAI1–deficient due to compound heterozygous mutations, and consequently had immotile cilia and no outer dynein arm, were transduced by the lentivirus. Cilia beating was recorded and electron microscopy of the cilia was performed. Transcription and translation of the transduced DNAI1 gene were detected in human cells treated with the lentivirus. In addition, immotile cilia recovered a normal beat and outer dynein arms reappeared. We demonstrated that it is possible to obtain a normalization of ciliary beat frequency of deficient human airway epithelial cells by using a lentivirus to transduce cells with the therapeutic gene. This preliminary step constitutes a conceptual proof that is indispensable in the perspective of Primary Ciliary Dyskinesia's in vivo gene therapy. This is the first time that recovery of cilia beating is demonstrated in this disease.

Author Summary

This manuscript reports on a successful gene therapy attempt on human airway epithelial cells of a patient suffering from Primary Ciliary Dyskinesia. In this autosomal recessive disease, cilia of the epithelial cells that border the upper and lower respiratory tracks are not functioning. As a result, patients suffer from recurrent airway infections leading progressively to respiratory insufficiency. There is no treatment as of today that could restore normal ciliary beating. In this report, we showed that it is feasible to transfer a therapeutic gene to human airway epithelial cells with a lentivirus. This transferred gene is transcribed and expressed. Moreover, defective cells that had immotile cilia due to compound heterozygous mutations in the DNAI1 gene recovered ciliary beating after treatment with a lentivirus containing a normal DNAI1 gene. This is the first report on gene therapy in Primary Ciliary Dyskinesia. Since lentivirus is able to insert therapeutic genes into the cell genome, this result may have impact on in vivo gene therapy in this disease and in diseases related to human epithelial airway cells such as cystic fibrosis.

In animal models of asthma, interleukin-13 (IL-13) induces goblet cell metaplasia, eosinophil infiltration of the bronchial mucosa, and bronchial hyperreactivity, but the basis of its effects on airway epithelia remain unknown. Lesions of the epithelial barrier, frequently observed in asthma and other chronic lung inflammatory diseases, are repaired through proliferation, migration, and differentiation of epithelial cells. An inflammatory process may then, therefore, influence epithelial regeneration. We have thus investigated the effect of IL-13 on mucociliary differentiation of human nasal epithelial cells in primary culture. We show that IL-13 alters ciliated cell differentiation and increases the proportion of secretory cells. IL-13 downregulates the actin-binding protein ezrin and other cytoskeletal components. IL-13 also impairs lateral cell contacts and interferes with the apical localization of ezrin seen in differentiated ciliated cells. In addition, an IL-4 antagonistic mutant protein (Y124D), which binds to the IL-4 receptor α subunit, a common chain of IL-4 and IL-13 receptors, inhibits IL-13’s effects. IL-13 also decreases ciliary beat frequency in a time- and dose-dependent manner. These results suggest that, in human allergic asthmatic responses, IL-13 affects both ciliated and secretory cell differentiation, leading to airway damage and obstruction.

Background

Immotile cilia syndrome (ICS) or primary ciliary dyskinesia (PCD) is an autosomal recessive disorder in humans in which the beating of cilia and sperm flagella is impaired. Ciliated epithelial cell linings are present in many tissues. To understand ciliary assembly and motility, it is important to isolate those genes involved in the process.

Results

Total RNA was isolated from cultured ciliated nasal epithelial cells after in vitro ciliogenesis and expressed sequenced tags (ESTs) were generated. The functions and locations of 63 of these ESTs were derived by BLAST from two public databases. These ESTs are grouped into various classes. One group has high homology not only with the mitochondrial genome but also with one or more chromosomal DNAs, suggesting that very similar genes, or genes with very similar domains, are expressed from both mitochondrial and nuclear DNA. A second class comprises genes with complete homology with part of a known gene, suggesting that they are the same genes. A third group has partial homology with domains of known genes. A fourth group, constituting 33% of the ESTs characterized, has no significant homology with any gene or EST in the database.

Conclusions

We have shown that sufficient information about the location of ESTs could be derived electronically from the recently completed human genome sequences. This strategy of EST localization should be significantly useful for mapping and identification of new genes in the forthcoming human genome sequences with the vast number of ESTs in the dbEST database.