Reducing exacerbations in children and adults with primary ciliary dyskinesia using erdosteine and/or azithromycin therapy (REPEAT trial): study protocol for a multicentre, double-blind, double-dummy, 2×2 partial factorial, randomised controlled trial.

INTRODUCTION: Primary ciliary dyskinesia (PCD) is a rare, progressive, inherited ciliopathic disorder, which is incurable and frequently complicated by the development of bronchiectasis. There are few randomised controlled trials (RCTs) involving children and adults with PCD and thus evidence of efficacy for interventions are usually extrapolated from people with cystic fibrosis. Our planned RCT seeks to address some of these unmet needs by employing a currently prescribed (but unapproved for long-term use in PCD) macrolide antibiotic (azithromycin) and a novel mucolytic agent (erdosteine). The primary aim of our RCT is to determine whether regular oral azithromycin and erdosteine over a 12-month period reduces acute respiratory exacerbations among children and adults with PCD. Our primary hypothesis is that: people with PCD who regularly use oral azithromycin and/or erdosteine will have fewer exacerbations than those receiving the corresponding placebo medications. Our secondary aims are to determine the effect of the trial medications on PCD-specific quality-of-life (QoL) and other clinical outcomes (lung function, time-to-next exacerbation, hospitalisations) and nasopharyngeal bacterial carriage and antimicrobial resistance. METHODS AND ANALYSIS: We are currently undertaking a multicentre, double-blind, double-dummy RCT to evaluate whether 12 months of azithromycin and/or erdosteine is beneficial for children and adults with PCD. We plan to recruit 104 children and adults with PCD to a parallel, 2×2 partial factorial superiority RCT at five sites across Australia. Our primary endpoint is the rate of exacerbations over 12 months. Our main secondary outcomes are QoL, lung function and nasopharyngeal carriage by respiratory bacterial pathogens and their associated azithromycin resistance. ETHICS AND DISSEMINATION: Our RCT is conducted in accordance with Good Clinical Practice and the Australian legislation and National Health and Medical Research Council guidelines for ethical conduct of Research, including that for First Nations Australians. TRIAL REGISTRATION NUMBER: ACTRN12619000564156.

A retrospective review of Achromobacter species and antibiotic treatments in patients with primary ciliary dyskinesia.

Objectives: Primary ciliary dyskinesia (PCD) is a rare congenital disease with defective mucociliary clearance causing frequent and often persistent pulmonary infections. Achromobacter species are opportunistic pathogens renowned for the difficulty of effective treatments and deteriorating effects on lung function. We aimed to describe the occurrence, treatment, and rate of successful eradication of Achromobacter species in patients with PCD. Methods: We retrospectively reviewed 18 years of historical microbiological samples and 10 years of electronic health records for PCD patients in Denmark. Results: We included 136 patients. Twenty-six patients had isolates of Achromobacter species. On average, 5% of the cohort had at least one annual isolate. Infections became persistent in 38% with a median length of 6.6 years leading to a significant number of antibiotic treatments. Resistance toward tobramycin and ciprofloxacin was prevalent. Overall, successful eradication was achieved in 62% of patients. We found the course of lung function significantly worse during persistent Achromobacter species infection than during the two preceding years, but not different to the course in unaffected age-matched controls. Conclusion The prevalence of Achromobacter species in patients with PCD is in line with what has been reported in cystic fibrosis and can occur transiently, intermittently, or develop into a serious persistent lung infection associated with long-term antibiotic treatment.

Higher throughput drug screening for rare respiratory diseases: readthrough therapy in primary ciliary dyskinesia.

