The disease-specific clinical trial network for primary ciliary dyskinesia: PCD-CTN.

Primary ciliary dyskinesia (PCD) is a rare genetic disorder characterised by impaired mucociliary clearance leading to irreversible lung damage. In contrast to other rare lung diseases like cystic fibrosis (CF), there are only few clinical trials and limited evidence-based treatments. Management is mainly based on expert opinions and treatment is challenging due to a wide range of clinical manifestations and disease severity. To improve clinical and translational research and facilitate development of new treatments, the clinical trial network for PCD (PCD-CTN) was founded in 2020 under the framework of the European Reference Network (ERN)-LUNG PCD Core. Applications from European PCD sites interested in participating in the PCD-CTN were requested. Inclusion criteria consisted of patient numbers, membership of ERN-LUNG PCD Core, use of associated standards of care, experience in PCD and/or CF clinical research, resources to run clinical trials, good clinical practice (GCP) certifications and institutional support. So far, applications from 22 trial sites in 18 European countries have been approved, including >1400 adult and >1600 paediatric individuals with PCD. The PCD-CTN is headed by a coordinating centre and consists of a steering and executive committee, a data safety monitoring board and committees for protocol review, training and standardisation. A strong association with patient organisations and industrial companies are further cornerstones. All participating trial sites agreed on a code of conduct. As CTNs from other diseases have demonstrated successfully, this newly formed PCD-CTN operates to establish evidence-based treatments for this orphan disease and to bring new personalised treatment approaches to patients.

Lower airway clinical outcome measures for use in primary ciliary dyskinesia research: a scoping review.

OBJECTIVES: Disease-specific, well-defined and validated clinical outcome measures are essential in designing research studies. Poorly defined outcome measures hamper pooling of data and comparisons between studies. We aimed to identify and describe pulmonary outcome measures that could be used for follow-up of patients with primary ciliary dyskinesia (PCD). METHODS: We conducted a scoping review by systematically searching MEDLINE, Embase and the Cochrane Database of Systematic Reviews online databases for studies published from 1996 to 2020 that included ≥10 PCD adult and/or paediatric patients. RESULTS: We included 102 studies (7289 patients). 83 studies reported on spirometry, 11 on body plethysmography, 15 on multiple-breath washout, 36 on high-resolution computed tomography (HRCT), 57 on microbiology and 17 on health-related quality of life. Measurement and reporting of outcomes varied considerably between studies (e.g. different scoring systems for chest HRCT scans). Additionally, definitions of outcome measures varied (e.g. definition of chronic colonisation by respiratory pathogen), impeding direct comparisons of results. CONCLUSIONS: This review highlights the need for standardisation of measurements and reporting of outcome measures to enable comparisons between studies. Defining a core set of clinical outcome measures is necessary to ensure reproducibility of results and for use in future trials and prospective cohorts.

Update on Respiratory Fungal Infections in Cystic Fibrosis Lung Disease and after Lung Transplantation.

Cystic fibrosis is the most common autosomal-recessive metabolic disease in the Western world. Impaired trans-membrane chloride transport via the cystic fibrosis transmembrane conductance regulator (CFTR) protein causes thickened body fluids. In the respiratory system, this leads to chronic suppurative cough and recurrent pulmonary infective exacerbations, resulting in progressive lung damage and respiratory failure. Whilst the impact of bacterial infections on CF lung disease has long been recognized, our understanding of pulmonary mycosis is less clear. The range and detection rates of fungal taxa isolated from CF airway samples are expanding, however, in the absence of consensus criteria and univocal treatment protocols for most respiratory fungal conditions, interpretation of laboratory reports and the decision to treat remain challenging. In this review, we give an overview on fungal airway infections in CF and CF-lung transplant recipients and focus on the most common fungal taxa detected in CF, Aspergillus fumigatus, Candida spp., Scedosporium apiospermum complex, Lomentospora species, and Exophiala dermatitidis, their clinical presentations, common treatments and prophylactic strategies, and clinical challenges from a physician's point of view.

An optimized, robust and reproducible protocol to generate well-differentiated primary nasal epithelial models from extremely premature infants.

