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.
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•The CRISPR/Cas9 system enables genome editing in intestinal stem cell organoids•cAMP-induced swelling is lost in CFTR mutant organoids of cystic fibrosis patients•CRISPR/Cas9-mediated repair of the CFTR locus restores organoid swelling
Correction of a disease-causing CFTR mutation in cultured intestinal stem cells from cystic fibrosis patients is demonstrated using the CRISPR/Cas9 system.
Severe childhood infection has a dose-dependent association with adult cardiovascular events and with adverse cardiometabolic phenotypes. The relationship between cardiovascular outcomes and less ...severe childhood infections is unclear. To investigate the relationship between common, non-hospitalised infections, antibiotic exposure, and preclinical vascular phenotypes in young children. A Dutch prospective population-derived birth cohort study. Recent antibiotic exposure was associated with adverse cardiovascular phenotypes; each antibiotic prescription in the 3 and 6 months prior to vascular assessment was associated with an 18.1 mum (95% confidence interval, 4.5-31.6, p = 0.01) and 10.7 mum (0.8-20.5, p = 0.03) increase in CIMT, respectively. Each additional antibiotic prescription in the preceding 6 months was associated with an 8.3 mPa.sup.-1 decrease in carotid distensibility (-15.6- -1.1, p = 0.02). Any parent-reported febrile episode (compared to none) showed weak evidence of association with diastolic BP (1.6 mmHg increase, 0.04-3.1, p = 0.04). GP-diagnosed infections were not associated with vascular phenotypes. Recent antibiotics are associated with adverse vascular phenotypes in early childhood. Mechanistic studies may differentiate antibiotic-related from infection-related effects and inform preventative strategies.
Combination treatment with the cystic fibrosis transmembrane conductance regulator (CFTR) modulators tezacaftor (VX-661) and ivacaftor (VX-770) was designed to target the underlying cause of disease ...in patients with cystic fibrosis.
In this phase 3, randomized, double-blind, multicenter, placebo-controlled, parallel-group trial, we evaluated combination therapy with tezacaftor and ivacaftor in patients 12 years of age or older who had cystic fibrosis and were homozygous for the CFTR Phe508del mutation. Patients were randomly assigned in a 1:1 ratio to receive either 100 mg of tezacaftor once daily and 150 mg of ivacaftor twice daily or matched placebo for 24 weeks. The primary end point was the absolute change in the percentage of the predicted forced expiratory volume in 1 second (FEV
) through week 24 (calculated in percentage points); relative change in the percentage of the predicted FEV
through week 24 (calculated as a percentage) was a key secondary end point.
Of the 510 patients who underwent randomization, 509 received tezacaftor-ivacaftor or placebo, and 475 completed 24 weeks of the trial regimen. The mean FEV
at baseline was 60.0% of the predicted value. The effects on the absolute and relative changes in the percentage of the predicted FEV
in favor of tezacaftor-ivacaftor over placebo were 4.0 percentage points and 6.8%, respectively (P<0.001 for both comparisons). The rate of pulmonary exacerbation was 35% lower in the tezacaftor-ivacaftor group than in the placebo group (P=0.005). The incidence of adverse events was similar in the two groups. Most adverse events were of mild severity (in 41.8% of patients overall) or moderate severity (in 40.9% overall), and serious adverse events were less frequent with tezacaftor-ivacaftor (12.4%) than with placebo (18.2%). A total of 2.9% of patients discontinued the assigned regimen owing to adverse events. Fewer patients in the tezacaftor-ivacaftor group than in the placebo group had respiratory adverse events, none of which led to discontinuation.
The combination of tezacaftor and ivacaftor was efficacious and safe in patients 12 years of age or older who had cystic fibrosis and were homozygous for the CFTR Phe508del mutation. (Funded by Vertex Pharmaceuticals; EVOLVE ClinicalTrials.gov number, NCT02347657 .).
Respiratory syncytial virus (RSV) infection is an important cause of infant mortality. Here, we estimated the potential impact of maternal vaccination against RSV on life-threatening RSV infection in ...infants.
