Leukocyte telomere length and attrition in association with disease severity in cystic fibrosis patients.

Cystic fibrosis (CF) is characterized by chronic airway inflammation and premature aging. The link with leukocyte telomere length (LTL) as a marker of biological aging is unclear. We studied disease severity and LTL in 168 CF patients of which 85 patients had a second retrospective LTL assessment. A higher FEV1 was associated with longer LTL, with a stronger effect in men (5.08% longer LTL) compared to women (0.41% longer LTL). A higher FEV1/FVC ratio was associated with 7.05% (P=0.017) longer LTL in men. CF asthma, as defined by the treatment with inhaled corticosteroids, was associated with -6.65% shorter LTL (P=0.028). Men homozygous for the ΔF508 genotype showed a -10.48% (P=0.026) shorter LTL compared to heterozygotes. A genotype-specific non-linear association between LTL shortening and chronological age was observed. Stronger age-related LTL shortening was observed in patients homozygous for the ΔF508 genotype (P-interaction= 0.044). This work showed that disease severity in CF patients negatively influences LTL, with slightly more pronounced effects in men. The homozygous genotype for ΔF508 may play a role in LTL attrition in CF patients. Understanding factors in CF patients that accelerate biological aging provides insights into mechanisms that can extend the overall life quality in CF-diseased.

Standards for the care of people with cystic fibrosis (CF); Planning for a longer life.

This is the final of four papers updating standards for the care of people with CF. That this paper "Planning a longer life" was considered necessary, highlights how much CF care has progressed over the past decade. Several factors underpin this progress, notably increased numbers of people with CF with access to CFTR modulator therapy. As the landscape for CF changes, so do the hopes and aspirations of people with CF and their families. This paper reflects the need to consider people with CF not as a "problem" to be solved, but as a success, a potential and a voice to be heard. People with CF and the wider CF community have driven this approach, reflecting many of the topics in this paper. This exercise involved wide stakeholder engagement. People with CF are keen to contribute to research priorities and be involved in all stages of research. People with CF want healthcare professionals to respect them as individuals and consider the impact of our actions on the world around us. Navigating life presents challenges to all, but for people with CF these challenges are heightened and complex. In this paper we highlight the concerns and life moments that impact people with CF, and events that the CF team should aim to support, including the challenges around having a family. People with CF and their care teams must embrace the updated standards outlined in these four papers to enjoy the full potential for a healthier life.

Repeatability and reproducibility of the Forskolin-induced swelling (FIS) assay on intestinal organoids from people with Cystic Fibrosis.

BACKGROUND: The forskolin-induced swelling (FIS) assay measures CFTR function on patient-derived intestinal organoids (PDIOs) and may guide treatment selection for individuals with Cystic Fibrosis (CF). The aim of this study is to demonstrate the repeatability and reproducibility of the FIS assay following a detailed Standard Operating Procedure (SOP), thus advancing the validation of the assay for precision medicine (theranostic) applications. METHODS: Over a 2-year period, FIS responses to CFTR modulators were measured in four European labs. PDIOs from six subjects with CF carrying different CFTR genotypes were used to assess the repeatability and reproducibility across the dynamic range of the assay. RESULTS: Technical, intra-assay repeatability was high (Lin's concordance correlation coefficient (CCC) 0.95-0.98). Experimental, within-subject repeatability was also high within each lab (CCCs all >0.9). Longer-term repeatability (>1 year) showed more variability (CCCs from 0.67 to 0.95). The reproducibility between labs was also high (CCC ranging from 0.92 to 0.97). Exploratory analysis also found that between-lab percentage of agreement of dichotomized CFTR modulator outcomes for predefined FIS thresholds ranged between 78 and 100 %. CONCLUSIONS: The observed repeatability and reproducibility of the FIS assay within and across different labs is high and support the use of FIS as biomarker of CFTR function in the presence or absence of CFTR modulators.

Positioning the preventive potential of microbiome treatments for cystic fibrosis in the context of current therapies.