Extremely premature infants are prone to severe respiratory infections, and the mechanisms underlying this exceptional susceptibility are largely unknown. Nasal epithelial cells (NEC) represent the first-line of defense and adult-derived ALI cell culture models show promising results in mimicking in vivo physiology. Therefore, the aim of this study was to develop a robust and reliable protocol for generating well-differentiated cell culture models from NECs of extremely premature infants. Nasal brushing was performed in 13 extremely premature infants at term corrected age and in 11 healthy adult controls to obtain NECs for differentiation at air-liquid interface (ALI). Differentiation was verified using imaging and functional analysis. Successful isolation and differentiation was achieved for 5 (38.5%) preterm and 5 (45.5%) adult samples. Preterm and adult ALI-cultures both showed well-differentiated morphology and ciliary function, however, preterm cultures required significantly longer cultivation times for acquiring full differentiation (44 ± 3.92 vs. 23 ± 1.83 days; p < 0.0001). Moreover, we observed that recent respiratory support may impair successful NECs isolation. Herewithin, we describe a safe, reliable and reproducible method to generate well-differentiated ALI-models from NECs of extremely premature infants. These models provide a valuable foundation for further studies regarding immunological and inflammatory responses and respiratory disorders in extremely premature infants.

Clinical phenotype and current diagnostic criteria for primary ciliary dyskinesia.

INTRODUCTION: Primary ciliary dyskinesia (PCD) is a rare, mostly autosomal-recessive disorder of motile cilia, characterized by chronic lung disease, rhinosinusitis, hearing impairment, and subfertility. PCD is still often missed or diagnosed late since symptoms overlap with common respiratory complaints, but should be considered if one or more of the cardinal clues are present. Areas covered: We provide an overview on clinical presentations of PCD and clues for when to consider PCD, these include unexplained neonatal respiratory distress, persistent rhinitis from the first days of life, situs anomalies, or otorrhoea following tympanostomy tube insertion. Diagnosis is on the basis of clinical suspicion, and an algorithm of nasal nitric oxide, ciliary beat pattern and frequency, transmission electron microscopy, immunofluorescence of ciliary proteins and genetic studies. However, there is no one gold-standard test as yet. We reviewed the current literature based on PubMed and Ovid databases literature search. Expert commentary: There is a need for increased awareness about PCD beyond specialist respiratory clinicians and a need for standardization of PCD diagnostics internationally. Early diagnosis means that inappropriate treatment based on misdiagnosed conditions can be avoided, and the onset of bronchiectasis may be delayed.

Update on paediatric obstructive sleep apnoea.

Obstructive sleep apnoea (OSA) is one of the most common causes of sleep-disordered breathing (SDB) in children. It is associated with significant morbidity, potentially impacting on long-term neurocognitive and behavioural development, as well as cardiovascular outcomes and metabolic homeostasis. The low grade systemic inflammation and increased oxidative stress seen in this condition are believed to underpin the development of these OSA-related morbidities. The significant variance in degree of end organ morbidity in patients with the same severity of OSA highlights the importance of the interplay of genetic and environmental factors in determining the overall OSA phenotype. This review seeks to summarize the current understanding of the aetiology and mechanisms underlying OSA, its risk factors, diagnosis and treatment.

Proceedings of the COST action BM1407 inaugural conference BEAT-PCD: translational research in primary ciliary dyskinesia - bench, bedside, and population perspectives.

Primary ciliary dyskinesia (PCD) is a rare heterogenous condition that causes progressive suppurative lung disease, chronic rhinosinusitis, chronic otitis media, infertility and abnormal situs. 'Better Experimental Approaches to Treat Primary Ciliary Dyskinesia' (BEAT-PCD) is a network of scientists and clinicians coordinating research from basic science through to clinical care with the intention of developing treatments and diagnostics that lead to improved long-term outcomes for patients. BEAT-PCD activities are supported by EU Framework Programme Horizon 2020 funded COST Action (BM1407). The Inaugural Conference of BEAT-PCD was held in December 2015 in Southampton, UK. The conference attracted ninety-six scientists, clinicians, allied health professionals, industrial partners and patient representatives from twenty countries. We aimed to identify the needs for PCD research and clinical care, particularly focussing on basic science, epidemiology, diagnostic testing, clinical management and clinical trials. The multidisciplinary conference provided an interactive platform for exchanging ideas through a program of lectures, poster presentations, breakout sessions and workshops. This allowed us to develop plans for collaborative studies. In this report, we summarize the meeting, highlight developments, and discuss open questions thereby documenting ongoing developments in the field of PCD research.

IgE/FcεRI-Mediated Antigen Cross-Presentation by Dendritic Cells Enhances Anti-Tumor Immune Responses.