BACKGROUND: Development of therapeutic approaches for rare respiratory diseases is hampered by the lack of systems that allow medium-to-high-throughput screening of fully differentiated respiratory epithelium from affected patients. This is a particular problem for primary ciliary dyskinesia (PCD), a rare genetic disease caused by mutations in genes that adversely affect ciliary movement and consequently mucociliary transport. Primary cell culture of basal epithelial cells from nasal brush biopsies followed by ciliated differentiation at the air-liquid interface (ALI) has proven to be a useful tool in PCD diagnostics but the technique's broader utility, including in pre-clinical PCD research, has been restricted by the limited number of basal cells that can be expanded from such biopsies. METHODS: We describe an immunofluorescence screening method, enabled by extensive expansion of basal cells from PCD patients and the directed differentiation of these cells into ciliated epithelium in miniaturised 96-well transwell format ALI cultures. As proof-of-principle, we performed a personalised investigation in a patient with a rare and severe form of PCD (reduced generation of motile cilia), in this case caused by a homozygous nonsense mutation in the MCIDAS gene. RESULTS: Initial analyses of ciliary ultrastructure, beat pattern and beat frequency in the 96-well transwell format ALI cultures indicate that a range of different PCD defects can be retained in these cultures. The screening system in our proof-of-principal investigation allowed drugs that induce translational readthrough to be evaluated alone or in combination with nonsense-mediated decay inhibitors. We observed restoration of basal body formation but not the generation of cilia in the patient's nasal epithelial cells in vitro. CONCLUSION: Our study provides a platform for higher throughput analyses of airway epithelia that is applicable in a range of settings and suggests novel avenues for drug evaluation and development in PCD caused by nonsense mutations.

Rational use of mucoactive medications to treat pediatric airway disease.

Many airway diseases in children, notably bronchiolitis, cystic fibrosis (CF), non-CF bronchiectasis including primary ciliary dyskinesia, pneumonia, and severe asthma are associated with retention of airway secretions. Medications to improve secretions clearance, the mucoactive medications, are employed to treat these diseases with varying degrees of success. This manuscript reviews evidence for the use of these medications and future directions of study.

Development of bacterial resistance during treatment with topical gentamicin for chronic rhinosinusitis in patients with cystic fibrosis and primary ciliary dyskinesis. Retrospective case series.

BACKGROUND: The management of chronic rhinosinusitis (CRS) in patients with cystic fibrosis (CF) and primary ciliary dyskinesia (PCD) is still a challenge. At our institution we have used gentamycin nasal spray, extemporaneously produced, for prophylactic treatment of moderate-to-severe CRS. The aim of this study was to investigate the gentamycin susceptibility of bacteria in sputum samples in CF and PCD patients treated for CRS. METHODOLOGY: Patients with CF and PCD who were prescribed gentamycin nasal spray for CRS and had sputum bacterial cultures taken pre-treatment and followed-up at least once after ≥6 months were retrospectively included. Microbiological data were descriptively analysed in terms of bacterial species and resistance to gentamycin. RESULTS: A case series of 17 CF and 12 PCD patients passed the inclusion criteria. Of those cases, three (18%) CF patients and one (8%) PCD patient developed resistance to gentamycin during treatment with gentamycin nasal spray. In all four cases, the resistant bacterial isolates were <i>P. aeruginosa</i>. Additionally, two CF patients already had <i>P. aeruginosa </i> isolates resistant to gentamycin in the pre-treatment culture. In further two CF patients, the multi-resistant <i>Burgdorferi cepacia </i>complex, including gentamycin resistance, was identified. <i>P. aeruginosa </i> and <i>S. aureus </i> in CF and <i>P. aeruginosa</i> and <i>H. influenza </i> in PCD were the predominant bacterial species. CONCLUSIONS: The study showed that there was moderate incidence of gentamycin resistance in CF and PCD patients at our institution. However, further prospective studies are needed to confirm the outcomes.

Efficacy and safety of azithromycin maintenance therapy in primary ciliary dyskinesia (BESTCILIA): a multicentre, double-blind, randomised, placebo-controlled phase 3 trial.

BACKGROUND: Use of maintenance antibiotic therapy with the macrolide azithromycin is increasing in a number of chronic respiratory disorders including primary ciliary dyskinesia (PCD). However, evidence for its efficacy in PCD is lacking. We aimed to determine the efficacy and safety of azithromycin maintenance therapy for 6 months in patients with PCD. METHODS: The Better Experimental Screening and Treatment for Primary Ciliary Dyskinesia (BESTCILIA) trial was a multicentre, double-blind, parallel group, randomised, placebo-controlled phase 3 trial done at 6 European PCD clinics (tertiary paediatric care centres and university hospitals in Denmark, Germany, Netherlands, Switzerland, and UK). Patients with a confirmed diagnosis of PCD, aged 7-50 years old, and predicted FEV1 greater than 40% were recruited. Participants were randomly assigned (1:1), stratified by age and study site, via a web-based randomisation system to azithromycin 250 mg or 500 mg as tablets according to bodyweight (

Azithromycin is the answer in paediatric respiratory medicine, but what was the question?