We developed a mathematical model for maternal vaccine-induced antibody dynamics and used characteristics of a maternal RSV vaccine currently in phase 3 of clinical development. The model was applied to data from two cohorts of children younger than 12 months with RSV-related paediatric intensive care unit (PICU) admission in the United Kingdom (n = 370) and the Netherlands (n = 167), and a cohort of 211 children younger than 12 months with RSV-related in-hospital death from 20 countries worldwide.
Our model predicted that, depending on vaccine efficiency, maternal vaccination at 30 weeks’ gestational age could have prevented 62–75% of RSV-related PICU admissions in the United Kingdom and 76–87% in the Netherlands. For the global mortality cohort, the model predicted that maternal vaccination could have prevented 29–48% of RSV-related in-hospital deaths. Preterm children and children with comorbidities were predicted to benefit less than (healthy) term children.
Maternal vaccination against RSV may substantially decrease life-threatening RSV infections in infants.
Background
Food allergy significantly impairs health‐related quality of life (HRQL). Currently, it is still unknown whether diagnostic interventions for food allergy improve HRQL. We aim to assess ...the impact of diagnostic interventions for food allergy on HRQL.
Methods
A systematic search was performed in MEDLINE, Embase, Cochrane Library, and CINAHL focused on patients with a (suspected) food allergy who underwent diagnostic interventions (ie, skin prick test, specific IgE, or oral food challenges OFC) and in whom HRQL was assessed. The mean difference between HRQL before and after the diagnostic intervention was calculated. A minimal clinically important difference of 0.5 was considered clinically relevant for the food allergy quality of life questionnaire.
Results
Seven of 1465 original identified publications were included in which the impact of an OFC on HRQL was investigated (total patients n = 1370). No other diagnostic interventions were investigated. Food allergy‐specific parent‐reported HRQL improved significantly after an OFC irrespective of the outcome in children with a suspected food allergy in two publications. The change was considered clinically relevant in one of two publications. In addition, parent‐reported HRQL improved after an OFC to assess the eliciting dose in children with a confirmed food allergy. The parental burden was significantly reduced after an OFC to assess resolution of food allergy. A meta‐analysis could not be performed due to the limited numbers of, and considerable heterogeneity between, eligible publications.
Conclusion
An OFC is associated with an improved food allergy‐specific HRQL and a reduced parental burden of food allergy.
Cystic fibrosis (CF) is caused by mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. The combination of the CFTR modulators elexacaftor, tezacaftor, and ivacaftor (ETI) ...enables the effective rescue of CFTR function in people with the most prevalent F508del mutation. However, the functional restoration of rare CFTR variants remains unclear. Here, we use patient-derived intestinal organoids (PDIOs) to identify rare CFTR variants and potentially individuals with CF that might benefit from ETI. First, steady-state lumen area (SLA) measurements were taken to assess CFTR function and compare it to the level observed in healthy controls. Secondly, the forskolin-induced swelling (FIS) assay was performed to measure CFTR rescue within a lower function range, and to further compare it to ETI-mediated CFTR rescue in CFTR genotypes that have received market approval. ETI responses in 30 PDIOs harboring the F508del mutation served as reference for ETI responses of 22 PDIOs with genotypes that are not currently eligible for CFTR modulator treatment, following European Medicine Agency (EMA) and/or U.S. Food and Drug Administration (FDA) regulations. Our data expand previous datasets showing a correlation between in vitro CFTR rescue in organoids and corresponding in vivo ppFEV1 improvement upon a CFTR modulator treatment in published clinical trials, and suggests that the majority of individuals with rare CFTR variants could benefit from ETI. CFTR restoration was further confirmed on protein levels using Western blot. Our data support that CFTR function measurements in PDIOs with rare CFTR genotypes can help to select potential responders to ETI, and suggest that regulatory authorities need to consider providing access to treatment based on the principle of equality for people with CF who do not have access to treatment.
Organoids are self‐organizing 3D structures grown from stem cells that recapitulate essential aspects of organ structure and function. Here, we describe a method to establish long‐term‐expanding ...human airway organoids from broncho‐alveolar resections or lavage material. The pseudostratified airway organoids consist of basal cells, functional multi‐ciliated cells, mucus‐producing secretory cells, and CC10‐secreting club cells. Airway organoids derived from cystic fibrosis (CF) patients allow assessment of CFTR function in an organoid swelling assay. Organoids established from lung cancer resections and metastasis biopsies retain tumor histopathology as well as cancer gene mutations and are amenable to drug screening. Respiratory syncytial virus (RSV) infection recapitulates central disease features, dramatically increases organoid cell motility via the non‐structural viral NS2 protein, and preferentially recruits neutrophils upon co‐culturing. We conclude that human airway organoids represent versatile models for the in vitro study of hereditary, malignant, and infectious pulmonary disease.