Epidemiologic studies discovered an inverse association between immunoglobulin E (IgE)-mediated allergies and cancer, implying tumor-protective properties of IgE. However, the underlying immunologic mechanisms remain poorly understood. Antigen cross-presentation by dendritic cells (DCs) is of key importance for anti-tumor immunity because it induces the generation of cytotoxic CD8+ T lymphocytes (CTLs) with specificity for tumor antigens. We demonstrate that DCs use IgE and FcεRI, the high-affinity IgE receptor, for cross-presentation and priming of CTLs in response to free soluble antigen at low doses. Importantly, IgE/FcεRI-mediated cross-presentation is a distinct receptor-mediated pathway because it does not require MyD88 signals or IL-12 induction in DCs. Using passive immunization with tumor antigen-specific IgE and DC-based vaccination experiments, we demonstrate that IgE-mediated cross-presentation significantly improves anti-tumor immunity and induces memory responses in vivo. Our findings suggest a cellular mechanism for the tumor-protective features of IgE and expand the known physiological functions of this immunoglobulin.

Accuracy of digital mRNA profiling of oesophageal biopsies as a novel diagnostic approach to eosinophilic oesophagitis.

BACKGROUND: Quantification of tissue eosinophils remains the golden standard in diagnosing eosinophilic oesophagitis (EoE), but this approach suffers from poor specificity. It has been recognized that histopathological changes that occur in patients with EoE are associated with a disease-specific tissue transcriptome. OBJECTIVE: We hypothesized that digital mRNA profiling targeted at a set of EoE-specific and Th2 inflammatory genes in oesophageal biopsies could help differentiate patients with EoE from those with reflux oesophagitis (RE) or normal tissue histology (NH). METHODS: The mRNA expression levels of 79 target genes were defined in both proximal and distal biopsies of 196 patients with nCounter® (Nanostring) technology. According to clinicopathological diagnosis, these patients were grouped in a training set (35 EoE, 30 RE, 30 NH) for building of a three-class prediction model using the random forest method, and a blinded predictive set (n = 47) for model validation. RESULTS: A diagnostic model built on ten differentially expressed genes was able to differentiate with 100% sensitivity and specificity between conditions in the training set. In a blinded predictive set, this model was able to correctly predict EoE in 14 of 18 patients in distal (sensitivity 78%, 95% CI 52-93%) and 16 of 18 patients in proximal biopsies (sensitivity 89%, 95% CI 64-98%), without false-positive diagnosis of EoE in RE or NH patients (specificity 100%, 95% CI 85-100%). Sensitivity was increased to 94% (95% CI 71-100%) when either the best predictive distal or proximal biopsy was used. CONCLUSION AND CLINICAL RELEVANCE: We conclude that mRNA profiling of oesophageal tissue is an accurate diagnostic strategy in detecting EoE.

Preventive sublingual immunotherapy in preschool children: first evidence for safety and pro-tolerogenic effects.

BACKGROUND: Prevention of new IgE sensitizations has been described during allergen-specific immunotherapy. However, prospective data using a preventive approach in very young children who would benefit most are missing. We initiated a prospective pilot study investigating the safety, immunomodulatory, and sensitization-preventive effect of sublingual immunotherapy (SLIT) in mono/oligoclonally sensitized, clinically asymptomatic children 2-5 yr of age. METHODS: In this double-blinded, randomized, placebo-controlled pilot study, 31 mono-/oligosensitized children to house-dust mite or grass pollen were included. SLIT with the respective source (n = 15) or placebo (n = 16) was applied. After dose-up-phase therapy was continued for 2 yr. Parents recorded clinical events, vaccinations, and drug intake in a diary. Skin prick testing and specific IgE and IgG measurements were recorded at baseline, 12 and 24 months. At the same time, allergen-specific proliferation and IL10- and TGFß-dependent Treg function were measured. RESULTS: Preventive application of SLIT in young children was safe (no relevant side effects in 21,170 single applications). After 12 and 24 months of treatment, the rate of allergen-specific sensitization (specific IgE and SPT reactivity) was comparable in the treatment and the placebo group. However, verum-treated patients displayed a significant up-regulation of allergen-specific IgG (p < 0.05). Furthermore, IL10-dependent inhibition (p < 0.05) was observed in vitro in the treatment group but not in the placebo group. CONCLUSION: Preventive SLIT is safe in children 2-5 yr of age and induces regulatory mechanisms involving allergen-specific IgG and IL10. Based on this pilot study, large-scale trials will need to investigate the modulation of sensitization and clinically relevant allergy.

A Th17- and Th2-skewed cytokine profile in cystic fibrosis lungs represents a potential risk factor for Pseudomonas aeruginosa infection.