The first clinical indication of non-antibiotic benefits of macrolides was in the Far East, in adults with diffuse panbronchiolitis. This condition is characterised by chronic airway infection, often with Pseudomonas aeruginosa, airway inflammation, bronchiectasis and a high mortality. Low dose erythromycin, and subsequently other macrolides, led in many cases to complete remission of the condition, and abrogated the neutrophilic airway inflammation characteristic of the disease. This dramatic finding sparked a flurry of interest in the many hundreds of macrolides in nature, especially their anti-inflammatory and immunomodulatory effects. The biggest subsequent trials of azithromycin were in cystic fibrosis, which has obvious similarities to diffuse panbronchiolitis. There were unquestionable improvements in lung function and pulmonary exacerbations, but compared to diffuse panbronchiolitis, the results were disappointing. Case reports, case series and some randomised controlled trials followed in other conditions. Three trials of azithromycin in preschool wheeze gave contradictory results; a trial in pauci-inflammatory adult asthma, and a trial in non-cystic fibrosis bronchiectasis both showed a significant reduction in exacerbations, but none matched the dramatic results in diffuse panbronchiolitis. There is clearly a huge risk of antibacterial resistance if macrolides are used widely and uncritically in the community. In summary, Azithromycin is not the answer to anything in paediatric respiratory medicine; the paediatric respiratory community needs to refocus on the dramatic benefits of macrolides in diffuse panbronchiolitis, use modern - omics technologies to determine the endotypes of inflammatory diseases and discover in nature or synthesise designer macrolides to replicate the diffuse panbronchiolitis results. We must now find out how to do better!

The effect of l-Arginine on Ciliary Beat Frequency in PCD patients, non-PCD respiratory patients and healthy controls.

OBJECTIVES: Few studies have examined the potentially therapeutic effect of increasing the production of endogenous nitric oxide (NO) in Primary Ciliary Dyskinesia (PCD) and other chronic respiratory conditions. Nasal NO is low in PCD and has been found to correlate with compromised Ciliary Beat Frequency (CBF). In this study we assessed the effect of increasing l-Arginine, as the substrate of NO synthases, on CBF in biopsies of human respiratory ciliated epithelium. METHODOLOGY: A total of 28 suspect cases with chronic respiratory manifestations referred for PCD diagnostic testing and 8 healthy controls underwent nasal brushing. Obtained epithelial cells were divided between three culture medium 199 solutions, containing different levels of l-Arginine (0.33 mM as baseline, 1 mM and 10 Mm as increased levels). CBF measurements were obtained at 37 °C and 25 °C at 1, 3 and 24 h after sample acquisition. RESULTS: Among a total of 36 recruited subjects, 8 had PCD confirmed (PCD n = 8), 20 had PCD excluded (non-PCD n = 20) and 8 were healthy controls (Healthy Controls = 8). Among PCD subjects, ciliary motility was characterized by rotational (n = 5) or dyskinetic (n = 3) beating. At 37 °C, compared to baseline, higher levels of l-Arginine resulted in up to 9% CBF increase at 1 h (p = 0.007), up to 9% CBF increase at 3 h (p < 0.001) and up to 12% CBF increase at 24 h (p = 0.002). Similar although smaller scale increases were recorded at 25 °C. The effect of l-Arginine was time dependent (interaction p = 0.002) and was similar in PCD patients, non-PCD chronic respiratory patients and healthy controls (interaction p = 0.800). CONCLUSIONS: l-Arginine increases CBF and merits to be evaluated as a potential stimulator of mucociliary clearance in chronic respiratory conditions and congenital ciliary disorders with residual motility. Larger human studies are needed to confirm these findings.

Ciliopathy proteins regulate paracrine signaling by modulating proteasomal degradation of mediators.