Synopsis
To date, persistent in vitro culture of adult human lung epithelium remains elusive. In this methods resource article, culture conditions to maintain three‐dimensional pulmonary tissue long‐term are reported and applied to recapitulate related diseases.
Culture conditions for long‐term expansion of healthy, hereditary disease and malignant human airway epithelial organoids.
Airway organoids are amenable for medium‐throughput drug screening.
Airway organoids readily allow modelng of viral infection.
Three‐dimensional human pulmonary tissue culture allows for investigation of hereditary diseases.
Invariant Natural Killer T (iNKT) cells respond to the ligation of lipid antigen-CD1d complexes via their T-cell receptor and are implicated in various immunometabolic diseases. We considered that ...immunometabolic factors might affect iNKT cell function. To this end, we investigated iNKT cell phenotype and function in a cohort of adolescents with chronic disease and immunometabolic abnormalities. We analyzed peripheral blood iNKT cells of adolescents with cystic fibrosis (CF, n = 24), corrected coarctation of the aorta (CoA, n = 25), juvenile idiopathic arthritis (JIA, n = 20), obesity (OB, n = 20), and corrected atrial septal defect (ASD, n = 25) as controls. To study transcriptional differences, we performed RNA sequencing on a subset of obese patients and controls. Finally, we performed standardized co-culture experiments using patient plasma, to investigate the effect of plasma factors on iNKT cell function. We found comparable iNKT cell numbers across patient groups, except for reduced iNKT cell numbers in JIA patients. Upon ex-vivo activation, we observed enhanced IFN-γ/IL-4 cytokine ratios in iNKT cells of obese adolescents versus controls. The Th1-skewed iNKT cell cytokine profile of obese adolescents was not explained by a distinct transcriptional profile of the iNKT cells. Co-culture experiments with patient plasma revealed that across all patient groups, obesity-associated plasma factors including LDL-cholesterol, leptin, and fatty-acid binding protein 4 (FABP4) coincided with higher IFN-γ production, whereas high HDL-cholesterol and insulin sensitivity (QUICKI) coincided with higher IL-4 production. LDL and HDL supplementation in co-culture studies confirmed the effects of lipoproteins on iNKT cell cytokine production. These results suggest that circulating immunometabolic factors such as lipoproteins may be involved in Th1 skewing of the iNKT cell cytokine response in immunometabolic disease.
Coffee and tea are commonly consumed during pregnancy. While several of their components, like caffeine, have strong pharmacological effects, the effect on the unborn fetus remains unclear. Caffeine ...intake has been associated with abortion, preterm birth and fetal growth restriction, but a general consensus on caffeine restriction is still lacking. We aimed to investigate antenatal coffee, tea and caffeine consumption and the effect on birth weight and length, gestational age at birth and hypertensive disorders in pregnancy.
A total of 936 healthy pregnancies from the WHISTLER birth cohort with data on coffee and tea consumption were included. Maternal and child characteristics as well as antenatal coffee and tea consumption were obtained through postpartum questionnaires. Reported consumption was validated using available preconceptional data. Caffeine intake was calculated from coffee and tea consumption. Linear and logistic regression was used to assess the association with birth outcome and hypertensive disorders.
After adjustment for smoking and maternal age, a daily consumption of more than 300mg of caffeine compared to less than 100mg of caffeine was significantly associated with an increased gestational age (linear regression coefficient = 2.00 days, 95%CI = 0.12-4.21, P = 0.03). Tea consumption was significantly related to a higher risk of pregnancy induced hypertension (OR = 1.13, 95%CI = 1.04-1.23, P = 0.004). No associations concerning coffee consumption or birth weight and birth length were observed.
Daily caffeine consumption of more than 300mg is possibly associated with an increase in gestational age at birth. A possible relation between high tea consumption and increased risk for pregnancy induced hypertension warrants further research. For most outcomes, we found no significant associations with coffee or tea intake.
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