Cornelis K. van der Ent

Functional Repair of CFTR by CRISPR/Cas9 in Intestinal Stem Cell Organoids of Cystic Fibrosis Patients

Single murine and human intestinal stem cells can be expanded in culture over long time periods as genetically and phenotypically stable epithelial organoids. Increased cAMP levels induce rapid swelling of such organoids by opening the cystic fibrosis transmembrane conductor receptor (CFTR). This response is lost in organoids derived from cystic fibrosis (CF) patients. Here we use the CRISPR/Cas9 genome editing system to correct the CFTR locus by homologous recombination in cultured intestinal stem cells of CF patients. The corrected allele is expressed and fully functional as measured in clonally expanded organoids. This study provides proof of concept for gene correction by homologous recombination in primary adult stem cells derived from patients with a single-gene hereditary defect.

A functional CFTR assay using primary cystic fibrosis intestinal organoids

We recently established conditions allowing for long-term expansion of epithelial organoids from intestine, recapitulating essential features of the in vivo tissue architecture. Here we apply this technology to study primary intestinal organoids of people suffering from cystic fibrosis, a disease caused by mutations in CFTR, encoding cystic fibrosis transmembrane conductance regulator. Forskolin induces rapid swelling of organoids derived from healthy controls or wild-type mice, but this effect is strongly reduced in organoids of subjects with cystic fibrosis or in mice carrying the Cftr F508del mutation and is absent in Cftr-deficient organoids. This pattern is phenocopied by CFTR-specific inhibitors. Forskolin-induced swelling of in vitro–expanded human control and cystic fibrosis organoids corresponds quantitatively with forskolin-induced anion currents in freshly excised ex vivo rectal biopsies. Function of the CFTR F508del mutant protein is restored by incubation at low temperature, as well as by CFTR-restoring compounds. This relatively simple and robust assay will facilitate diagnosis, functional studies, drug development and personalized medicine approaches in cystic fibrosis.

Successful treatment of allergic bronchopulmonary aspergillosis with recombinant anti-IgE antibody

Allergic bronchopulmonary aspergillosis (ABPA) can cause severe worsening of the respiratory condition in patients with cystic fibrosis. Treatment can result in steroid dependency and serious adverse events. A dramatic and rapid improvement of respiratory symptoms and lung function after a single dose of anti-IgE antibody (omalizumab) in a 12-year-old girl with cystic fibrosis and ABPA is described. This is the first report of this experimental treatment. It suggests an important role for IgE in the pathogenesis of ABPA and offers new therapeutic possibilities.

Characterizing responses to CFTR-modulating drugs using rectal organoids derived from subjects with cystic fibrosis

Rectal organoids from subjects with cystic fibrosis can be used to assess responses to drugs that modulate CFTR. Mini-guts for personalized cystic fibrosis therapy Cystic fibrosis is caused by mutations in the CFTR gene that severely reduce the function of the CFTR protein. New drugs for treating cystic fibrosis modulate CFTR protein function, but drug efficacy is dependent on which CFTR mutation a patient carries. Dekkers et al. now show that the efficacy of these drugs can be individually assessed in a laboratory test using epithelial cells cultured as mini-guts from rectal biopsies from subjects with cystic fibrosis. The authors show that the drug responses observed in mini-guts or rectal organoids can be used to predict which patients may be potential responders to the drug. This preclinical test may help to quickly identify responders to CFTR-modulating drug therapy even when patients carry very rare CFTR mutations. Identifying subjects with cystic fibrosis (CF) who may benefit from cystic fibrosis transmembrane conductance regulator (CFTR)–modulating drugs is time-consuming, costly, and especially challenging for individuals with rare uncharacterized CFTR mutations. We studied CFTR function and responses to two drugs—the prototypical CFTR potentiator VX-770 (ivacaftor/KALYDECO) and the CFTR corrector VX-809 (lumacaftor)—in organoid cultures derived from the rectal epithelia of subjects with CF, who expressed a broad range of CFTR mutations. We observed that CFTR residual function and responses to drug therapy depended on both the CFTR mutation and the genetic background of the subjects. In vitro drug responses in rectal organoids positively correlated with published outcome data from clinical trials with VX-809 and VX-770, allowing us to predict from preclinical data the potential for CF patients carrying rare CFTR mutations to respond to drug therapy. We demonstrated proof of principle by selecting two subjects expressing an uncharacterized rare CFTR genotype (G1249R/F508del) who showed clinical responses to treatment with ivacaftor and one subject (F508del/R347P) who showed a limited response to drug therapy both in vitro and in vivo. These data suggest that in vitro measurements of CFTR function in patient-derived rectal organoids may be useful for identifying subjects who would benefit from CFTR-correcting treatment, independent of their CFTR mutation.

