Cell proliferation in bronchial epithelium and submucosal glands of cystic fibrosis patients.

Integrative gene therapy typically requires dividing cells. This requirement has been perceived as an impediment for gene transfer to mature, uninjured airways where proliferation rates are very low. In diseases such as cystic fibrosis (CF) that may be candidates for integrative gene therapy, airway cell turnover is not known but may be increased as a result of chronic inflammation. To determine if cells in airway surface epithelium and submucosal glands of CF patients proliferate at an increased rate, paraffin sections of bronchial segments removed from CF patients (n = 6) at the time of lung transplantation or rapid autopsy and from non-CF patients (n = 4) undergoing lung resection or transplantation were immunostained with PC10, a monoclonal antibody to proliferating cell nuclear antigen (PCNA), a marker of proliferating cells. The PCNA index (percentage of nuclei immunostaining for PCNA) in CF bronchial surface epithelium was 17.0 +/- 4.6% (mean +/- SEM), substantially greater than in non-CF airways (less than 0.2%). Within submucosal glands, PCNA-positive cells were more prevalent in the collecting ducts of CF patients than in those of normal subjects, but only rare mucous or serous cells were PCNA positive. These studies show that airway epithelial cell proliferation rates can be very high in inflamed CF airways. This prevalence of proliferating cells suggests that CF airway epithelium and submucosal gland ducts may be amenable to gene transfer using vectors, such as retroviruses, that require cell replication for stable integrative expression. Further studies are needed to evaluate cell proliferation in CF airways with less extensive airway injury

Primary ciliary dyskinesia: Recent advances in diagnostics, genetics, and characterization of clinical disease

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous recessive disorder of motile cilia that leads to oto-sino-pulmonary diseases and organ laterality defects in approximately 50% of cases. The estimated incidence of PCD is approximately 1 per 15,000 births, but the prevalence of PCD is difficult to determine, primarily because of limitations in diagnostic methods that focus on testing ciliary ultrastructure and function. Diagnostic capabilities have recently benefitted from (1) documentation of low nasal nitric oxide production in PCD and (2) discovery of biallelic mutations in multiple PCD-causing genes. The use of these complementary diagnostic approaches shows that at least 30% of patients with PCD have normal ciliary ultrastructure. More accurate identification of patients with PCD has also allowed definition of a strong clinical phenotype, which includes neonatal respiratory distress in >80% of cases, daily nasal congestion and wet cough starting soon after birth, and early development of recurrent/chronic middle-ear and sinus disease. Recent studies, using advanced imaging and pulmonary physiologic assessments, clearly demonstrate early onset of lung disease in PCD, with abnormal air flow mechanics by age 6-8 years that is similar to cystic fibrosis, and age-dependent onset of bronchiectasis. The treatment ofPCDis not standardized, and there are no validated PCD-specific therapies. Most patients with PCD receive suboptimal management, which should include airway clearance, regular surveillance of pulmonary function and respiratory microbiology, and use of antibiotics targeted to pathogens. The PCD Foundation is developing a network of clinical centers, which should improve diagnosis and management of PCD

Use of telavancin in adolescent patients with cystic fibrosis and prior intolerance to vancomycin: A case series

The most common pathogen in pediatric cystic fibrosis (CF) patients is Staphylococcus aureus, and drug-resistant species are associated with negative outcomes. Methicillin-resistant Staphylococcus aureus (MRSA) is notoriously hard to treat because many antibiotics are not FDA approved for children and drug allergies or intolerances can prohibit the use of others. Telavancin is currently indicated for hospital-acquired pneumonia and ventilator-associated pneumonia caused by MRSA, but it has not been studied in patients with CF or in pediatrics. As a semi-synthetic derivative of vancomycin, it is unknown if cross-reactivity with telavancin occurs in patients with vancomycin hypersensitivity or intolerance. In this case series, we describe three adolescent patients with CF and previous intolerance to vancomycin who received telavancin for bronchopulmonary exacerbations

Investigation of the possible role of a novel gene, DPCD, in primary ciliary dyskinesia