Cilia are critical mediators of paracrine signaling; however, it is unknown whether proteins that contribute to ciliopathies converge on multiple paracrine pathways through a common mechanism. Here, we show that loss of cilopathy-associated proteins Bardet-Biedl syndrome 4 (BBS4) or oral-facial-digital syndrome 1 (OFD1) results in the accumulation of signaling mediators normally targeted for proteasomal degradation. In WT cells, several BBS proteins and OFD1 interacted with proteasomal subunits, and loss of either BBS4 or OFD1 led to depletion of multiple subunits from the centrosomal proteasome. Furthermore, overexpression of proteasomal regulatory components or treatment with proteasomal activators sulforaphane (SFN) and mevalonolactone (MVA) ameliorated signaling defects in cells lacking BBS1, BBS4, and OFD1, in morphant zebrafish embryos, and in induced neurons from Ofd1-deficient mice. Finally, we tested the hypothesis that other proteasome-dependent pathways not known to be associated with ciliopathies are defective in the absence of ciliopathy proteins. We found that loss of BBS1, BBS4, or OFD1 led to decreased NF-κB activity and concomitant IκBß accumulation and that these defects were ameliorated with SFN treatment. Taken together, our data indicate that basal body proteasomal regulation governs paracrine signaling pathways and suggest that augmenting proteasomal function might benefit ciliopathy patients.

A case of primary ciliary dyskinesia with pulmonary arterial hypertension responding to oral sildenafil.

Primary ciliary dyskinesia (PCD) is an autosomal recessive heterogeneous group of conditions with variable clinical findings like recurrent respiratory tract infections, bronchiectasis, situs inversus, singly or in various combinations. Development of Pulmonary arterial hypertension can be a late complication of this disease. Here we present a case of PCD with recurrent respiratory tract infections, bronchiectasis and severe PAH, who responded to treatment with Oxygen, IV broad spectrum antibiotics and oral sildenafil.

[Recombinant human DNase in conditions other than cystic fibrosis]. / Rekombinant humant deoxyribonuklease til andet end cystisk fibrose.

Recombinant human DNase (rhDNase) reduces viscosity of sputum. Effect has been documented in cystic fibrosis and postoperatively in paediatric heart disease. Single dose treatment with rhDNase in paediatric asthma has no effect. In respiratory syncytial virus infection, treatment with rhDNase may be associated with increased need for supplemental oxygen. In adults with idiopathic bronchiectasis, treatment with rhDNase leads to more pulmonary exacerbations and a greater decline in pulmonary function tests. There are no controlled studies on rhDNase in primary ciliary dyskinesia or atelectasis.

Zinc increases ciliary beat frequency in a calcium-dependent manner.

BACKGROUND: Dynamic regulation of respiratory ciliary beat frequency (CBF) is regulated by fluxes in intracellular calcium (Ca(2+)). P2X receptors (P2XR) are extracellular ATP-gated, Ca(2+)-permeable, nonselective cation channels. Zinc increases intracellular Ca(2+) in a sodium (Na(+))-free environment through activation of P2XR channels. We hypothesize that topical zinc increases CBF in a Ca(2+)-dependent fashion as a result of this mechanism. METHODS: The apical surface of mouse sinonasal air-liquid interface cultures were bathed in zinc in a Na(+)-free solution with or without Ca(2+). High-speed digital video imaging captured and analyzed CBF at a sampling rate of 100 frames/s. RESULTS: CBF significantly increased fourfold over baseline from 5.99 +/- 3.16 Hz to 22.4 +/- 4.33 Hz in the presence of zinc chloride (50 micromoles) and calcium chloride (3 mM). This effect is abolished in the presence of extracellular Na(+) and was pH dependent. CONCLUSION: Zinc stimulates CBF in the presence of Ca(2+) likely through activation of P2X receptors. Thus, zinc represents a promising agent for stimulation of mucociliary clearance.

[A new breath actuated dry powder inhaler (Auto-Jethaler)]. / Ein neuer atemzuggesteuerter Pulverinhalator (Auto-Jethaler).