EMERGENCE AND EPIDEMIC OCCURRENCE OF ENTEROVIRUS 68 RESPIRATORY INFECTIONS IN THE NETHERLANDS IN 2010

Following an increase in detection of enterovirus 68 (EV68) in community surveillance of respiratory infections in The Netherlands in 2010, epidemiological and virological analyses were performed to investigate the possible public health impact of EV68 infections. We retrospectively tested specimens collected from acute respiratory infections surveillance and through three children cohort studies conducted in The Netherlands from 1994 through 2010. A total of 71 of 13,310 (0.5%) specimens were positive for EV68, of which 67 (94%) were from symptomatic persons. Twenty-four (34%) of the EV68 positive specimens were collected during 2010. EV68-positive patients with respiratory symptoms showed significantly more dyspnea, cough and bronchitis than EV68-negative patients with respiratory symptoms. Phylogenetic analysis showed an increased VP1 gene diversity in 2010, suggesting that the increased number of EV68 detections in 2010 reflects a real epidemic. Clinical laboratories should consider enterovirus diagnostics in the differential diagnosis of patients presenting with respiratory symptoms.

Maintenance azithromycin treatment in pediatric patients with cystic fibrosis: long-term outcomes related to macrolide resistance and pulmonary function.

Background: Maintenance azithromycin therapy may improve pulmonary function in patients with cystic fibrosis (CF) with Pseudomonas aeruginosa infection because of its antiinflammatory properties. However, azithromycin therapy might increase macrolide resistance in Staphylococcus aureus cultured from respiratory secretions. We studied the emergence of macrolide resistance in S. aureus and correlated this to pulmonary function decline in pediatric patients with CF on daily azithromycin therapy. Methods: Respiratory cultures of 100 patients with CF were analyzed for S. aureus colonization and its resistance pattern before and during 3 years after initiation of azithromycin maintenance therapy. Mean annual change in forced expiratory volume as percent of predicted (FEV1 %) was calculated to compare pulmonary function before and after azithromycin therapy. Results: Staphylococcal colonization did not significantly decrease after initiation of azithromycin (50% versus 48%). Before start of therapy, 10% of patients with staphylococcal colonization had macrolide-resistant strains. Staphylococcal resistance increased to 83% in the first year; 97% in the second and 100% in the third year after initiation of azithromycin therapy (P < 0.001). Half of macrolide-resistant S. aureus comprised the macrolide–lincosamide–streptogramin phenotype. Percent forced expiratory volume in 1 second improved in the first year after initiation of azithromycin (mean annual change: −4.75% before versus +3.09% after initiation; P < 0.01) but decreased during the second and third years after initiation (−5.15% and −3.65%, respectively). Emergence of macrolide-resistant S. aureus was not related to pulmonary function decline. Conclusion: Maintenance azithromycin therapy in patients with CF leads to macrolide resistance in nearly all S. aureus carriers. Pulmonary function improvement after initiation of azithromycin therapy seems to be temporary and appears not to be related to macrolide resistance of S. aureus.

Respiratory Pathogens in Children with and without Respiratory Symptoms

Objectives To investigate the occurrence of respiratory pathogens in samples from children with and without respiratory symptoms and to identify whether age and/ or coinfections modify the impact of respiratory pathogens on symptoms. Study design In a prospective longitudinal study, 18 children were sampled biweekly for respiratory pathogens, irrespective of respiratory symptoms. Polymerase chain reaction was performed for 13 respiratory pathogens. Episodes were defined “asymptomatic” if no symptoms of any respiratory tract illness were present between 1 week before and 1 week after sampling. Results A total of 230 samples were collected. In 56% of the symptomatic episodes, a pathogen was detected, compared with 40% of the asymptomatic episodes (P = .03). Rhinovirus and coronaviruses were most prevalent in both symptomatic and asymptomatic episodes. In the youngest children, 9% of the pathogen-positive episodes were asymptomatic, compared with 36% in the oldest children (P = .01). Multiple pathogens were found in 17% of the symptomatic episodes and in 3% of the asymptomatic episodes (P = .02). Conclusions Respiratory pathogens are frequently detected in samples from children with no respiratory symptoms. Symptomatic cases occurred more often in younger children and with detections of more than 1 respiratory pathogen.