RATIONALE: Cystic fibrosis (CF) is characterized by progressive pulmonary inflammation that is infection-triggered. Pseudomonas aeruginosa represents a risk factor for deterioration of lung function and reduced life expectancy. OBJECTIVES: To assess T-cell cytokine/chemokine production in clinically stable children with CF and evaluate the association between T-cell subtypes and susceptibility for infection with P. aeruginosa. METHODS: T-cell cytokine/chemokine profiles were measured in bronchoalveolar lavage fluid (BALF) from children with CF (n = 57; 6.1 ± 5.9 yr) and non-CF control subjects (n = 18; 5.9 ± 4.3 yr). Memory responses to Aspergillus fumigatus and P. aeruginosa were monitored. High-resolution computed tomography-based Helbich score was assessed. In a prospective observational trial the association between BALF cytokine/chemokine profiles and subsequent infection with P. aeruginosa was studied. MEASUREMENTS AND MAIN RESULTS: Th1- (INF-γ), Th2- (IL-5, IL-13), Th17- (IL-17A), and Th17-related cytokines (IL-1ß, IL-6) were significantly up-regulated in airways of patients with CF. IL-17A, IL-13, and IL-5 were significantly higher in BALF of symptomatic as compared with clinically asymptomatic patients with CF. IL-17A and IL-5 correlated with the percentage of neutrophils in BALF (r = 0.41, P < 0.05 and r = 0.46, P < 0.05, respectively). Th17- (IL-17A, IL-6, IL-1ß, IL-8) and Th2-associated cytokines and chemokines (IL-5, IL-13, TARC/CCL17), but not IFN-γ levels, significantly correlated with high-resolution computed tomography changes (Helbich score; P < 0.05). P. aeruginosa- and A. fumigatus-specific T cells from patients with CF displayed significantly higher IL-5 and IL-17A mRNA expression. IL-17A and TARC/CCL17 were significantly augmented in patients that developed P. aeruginosa infection within 24 months. CONCLUSIONS: We propose a role for Th17 and Th2 T cells in chronic inflammation in lungs of patients with CF. High concentrations of these cytokines/chemokines in CF airways precede infection with P. aeruginosa.

Fc-epsilon-RI, the high affinity IgE-receptor, is robustly expressed in the upper gastrointestinal tract and modulated by mucosal inflammation.

BACKGROUND: The role of the high affinity IgE receptor, FcεRI, in IgE-mediated immune responses of the gastrointestinal (GI) mucosa is poorly understood. Currently, a detailed characterization of FcεRI expression throughout the human gut is lacking. The aim of this study was to define the expression pattern of FcεRI in the GI tract. METHODS/PRINCIPAL FINDINGS: We compared FcεRI expression in children with gastritis/esophagitis (n = 10), celiac disease (n = 10), inflammatory bowel disease (IBD) (n = 9), and normal mucosa (n = 5). The α-subunit of FcεRI (FcεRIα), detected by immunohistochemistry, was found on cells infiltrating the mucosa of the esophagus, the stomach, and the duodenum, but was rarely detected in more distal sections of the GI tract. Accordingly, quantitative RT-PCR analysis on esophagus, stomach, duodenum, colon, and rectum biopsies revealed that FcεRIα and -ß expression levels decreased towards the distal intestine. mRNA transcripts of the common Fc-receptor-γ chain were present in the entire GI mucosa. Double-immunofluorescence staining of esophageal specimens confirmed that FcεRIα was expressed on intraepithelial mast cells and Langerhans cells. The mRNA expression levels of the α, ß, and γ subunits of FcεRI did not correlate with total serum IgE but were associated with mucosal inflammation. CONCLUSION/SIGNIFICANCE: Our data define the upper GI tract as the main site for IgE-mediated immune activation via FcεRI. Tissue mRNA levels of FcεRIα are regulated by inflammatory conditions rather than serum IgE, indicating that FcεRI might also play a role in pathologies other than allergy.

How to connect an IgE-driven response with CTL activity?

One of the goals of cell-based immune therapy in cancer is the induction of tumor-specific cytotoxic T-lymphocyte (CTL) responses. To achieve this objective, the ability of dendritic cells (DC) to cross-present tumor antigens can be exploited. One of the most efficient pathways for the induction of CTLs by cross-presentation is mediated by immunoglobulins of the IgG class, which are used by DCs to sample antigen in the form of immune complexes via Fc-gamma receptors. Could DCs use an IgE-mediated cross-presentation mechanism in a comparable manner to induce CTLs? We here discuss the potential of two human IgE Fc receptors, FcεRI and FcεRII, to serve as antigen uptake receptors for IgE-mediated cross-presentation. We conclude that the existence of an IgE-mediated cross-presentation pathway would provide a direct link between IgE-driven immune responses and CTL activity.