BACKGROUND: The aim was to examine the handling of the recently developed breath actuated dry powder inhaler Auto-Jethaler (PulmoTec GmbH/Höchstädt, launch by Ratiopharm and CT Berlin). METHOD: 75 patients suffering from asthma, cystic fibrosis or primary ciliary dysfunction (age: 3 to 34 years; 29 female, 46 male) with mild or moderate bronchial obstruction took part in the study. Lung function testing including body plethysmography was performed to measure bronchial obstruction independent of effort. Peak Inspiratory Flow (PIF) was measured using a Fleisch pneumotachograph equipped with or without a Auto-Jethaler. Instead of the commercially available drug tablet a stainless steal ring device of equivalent resistance, easy to disinfect, was used. Actuation of the rotation mechanism which was triggered by inspiration at an inspiratory flow of about 40 L/min was accompanied by a rattling noise. Aims of the study were to examine, whether the subjects were able to handle the new device, and to measure PIF without and via Auto-Jethaler. RESULTS: Handling of the Auto-Jethaler was found to be easy. All patients managed to reach or surpass the critical value of 40 L/min, even those with mild to moderate bronchial obstruction. PIF without Jethaler was 85 to 599 L/min, via Auto-Jethaler 40 to 215 L/min. PIF was significantly age dependent (p<0.001). CONCLUSIONS: These findings suggest that the Auto-Jethaler will be an appropriate device for drug administration in children older than 3 years provided that they understand the inspiratory breathing manoeuvre.

A prospective study of respiratory ciliary structure and function after stem cell transplantation.

We prospectively investigated the morphological and ciliary function abnormalities in 19 consecutive Chinese patients undergoing hemopoietic stem cell transplantation (HSCT) and studied their relationship with pulmonary complications. The percentage of structural ciliary abnormalities preceding HSCT was comparable to normal controls, but increased up to 1-year post-HSCT. However, the abnormalities did not correlate with ciliary or pulmonary function. Ciliary beat frequency (CBF) for patients undergoing autologous and allogeneic SCT was lower than that of matched controls, with a further decline at one year. There was, however, no significant change in pulmonary function for the whole cohort. There was considerable variation in CBF and ciliary abnormalities in all cases during 3-month interval assessments. Regular ciliary assessment did not predict the only two patients who eventually suffered from bronchiolitis obliterans (BO). We conclude that structural and functional ciliary abnormalities are common in recipients of HSCT, and predict post-HSCT deterioration. However, there is no evidence to show that CBF monitoring may be of prospective benefit.

A novel in vitro model for screening and evaluation of anti-asthenopia drugs.

Patients suffering asthenopia are steadily increasing with an expanding use of visual display terminals such as computers. An attempt was made to develop an in vitro model for asthenopia. Ciliary muscle removed from eyeballs of a rabbit was stimulated with acethylcholine, resulting in contraction of the muscle. Repeated stimulations caused decreased contraction, which may be related to fatiguing of ciliary muscle and hence asthenopia. Treatment of the repeatedly stimulated muscle with cyanocobalamin restored contraction dose-dependently. Thus, the model developed in this study can be used to screen drug candidates for treating asthenopia.

[A case of immotile-dyskinetic cilia syndrome responding to clenbuterol hydrochloride and azithromycin].

In this case, a 30-year-old man had been treated for chronic sinusitis and bronchiectasis since 2000, and presented at our outpatient clinic in May 2001 with chief complaints of massive yellow sputum expectoration and dyspnea. After he was admitted by our hospital, Pseudomonas aeruginosa bacteria were isolated at the rate of 10(8)/ml from his sputum culture. In electron-microscopic observation, the cilia of the bronchial epithelium were found to lack dynein arms. Semen examination revealed decreased sperm motility. Thus, the following diagnosis was made: diffuse bronchiectasis associated with the immotile-dyskinetic cilia syndrome, complicated with a P. aeruginosa infection. After the airway infection was ameliorated, 40 mg/day of clenbuterol hydrochloride was administered in combination with 250 mg of azithromycin, which was given twice a week, and which led to a markedly decreased frequency of exacerbation of airway infection. Moreover, chest CT scanning and respiratory function testing also indicated improvements. It was hypothesized that the decreased cilia motility due to P. aeruginosa-produced pyocyanin would be ameliorated with a b2 stimulant, and the inhibitory effect of a macrolide on the P. aeruginosa biofilm and production of pyocyanin would also be involved in the improvement of this case.