Primary ciliary dyskinesia (PCD) is an autosomal recessive disease caused by mutations that affect the proper function of cilia. Recently, deletion of DNA polymerase λ (Poll) in mice produced a phenotype characteristic of PCD (Kobayashi et al., 2002, Mol. Cell. Biol. 22:2769-2776). Because it is unclear how a mutation in a DNA polymerase would result in a specific defect in axonemes, the targeting construct was examined further. Analysis of the genomic region surrounding the Poll gene revealed an uncharacterized gene, named Dpcd, that is predicted to be transcribed from the opposite strand relative to Poll. The deletion of Poll would also remove the first exon of Dpcd. Because it is possible that the PCD phenotype observed is due to the absence of either gene, the expression of these genes during ciliogenesis of human airway epithelial cells was examined. Northern analysis demonstrated that DPCD expression increases during ciliated cell differentiation; the expression of POLL decreases. To examine directly whether DPCD is mutated in cases of human PCD, the complete coding sequence of DPCD was sequenced from 51 unrelated PCD patients. No disease-causing mutations were confirmed; however, one variant could not be excluded. Therefore, DPCD remains a novel candidate gene for PCD

Destruction of Staphylococcus aureus and the impact of chlortetracycline on biomethane production during anaerobic digestion of chicken manure

Research was undertaken to ascertain the effect on biogas potential during the anaerobic digestion of chicken manure containing Staphylococcus aureus and chlortetracycline (antibiotic) from infected chicken flocks. S. aureus is a pathogenic bacteria in chicken flocks that is usually treated with the broad-spectrum antibiotic, chlortetracycline. Veterinary antibiotics are often prescribed in the poultry sector for on-farm use at the flock level to control disease; consequently, significant quantities of antibiotics are excreted from the bird into the manure. Subsequent anaerobic digestion of this chicken manure could lead to pathogens and antibiotics affecting the digestion process. Anaerobic digestion biochemical methane potential assays were completed at 35°C for 39 days, with some assays receiving S. aureus and some receiving S. aureus and chlortetracycline. No viable S. aureus cells were detected after Day 0 of the experiment. A further experiment utilising an order of magnitude greater concentration of S. aureus demonstrated a significant reduction (>400 fold) in S. aureus within 24 h when inoculated into anaerobic digestate, with no viable S. aureus cells detected by the end of 3 days. Furthermore, the efficacy of chlortetracycline was significantly reduced when applied to anaerobic digestate compared to water alone. Total biogas yields from chicken manure were significantly lowered by the addition of S. aureus, with and without chlortetracycline. However, there was no significant difference in methane yields between treatments. The cellulose control assays showed a lag phase in methane production after receiving chlortetracycline. In comparison, the absence of a lag phase when the antibiotic were added to chicken manure may have been due to the relatively high nitrogen content of the feedstock reducing the inhibition of chlortetracycline on methanogens. Therefore, this study demonstrates that the addition of S. aureus and chlortetracycline does not have a commercially relevant effect on the digestion of chicken manure

Relations between fusion cross sections and average angular momenta

We study the relations between moments of fusion cross sections and averages of angular momentum. The role of the centrifugal barrier and the target deformation in determining the effective barrier radius are clarified. A simple method for extracting average angular momentum from fusion cross sections is demonstrated using numerical examples as well as actual data.Comment: 16 REVTeX pages plus 8 included Postscript figures (uses the epsf macro); submitted to Phys. Rev. C; also available at http://nucth.physics.wisc.edu/preprint

Germline mutations in an intermediate chain dynein cause primary ciliary dyskinesia

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous, autosomal recessive disorder caused by abnormal ciliary ultrastructure and function, characterized clinically by otosino-pulmonary disease. Mutations in an intermediate chain dynein (DNAI1: IC78) have recently been described in PCD patients, with outer dynein arm (ODA) defects. The aims of the current study were to test for novel DNAI1 mutations in 13 PCD patients with ODA defects (from 7 unrelated families) and to assess genotype/phenotype correlations in patients and family members. A previously reported mutation (219+3insT) was detected in three PCD patients from two families. The opposite allele had the novel missense mutation G1874C (W568S) in both affected individuals from one family, and a nonsense mutation G1875A (W568X) in an affected individual from another family. The tryptophan at position 568 is a highly conserved residue in the WD-repeat region, and a mutation is predicted to lead to abnormal folding of the protein and loss of function. None of these mutations were found in 32 other PCD patients with miscellaneous ciliary defects. Mutations in DNAI1 are causative for PCD with ODA defects, and are likely the genetic origin of clinical disease in some PCD patients with ultrastructural defects in the ODA

