K.M. de Winter-de Groot

A bioassay using intestinal organoids to measure CFTR modulators in human plasma

Treatment efficacies of drugs depend on patient-specific pharmacokinetic and pharmacodynamic properties. Here, we developed an assay to measure functional levels of the CFTR potentiator VX-770 in human plasma and observed that VX-770 in plasma from different donors induced variable CFTR function in intestinal organoids. This assay can help to understand variability in treatment response to CFTR potentiators by functionally modeling individual pharmacokinetics.

Measurement of nasal nitric oxide: evaluation of six different sampling methods.

Background  Specific guidelines are developed for the measurement of bronchial FENO, however, nasal nitric oxide (nNO) measurement is not standardised yet, resulting in divergent nNO values. This study compares six different sampling methods for nNO as described in the literature, to analyse their outcome and short term and long term reproducibility.

81 The concordance between the microbiome of the upper and lower respiratory tract is investigated in infants with cystic fibrosis

Background Nasopharyngeal (NP) and oropharyngeal samples (OP) are commonly used to direct therapy for lower respiratory tract (LRT) infections in non-expectorating infants with Cystic Fibrosis (CF). The level of concordance between microbiota of the upper respiratory tract (URT) and LRT in young children is, however, largely unknown. Objective To examine the concordance between URT and LRT microbiota in infants with CF. Methods Paired bronchoalveolar lavage fluid (BAL), NP and OP samples were longitudinally collected from 17 CF patients at the age of 3 and 12 months. We studied the concordance between the URT and LRT microbial communities by 16S-rRNA-based sequencing. Results Bacterial diversity is lower in NP compared to the OP and BAL samples. On group level, OP microbiota profiles showed more similarity with BAL microbiota compared to NP microbiota. However, potential pathogens like staphylococci , Moraxella and Haemophilus influenzae were almost exclusively observed in the NP but not in OP. Moreover, intraindividual concordance between both NP and OP microbiota profiles and paired BAL microbiota was limited. Conclusion At a population level, OP microbiota profiles resemble lung microbiota more closely than NP microbiota, however, potential respiratory pathogens are often detected in NP but not in OP samples. More importantly, within-individual concordance between the URT and LRT microbiome in CF patients is limited, restricting the diagnostic value of URT sampling during LRT infections in infancy. Whether microbiota profiling of BAL samples will indeed more reliably guide treatment of infections in CF infants compared to conventional culture remains to be studied.

DYNAMIC DEVELOPMENT OF THE GUT MICROBIOTA IN CHILDREN WITH CYSTIC FIBROSIS AND HEALTHY CHILDREN DURING THE FIRST 18 MONTHS OF LIFE

The current therapeutic strategy to repair cystic fibrosis-causing defects in the chloride channel CFTR is to develop novel and better correctors (to improve folding) and potentiators (to improve function). Galapagos- AbbVie identified a novel potentiator GLPG1837 by compound screening on mutant CFTR. YFP-halide efflux assays and single channel measurements showed ∼2.5-fold improvement in channel activity by GLPG1837 compared to VX-770 (ivacaftor/Kalydeco) on G551D CFTR (1, 2). GLPG1837 successfully passed the Phase-2 clinical trials and proved to be the first potentiator after VX-770 to show competitive results on G551D patients. To identify potential differences in the mode of actions of these potentiators we studied their effects on CFTR folding and function. Biochemical radiolabeling experiments showed that mutations in the intracellular loop 2 (ICL2) disrupt domain assembly between TMD1 and NBD2, a late folding event in CFTR, but in most cases do not impair CFTR trafficking towards the cell surface. Protease-susceptibility assays showed that VX-770 improved late TMD1 folding of many ICL2 mutations, but GLPG1837 did not. YFP-halide efflux assays showed that these ICL2 mutants had varying effect on channel function, ranging from wild-type-like to function-defective mutants. GLPG1837 restored function of non-CF gating mutant E267K much better than VX-770. Residue E267 in ICL2 electrostatically interacts with K1060 in ICL4 to promote channel opening (3). This indicates that GLPG1837 is more efficient in compensating for this lost interaction. Altogether, our biochemical and functional data suggests that potentiators VX-770 and GLPG1837 have a different mode of action.

WS07.5 Gut microbiome in healthy children and children with cystic fibrosis during the first 18 months of life

Introduction: Progressive lung damage, associated with bacterial infection and neutrophil mediated inflammation, is the major cause of morbidity and death in CF. Ivacaftor improves lung function in CF patients with the G551D mutation and this may be associated with changes in airway bacterial community composition. Objectives: To determine the effect of ivacaftor on CF airway bacterial community composition and to examine the relationship with lung function and measures of systemic inflammation. Methods: Sputum and blood samples from CF patients (n = 20) were collected prior to and every three months (for 12 months) after initiation of ivacaftor therapy. Molecular based analysis (Illumina MiSeq) was used to define airway bacterial community composition. ELISA’s were used to quantify measures of systemic inflammation in blood (IL-8, IL-6 & CRP). Results: Mean relative abundance of Pseudomonas decreased after ivacaftor therapy whilst relative abundance of Streptococcus, Haemophilus, Rothia and Prevotella increased. There was no correlation between relative abundance of individual taxa and lung function. However, there was a significant inverse correlation between the relative abundance of both Streptococcus and Prevotella and IL-8 (p < 0.05). There was no association between relative abundance of individual taxa and IL-6 or CRP. Conclusion: Alterations in CF airway bacterial community composition post-ivacaftor therapy are associated with reduced levels of the potent neutrophil chemoattractant IL-8. This may result in decreased neutrophil influx into the airways and improve lung function by limiting neutrophil mediated inflammation. Acknowledgement: Funding: DEL NI Studentship & CFMATTERS (603038)

79 Development of the upper respiratory tract microbiome was investigated longitudinally in cystic fibrosis infants and controls 0–6 months of age

Background Cystic Fibrosis (CF) is characterized by early damage of the lungs by infections. The responsible pathogens find their ecological niche in the upper respiratory tract (URT). Information on microbial colonization dynamics in the URT of CF infants is lacking. Objective To examine colonization dynamics of URT microbiota in CF infants over time. Methods Case-control study. Questionnaires and nasopharyngeal (NP) samples were collected monthly from 20 CF patients and 45 controls. We studied the dynamics of URT microbiota from 0–6 months of life by 16SrRNA-based sequencing. Results The bacterial density of URT microbiota is lower in CF infants compared to controls, and inversely correlated with antibiotic use. Also, the microbial community composition is significantly different between groups over the first 6 months of life. Where in CF infants the URT microbiota is initially predominated by Corynebacterium and Staphylococcus followed by Streptococcus and Moraxella, in controls Moraxella, Haemophilus and another Corynebacterium sp. predominate throughout the first 6 months. Multivariate analyses show significantly more Staphylococcus , Pseudomonas , Enterobacteriaceae and Bacillus spp. and less Moraxella, Haemophilus, Dolosigranulum and specific Corynebacterium spp. in CF infants vs. Controls. Antibiotic use is independently associated with increased carriage of Gram-negative bacteria and reduced carriage of non-pathogenic commensals. Conclusion CF infants have a significantly different development of URT microbiota compared to controls from birth on. This information may help to improve targeted therapy, and allows surveillance of ecological side effects of antibiotics.