RSV Mediates Pseudomonas aeruginosa Binding to Cystic Fibrosis and Normal Epithelial Cells

Cystic fibrosis lung disease typically has a course of exacerbations and remissions, suggesting that external factors like viral infections can influence this course. Clinical data suggest synergism between respiratory syncytial virus (RSV) infections and Pseudomonas aeruginosa in cystic fibrosis (CF) lung disease. We studied the influence of RSV infection on adherence of P. aeruginosa to IB3-1, HEp-2, and A549 epithelial cell monolayers in vitro. RSV infection of epithelial cells as well as simultaneous addition of RSV and P. aeruginosa to noninfected cells both strongly enhanced the pseudomonal adherence to epithelial cells. The increased adherence varied from 1.2- to 8.2-fold in case of previous RSV infection, and from 1.7- to 16.1-fold in case of simultaneous addition. We observed direct binding of RSV to P. aeruginosa, and blocking of RSV with heparin eliminated the effect on increased adherence. This suggests that RSV possibly acts as a coupling agent between P. aeruginosa and epithelial cells. In conclusion, RSV enhances P. aeruginosa infection of respiratory epithelial cells. It suggests a role of specific viral–bacterial interactions in exacerbations of CF lung disease, which could have important implications on prevention and treatment.

Prevalence and impact of respiratory viral infections in young children with cystic fibrosis: prospective cohort study.

OBJECTIVE. We aimed to investigate differences in upper and lower respiratory tract symptoms in relation to respiratory viral infections detected with polymerase chain reaction assays in young children with cystic fibrosis and healthy control subjects. METHODS. In a 6-month winter period, 20 young children with cystic fibrosis and 18 age-matched, healthy, control subjects were contacted twice per week for detection of symptoms of an acute respiratory illness. If any symptom was present, then a home visit was made for physical examination and collection of nasopharyngeal swabs for viral analysis. In addition, parents were instructed to collect nasopharyngeal swabs every 2 weeks. RESULTS. Children with cystic fibrosis and healthy control subjects had similar frequencies of acute respiratory illnesses (3.8 ± 1.0 and 4.2 ± 1.7 episodes, respectively). Although there were no significant differences in upper respiratory tract symptoms, the children with cystic fibrosis had longer periods of lower respiratory tract symptoms (22.4 ± 22.2 vs 12.8 ± 13.8 days) and a higher mean severity score per episode (2.35 ± 0.64 vs 1.92 ± 0.46). In addition, similar increases in upper respiratory tract symptom scores were associated with significantly greater increases in lower respiratory tract symptom scores in children with cystic fibrosis. No differences in the seasonal occurrences and distributions of respiratory viruses were observed, with picornaviruses and coronaviruses being the most prevalent. CONCLUSIONS. Although there were no differences in the seasonal occurrences and distributions of polymerase chain reaction-detected respiratory viruses, acute respiratory illnesses were frequently associated with increased lower respiratory tract morbidity in young children with cystic fibrosis.

Intestinal Obstruction Syndromes in Cystic Fibrosis: Meconium Ileus, Distal Intestinal Obstruction Syndrome, and Constipation

Meconium ileus at birth, distal intestinal obstruction syndrome (DIOS), and constipation are an interrelated group of intestinal obstruction syndromes with a variable severity of obstruction that occurs in cystic fibrosis patients. Long-term follow-up studies show that today meconium ileus is not a risk factor for impaired nutritional status, pulmonary function, or survival. DIOS and constipation are frequently seen in cystic fibrosis patients, especially later in life; genetic, dietary, and other associations have been explored. Diagnosis of DIOS is based on suggestive symptoms, with a right lower quadrant mass confirmed on abdominal radiography, whereas symptoms of constipation are milder and of longer standing. In DIOS, early aggressive laxative treatment with oral laxatives (polyethylene glycol) or intestinal lavage with balanced osmotic electrolyte solution and rehydration is required, which now makes the need for surgical interventions rare. Constipation can generally be well controlled with polyethylene glycol maintenance treatment.