Cytoplasmic “ciliary inclusions” in isolation are not sufficient for the diagnosis of primary ciliary dyskinesia

Background: The diagnosis of primary ciliary dyskinesia (PCD) is difficult and requires a combination of clinical features, nasal nitric oxide testing, cilia ultrastructural analysis by electron microscopy (EM), and genetics. A recently described cytoplasmic ultrastructural change termed “ciliary inclusions” was reported to be diagnostic of PCD; however, no supporting evidence of PCD was provided. In this study, we sought to confirm, or refute, the diagnosis of PCD in subjects with “ciliary inclusions” on EM. Methods: Six subjects from five families with previous lab reports of “ciliary inclusions” on EMs of ciliated cells were identified and evaluated at a Genetic Disorders of Mucociliary Clearance Consortium site. We performed a detailed clinical history, nasal nitric oxide measurement, genetic testing including whole-exome sequencing (WES), and when possible, repeat ciliary EM study. Results: Only one of six subjects had multiple and persistent clinical features congruent with PCD. No subject had situs inversus. Only one of six subjects had a very low nasal nitric oxide level. No “ciliary inclusions” were found in three subjects who had a repeat ciliary EM, and ciliary axonemal ultrastructures were normal. Genetic testing, including WES, was negative for PCD-causing genes, and for pathogenic variants in gene pathways that might cause “ciliary inclusions,” such as ciliary biogenesis. Conclusion: “Ciliary Inclusions”, in isolation, are not sufficient to diagnosis PCD. If seen, additional studies should be done to pursue an accurate diagnosis

Cryo-electron tomography reveals ciliary defects underlying human RSPH1 primary ciliary dyskinesia

Cilia play essential roles in normal human development and health; cilia dysfunction results in diseases such as primary ciliary dyskinesia (PCD). Despite their importance, the native structure of human cilia is unknown, and structural defects in the cilia of patients are often undetectable or remain elusive because of heterogeneity. Here we develop an approach that enables visualization of human (patient) cilia at high-resolution using cryo-electron tomography of samples obtained noninvasively by nasal scrape biopsy. We present the native 3D structures of normal and PCD-causing RSPH1-mutant human respiratory cilia in unprecedented detail; this allows comparisons of cilia structure across evolutionarily distant species and reveals the previously unknown primary defect and the heterogeneous secondary defects in RSPH1-mutant cilia. Our data provide evidence for structural and functional heterogeneity in radial spokes, suggest a mechanism for the milder RSPH1 PCD phenotype and demonstrate that cryo-electron tomography can be applied to human disease by directly imaging patient samples

Identification of genetic variants in CFAP221 as a cause of primary ciliary dyskinesia

Primary ciliary dyskinesia (PCD) is a rare disorder that affects the biogenesis or function of motile cilia resulting in chronic airway disease. PCD is genetically and phenotypically heterogeneous, with causative mutations identified in over 40 genes; however, the genetic basis of many cases is unknown. Using whole-exome sequencing, we identified three affected siblings with clinical symptoms of PCD but normal ciliary structure, carrying compound heterozygous loss-of-function variants in CFAP221. Computational analysis suggests that these variants are the most damaging alleles shared by all three siblings. Nasal epithelial cells from one of the subjects demonstrated slightly reduced beat frequency (16.5 Hz vs 17.7 Hz, p = 0.16); however, waveform analysis revealed that the CFAP221 defective cilia beat in an aberrant circular pattern. These results show that genetic variants in CFAP221 cause PCD and that CFAP221 should be considered a candidate gene in cases where PCD is suspected but cilia structure and beat frequency appear normal