Antibiotics and cystic fibrosis transmembrane conductance regulator (CFTR) modulators play a pivotal role in cystic fibrosis (CF) treatment, but both have limitations. Antibiotics are linked to antibiotic resistance and disruption of the airway microbiome, while CFTR modulators are not widely accessible, and structural lung damage and pathogen overgrowth still occur. Complementary strategies that can beneficially modulate the airway microbiome in a preventive way are highly needed. This could be mediated via oral probiotics, which have shown some improvement of lung function and reduction of airway infections and exacerbations, as a cost-effective approach. However, recent data suggest that specific and locally administered probiotics in the respiratory tract might be a more targeted approach to prevent pathogen outgrowth in the lower airways. This review aims to summarize the current knowledge on the CF airway microbiome and possibilities of microbiome treatments to prevent bacterial and/or viral infections and position them in the context of current CF therapies.

Standards for the care of people with cystic fibrosis (CF); recognising and addressing CF health issues.

This is the third in a series of four papers updating the European Cystic Fibrosis Society (ECFS) standards for the care of people with CF. This paper focuses on recognising and addressing CF health issues. The guidance was produced with wide stakeholder engagement, including people from the CF community, using an evidence-based framework. Authors contributed sections, and summary statements which were reviewed by a Delphi consultation. Monitoring and treating airway infection, inflammation and pulmonary exacerbations remains important, despite the widespread availability of CFTR modulators and their accompanying health improvements. Extrapulmonary CF-specific health issues persist, such as diabetes, liver disease, bone disease, stones and other renal issues, and intestinal obstruction. These health issues require multidisciplinary care with input from the relevant specialists. Cancer is more common in people with CF compared to the general population, and requires regular screening. The CF life journey requires mental and emotional adaptation to psychosocial and physical challenges, with support from the CF team and the CF psychologist. This is particularly important when life gets challenging, with disease progression requiring increased treatments, breathing support and potentially transplantation. Planning for end of life remains a necessary aspect of care and should be discussed openly, honestly, with sensitivity and compassion for the person with CF and their family. CF teams should proactively recognise and address CF-specific health issues, and support mental and emotional wellbeing while accompanying people with CF and their families on their life journey.

Standards for the care of people with cystic fibrosis; establishing and maintaining health.

This is the second in a series of four papers updating the European Cystic Fibrosis Society (ECFS) standards for the care of people with CF. This paper focuses on establishing and maintaining health. The guidance is produced using an evidence-based framework and with wide stakeholder engagement, including people from the CF community. Authors provided a narrative description of their topic and statements, which were more directive. These statements were reviewed by a Delphi exercise, achieving good levels of agreement from a wide group for all statements. This guidance reinforces the importance of a multi-disciplinary CF team, but also describes developing models of care including virtual consultations. The framework for health is reinforced, including the need for a physically active lifestyle and the strict avoidance of all recreational inhalations, including e-cigarettes. Progress with cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapy is reviewed, including emerging adverse events and advice for dose reduction and interruption. This paper contains guidance that is pertinent to all people with CF regardless of age and eligibility for and access to modulator therapy.

Standards for the care of people with cystic fibrosis (CF): A timely and accurate diagnosis.

There is considerable activity with respect to diagnosis in the field of cystic fibrosis (CF). This relates primarily to developments in newborn bloodspot screening (NBS), more extensive gene analysis and improved characterisation of CFTR-related disorder (CFTR-RD). This is particularly pertinent with respect to accessibility to variant-specific therapy (VST), a transformational intervention for people with CF with eligible CFTR gene variants. This advance reinforces the need for a timely and accurate diagnosis. In the future, there is potential for trials to assess effectiveness of variant-specific therapy for CFTR-RD. The guidance in this paper reaffirms previous standards, clarifies a number of issues, and integrates emerging evidence. Timely and accurate diagnosis has never been more important for people with CF.

Standards of care for CFTR variant-specific therapy (including modulators) for people with cystic fibrosis.