Development and validation of a standardized ELISA for the detection of soluble Fc-epsilon-RI in human serum.

The aim of this study was to develop a standardized enzyme-linked immunosorbent assay (ELISA) for detection of human soluble Fc-epsilon-RI (sFcεRI), a serum isoform of the high affinity IgE receptor. A recombinant version of sFcεRI was produced in baculovirus and used as standard. ELISA plates were coated with anti-mouse IgG followed by incubation with the monoclonal capture antibody CRA1. This FcεRI-alpha-specific antibody binds to the stalk region of the protein and does not inhibit IgE-binding. After incubation with standards or serum samples, plates were incubated with chimeric IgE followed by detection with horseradish peroxidase conjugated anti-human IgE. Enzymatic activity was visualized with (3,3',5,5')-tetramethylbenzidine. Specificity was demonstrated by omission of capture or detection reagents. Units (U) of detection were established and the dynamic range of the assay was defined as 10-640 U/ml for a 1/5 serum dilution. Parameters of linearity (R(2)>0.999), matrix interference test (recovery of 70-110%), intra-assay variability (coefficient of variation (CV) <20%) and inter-assay variability (CV <20%) met acceptance criteria for immunoassay validation. Correlation analysis of serum units of sFcεRI measured with the new ELISA and serum IgE levels confirmed earlier published data describing a weak correlation of the two parameters in patients with elevated serum IgE while no correlation in patients with normal serum IgE or the total patient group was found. In summary, we established and validated a standardized ELISA for the detection of sFcεRI. This novel method now allows for comparative analysis of sFcεRI levels in health and disease.

A soluble form of the high affinity IgE receptor, Fc-epsilon-RI, circulates in human serum.

Soluble IgE receptors are potential in vivo modulators of IgE-mediated immune responses and are thus important for our basic understanding of allergic responses. We here characterize a novel soluble version of the IgE-binding alpha-chain of Fc-epsilon-RI (sFcεRI), the high affinity receptor for IgE. sFcεRI immunoprecipitates as a protein of ∼40 kDa and contains an intact IgE-binding site. In human serum, sFcεRI is found as a soluble free IgE receptor as well as a complex with IgE. Using a newly established ELISA, we show that serum sFcεRI levels correlate with serum IgE in patients with elevated IgE. We also show that serum of individuals with normal IgE levels can be found to contain high levels of sFcεRI. After IgE-antigen-mediated crosslinking of surface FcεRI, we detect sFcεRI in the exosome-depleted, soluble fraction of cell culture supernatants. We further show that sFcεRI can block binding of IgE to FcεRI expressed at the cell surface. In summary, we here describe the alpha-chain of FcεRI as a circulating soluble IgE receptor isoform in human serum.

High-affinity IgE receptors on dendritic cells exacerbate Th2-dependent inflammation.

The IgE-mediated and Th2-dependent late-phase reaction remains a mechanistically enigmatic and daunting element of human allergic inflammation. In this study, we uncover the FcεRI on dendritic cells (DCs) as a key in vivo component of this form of allergy. Because rodent, unlike human, DCs lack FcεRI, this mechanism could be revealed only by using a new transgenic mouse model with human-like FcεRI expression on DCs. In the presence of IgE and allergen, FcεRI(+) DCs instructed naive T cells to differentiate into Th2 cells in vitro and boosted allergen-specific Th2 responses and Th2-dependent eosinophilia at the site of allergen exposure in vivo. Thus, FcεRI on DCs drives the cascade of pathogenic reactions linking the initial allergen capture by IgE with subsequent Th2-dominated T cell responses and the development of late-phase allergic tissue inflammation.

Clinical practice: Allergen-specific immunotherapy in children: facts and FAQs.

Allergen-specific immunotherapy (SIT) in its various application forms represents the main treatment approach of IgE-mediated allergic diseases in adults and children. Despite this clear recommendation, many particularities of products, patient characteristics, and product availability in different countries hamper the use of allergen-specific immunotherapy in particular in children. The frequently asked questions by parents, patients, and physicians are the backbone of this review. Thus, the potentials and limitations of allergen-specific immunotherapy in children and adolescents will be highlighted. IgE-mediated allergic diseases are affecting about 20% of the population. They manifest commonly early in life, and hence, the use of SIT should be considered also early in the course of the disease.