Cystic fibrosis (CF) has entered the era of variant-specific therapy, tailored to the genetic variants in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. CFTR modulators, the first variant-specific therapy available, have transformed the management of CF. The latest standards of care from the European CF Society (2018) did not include guidance on variant-specific therapy, as CFTR modulators were becoming established as a novel therapy. We have produced interim standards to guide healthcare professionals in the provision of variant-specific therapy for people with CF. Here we provide evidence-based guidance covering the spectrum of care, established using evidence from systematic reviews and expert opinion. Statements were reviewed by key stakeholders using Delphi methodology, with agreement (≥80%) achieved for all statements after one round of consultation. Issues around accessibility are discussed and there is clear consensus that all eligible people with CF should have access to variant-specific therapy.

"Il faut continuer à poser des questions" patient reported outcome measures in cystic fibrosis: An anthropological perspective.

BACKGROUND: People with cystic fibrosis (pwCF) are central in the development of patient-led assessment tools. Qualitative analysis of a frequently used CF-specific patient-reported outcome measure (PROM) sought patient recommendations for development of a new quality of life (QoL) tool. METHODS: We performed an inventory of PROMs, symptom-report and QoL tools used in clinical trials within the European Cystic Fibrosis Society Clinical Trial Network (ECFS-CTN) and in routine clinical practice among Cystic Fibrosis Europe and ECFS members. A qualitative study using cognitive interviews with pwCF and their caregivers reviewed the Cystic Fibrosis Questionnaire (CFQ), the French initial form of the Cystic Fibrosis Questionnaire-Revised (CFQ-R). RESULTS: Survey results from 33 countries revealed over 70 tools used in routine clinical practice, utilized by clinical specialists (n=124), pwCF/parents/carers (n=49) and other allied health professionals (n=60). The CFQ-R was the main PROM used in clinical trials. The qualitative study enrolled 99 pwCF, 6 to 11 years (n=31); 12 to 18 years (n=38); >18 years (n=30) and 26 parents. Inductive thematic analysis based on the CFQ, revealed 19 key themes. Themes common across all cohorts included burden of treatment, impact of disease on day-to-day life, relationships/family, stress/mood, and nutrition. Themes unique to individual groups included, treatment when not symptomatic for the paediatric group; education/studies and planning for the future for adolescents, impact of anxiety and depression on day-to-day life for adults, and for parents, questions addressing anxiety and their role as carers. CONCLUSIONS: Patient-centeredness is paramount in development of an up-to-date PROM in the era of novel therapies.

The aging lung: tissue telomere shortening in health and disease.

BACKGROUND: Telomere shortening has been associated with several lung diseases. However, telomere length is generally measured in peripheral blood leucocytes rather than in lung tissue, where disease occurs. Consequently, telomere dynamics have not been established for the normal human lung nor for diseased lung tissue. We hypothesized an age- and disease-dependent shortening of lung tissue telomeres. METHODS: At time of (re-)transplantation or autopsy, 70 explant lungs were collected: from unused donors (normal, n = 13) and patients with cystic fibrosis (CF, n = 12), chronic obstructive pulmonary disease (COPD, n = 11), chronic hypersensitivity pneumonitis (cHP, n = 9), bronchiolitis obliterans syndrome (BOS) after prior transplantation (n = 11) and restrictive allograft syndrome (RAS) after prior transplantation (n = 14). Lungs were inflated, frozen and then scanned using CT. Four tissue cores from distinct lung regions were sampled for analysis. Disease severity was evaluated using CT and micro CT imaging. DNA was extracted from the samples and average relative telomere length (RTL) was determined using real-time qPCR. RESULTS: The normal lungs showed a decrease in RTL with age (p < 0.0001). Of the diseased lungs, only BOS and RAS showed significant RTL decrease with increasing lung age (p = 0.0220 and p = 0.0272 respectively). Furthermore, we found that RTL showed considerable variability between samples within both normal and diseased lungs. cHP, BOS and RAS lungs had significant shorter RTL in comparison with normal lungs, after adjustment for lung age, sex and BMI (p < 0.0001, p = 0.0051 and p = 0.0301 respectively). When investigating the relation between RTL and regional disease severity in CF, cHP and RAS, no association was found. CONCLUSION: These results show a progressive decline in telomere length with age in normal, BOS and RAS lungs. cHP, BOS and RAS lungs demonstrated shorter RTL compared to normal lungs. Lung tissue RTL does not associate with regional disease severity within the lung. Therefore, tissue RTL does not seem to fully reflect peripheral blood telomere length.

The Effect of Immunosuppression on Airway Integrity.

BACKGROUND: Insults to the airway epithelium play a key role in constrictive bronchiolitis after lung transplantation, the typical hallmark of chronic rejection. Our hypothesis is that immunosuppressives might affect airway integrity. METHODS: A biculture of human bronchial epithelial cells and lung microvascular endothelial cells was exposed to immunosuppressives (serum through levels) for 24 hours or 4 days. Cytotoxicity, transepithelial electrical resistance (TEER), and permeability was measured after exposure to monotherapies and combination therapies. Apoptosis, oxidative stress, inflammation (IL-8), real-time polymerase chain reaction for epithelial-to-mesenchymal transition and tight junction proteins were assessed in exposed cells. RESULTS: Mycophenolate mofetil (MMF) and combination therapies including MMF, at serum trough levels and higher, are toxic for the human bronchial epithelial cells after 4-day exposure. Moreover, already after 24 hours, TEER of cells exposed to MMF decreases and permeability increases. MMF did not induce apoptosis, oxidative stress, loss of tight junctions or production of IL-8 after 24 hours, but possibly induces epithelial-to-mesenchymal transition in epithelial cells. MMF was detectable at both sides of the biculture and was also present in bronchoalveolar lavage of lung transplantation patients. Other immunosuppressives were not toxic, neither changed TEER or permeability. CONCLUSIONS: Our findings suggest that MMF is present in the airways of lung transplant patients and might affect the structural integrity of the airway, which needs further investigation and validation in the clinical setting.

Interleukin-1α induced release of interleukin-8 by human bronchial epithelial cells in vitro: assessing mechanisms and possible treatment options.

Survival after lung transplantation is hampered by chronic lung allograft dysfunction (CLAD). Persistently elevated BAL-neutrophilia is observed in some patients despite treatment with azithromycin, which may be induced by IL-1α. Our aim is to establish an in vitro model, assess mechanistic pathways and test different therapeutic strategies of IL-1α-induced release of IL-8 by human bronchial epithelial cells. Bronchial epithelial cells (16HBE) were stimulated with IL-1α with or without azithromycin or dexamethasone. IL-8 protein was analyzed in cell supernatant. Different MAP kinases (p38, JNK, ERK1/2 , Iκß) and targets known to be involved in tumor formation (PI3K, Akt) were investigated. Finally, different treatment options were tested for their potential inhibitory effect. IL-1α induced IL-8 in bronchial epithelial cells, which was dose-dependently inhibited by dexamethasone but not by azithromycin. IL-1α induced p38 and Akt phosphorylation, but activation of these MAPK was not inhibited by dexamethasone. JNK, ERK1/2 , Iκß and PI3K were not activated. None of the tested drugs reduced the IL-1α induced IL-8 production. We established an in vitro model wherein steroids inhibit the IL-1α-induced IL-8 production, while azithromycin was ineffective. Despite using this simple in vitro model, we could not identify a new treatment option for azithromycin-resistant airway neutrophilia.

End-stage cystic fibrosis lung disease is characterised by a diverse inflammatory pattern: an immunohistochemical analysis.

BACKGROUND: Cystic fibrosis (CF) lung disease is characterised by vigorous airway inflammation eventually resulting in severe lung damage. This study aimed to describe the diversity of the inflammatory pattern in end-stage CF lungs by evaluating and quantifying which components of the innate and adaptive immunity are involved, and by assessing whether this is gender-specific. METHODS: CF explant lung tissue (n = 20) collected at time of transplantation and control tissue (n = 22) was sectioned (9 µm) and stained for neutrophils, eosinophils, mast cells, dendritic cells, macrophages, CD4 T cells, cytotoxic T cells and B cells. Quantification with special attention for immune cell location was performed. RESULTS: Neutrophils, mast cells, dendritic cells, macrophages, CD4 T and cytotoxic T cells were significantly increased in CF compared to controls and there was a disproportionate increase of neutrophils around the airways in CF. Large amounts of lymphoid follicles were found in the CF lung and they had a skewed B cell/T cell composition. Upon subdividing the CF patients into a male and female population, eosinophils, mast cells and CD4 T cells were increased specifically in CF females. In this subpopulation, lymphoid follicles had less B cells and more CD8 T cells. CONCLUSION: These data demonstrate a diverse inflammatory response in the CF lung, reflected by an increase of both myeloid and lymphoid immune cells. Inflammation in the CF lung appeared to be gender-specific in our population, as the significant increase of eosinophils, mast cells and CD4 T cells was especially notable in the female subpopulation.

Immunological diversity in phenotypes of chronic lung allograft dysfunction: a comprehensive immunohistochemical analysis.

Chronic rejection after organ transplantation is defined as a humoral- and cell-mediated immune response directed against the allograft. In lung transplantation, chronic rejection is nowadays clinically defined as a cause of chronic lung allograft dysfunction (CLAD), consisting of different clinical phenotypes including restrictive allograft syndrome (RAS) and bronchiolitis obliterans syndrome (BOS). However, the differential role of humoral and cellular immunity is not investigated up to now. Explant lungs of patients with end-stage BOS (n = 19) and RAS (n = 18) were assessed for the presence of lymphoid (B and T cells) and myeloid cells (dendritic cells, eosinophils, mast cells, neutrophils, and macrophages) and compared to nontransplant control lung biopsies (n = 21). All myeloid cells, with exception of dendritic cells, were increased in RAS versus control (neutrophils, eosinophils, and mast cells: all P < 0.05, macrophages: P < 0.001). Regarding lymphoid cells, B cells and cytotoxic T cells were increased remarkably in RAS versus control (P < 0.001) and in BOS versus control (P < 0.01). Interestingly, lymphoid follicles were restricted to RAS (P < 0.001 versus control and P < 0.05 versus BOS). Our data suggest an immunological diversity between BOS and RAS, with a more pronounced involvement of the B-cell response in RAS characterized by a structural organization of lymphoid follicles. This may impact future therapeutic approaches.

Humoral immunity in phenotypes of chronic lung allograft dysfunction: A broncho-alveolar lavage fluid analysis.

BACKGROUND: Recently, antibody mediated rejection (AMR) has been associated with a higher incidence of chronic lung allograft dysfunction (CLAD) and mortality after lung transplantation (LTx). We investigated markers related to AMR and matrix remodeling in CLAD, with special attention for its two phenotypes being bronchiolitis obliterans syndrome (BOS) and restrictive CLAD (rCLAD). METHODS: Immunoglobulins (IgA, IgE, IgG1-IgG4, total IgG and IgM) and complement (C4d and C1q) were quantified in lung lavage samples at the moment of BOS (n=15) or RAS (n=16) diagnosis; and were compared to stable transplant patients who served as control (n=14). Also, airway remodeling and metalloproteinases (MMPs) were investigated via zymography and gelatin degradation. The presence of DSA was additionally assessed in blood. RESULTS: Total IgG, IgG1-IgG4 and IgM were increased in rCLAD versus control (p<0.001) and BOS patients (p<0.01). IgA and IgE were increased in rCLAD compared to control (respectively p<0.05 and p<0.01), but not to BOS. Total IgG and IgE were increased in BOS versus control (respectively p<0.01 and p<0.05). Complement proteins were exclusively present in rCLAD and correlated positively with immunoglobulins. Additionally, in blood, DSA were more present in rCLAD (p=0.041). MMP-9 levels increased in RAS and BOS versus control (p<0.001) and MMP-9 induced gelatin degradation was only increased in BOS compared to control (p<0.01). CONCLUSION: We demonstrated increased levels of immunoglobulins and complement proteins dominantly present in rCLAD. This leads to the belief that antibodies and AMR might play a more important role in rCLAD compared to BOS. Therefore, anti B-cell therapy could offer beneficial therapeutic effects in patients diagnosed with rCLAD, which needs further research.

Morphometric Analysis of Explant Lungs in Cystic Fibrosis.

RATIONALE: After repeated cycles of lung infection and inflammation, patients with cystic fibrosis (CF) evolve to respiratory insufficiency. Although histology and imaging have provided descriptive information, a thorough morphometric analysis of end-stage CF lung disease is lacking. OBJECTIVES: To quantify the involvement of small and large airways in end-stage CF. METHODS: Multidetector computed tomography (MDCT) and micro-CT were applied to 11 air-inflated CF explanted lungs and 7 control lungs to measure, count, and describe the airway and parenchymal abnormalities in end-stage CF lungs. Selected abnormalities were further investigated with thin section histology. MEASUREMENTS AND MAIN RESULTS: On MDCT, CF explanted lungs showed an increased median (interquartile range) number (631 [511-710] vs. 344 [277-349]; P = 0.003) and size of visible airways (cumulative airway diameter 217 cm [209-250] vs. 91 cm [80-105]; P < 0.001) compared with controls. Airway obstruction was seen, starting from generation 6 and increasing to 40 to 50% of airways from generation 9 onward. Micro-CT showed that the total number of terminal bronchioles was decreased (2.9/ml [2.6-4.4] vs. 5.3/ml [4.8-5.7]; P < 0.001); 49% were obstructed, and the cross-sectional area of the open terminal bronchioles was reduced (0.093 mm(2) [0.084-0.123] vs. 0.179 mm(2) [0.140-0.196]; P < 0.001). On micro-CT, 41% of the obstructed airways reopened more distally. This remodeling was confirmed on histological analysis. Parenchymal changes were also seen, mostly in a patchy and peribronchiolar distribution. CONCLUSIONS: Extensive changes of dilatation and obstruction in nearly all airway generations were observed in end-stage CF lung disease.

CYFRA 21.1 in bronchoalveolar lavage of idiopathic pulmonary fibrosis patients.

AIM: Idiopathic pulmonary fibrosis (IPF) is one of the most aggressive forms of interstitial lung diseases, however, clinically relevant biomarkers of diagnosis or prognosis are lacking. In this study, we investigated the levels of a fragment of Cytokeratin 19 (CYFRA 21.1) in bronchoalveolar lavage (BAL) of IPF patients at time of diagnosis. We further evaluated associations between CYFRA 21.1, pulmonary function evolution, mortality, and BAL cell count. MATERIALS AND METHODS: Using the Lumipulse® G1200, CYFRA 21.1 was measured in BAL samples of 81 IPF patients and 9 controls. Based upon the median detected level (1.2 ng/mL) of CYFRA 21.1 in IPF patients, they were subdivided into an IPF CYFRA 21.1 low group (≤ 1.2 ng/mL) and IPF CYFRA 21.1 high group (> 1.2 ng/mL). RESULTS: The CYFRA 21.1 levels were significantly higher in BAL of IPF patients compared to controls (P = .0015).Worse survival was observed, but no changes in pulmonary function, for IPF patients with high CYFRA 21.1 levels versus patients with low CYFRA 21.1 levels [P = .030, HR: 0.41, (0.18-0.92)[. The CYFRA 21.1 level correlated with both neutrophils (%: R = 0.60, P < .0001; #: R = 0.47, P < .0001) and eosinophils (%: R = 0.38, P = .0005; #: R = 0.30, P < .0072). CONCLUSIONS: CYFRA 21.1 is increased in BAL of IPF patients. IPF patients with a high CYFRA 21.1 concentration have a worse survival. CYFRA 21.1 levels correlate with eosinophils and neutrophils. Further studies are warranted in using CYFRA 21.1 as a biomarker for IPF prognosis.