Do We Have a Proper Model?

It has been reported recently that the cystic fibrosis transmembrane conductance regulator (CFTR) besides transcellular chloride transport, also controls the paracellular permeability of bronchial epithelium. The aim of this study was to test whether overexpressing wtCFTR solely regulates paracellular permeability of cell monolayers. To answer this question we used a CFBE41o– cell line transfected with wtCFTR or mutant F508del-CFTR and compered them with parental line and healthy 16HBE14o– cells. Transepithelial electrical resistance (TER) and paracellular fluorescein flux were measured under control and CFTR-stimulating conditions. CFTR stimulation significant decreased TER in 16HBE14o– and also in CFBE41o– cells transfected with wtCFTR. In contrast, TER increased upon stimulation in CFBE41o– cells and CFBE41o– cells transfected with F508del-CFTR. Under non-stimulated conditions, all four cell lines had similar paracellular fluorescein flux. Stimulation increased only the paracellular permeability of the 16HBE14o– cell monolayers. We observed that 16HBE14o– cells were significantly smaller and showed a different structure of cell-cell contacts than CFBE41o– and its overexpressing clones. Consequently, 16HBE14o– cells have about 80% more cell-cell contacts through which electrical current and solutes can leak. Also tight junction protein composition is different in ‘healthy’ 16HBE14o– cells compared to ‘cystic fibrosis’ CFBE41o– cells. We found that claudin-3 expression was considerably stronger in 16HBE14o– cells than in the three CFBE41o– cell clones and thus independent of the presence of functional CFTR. Together, CFBE41o– cell line transfection with wtCFTR modifies transcellular conductance, but not the paracellular permeability. We conclude that CFTR overexpression is not sufficient to fully reconstitute transport in CF bronchial epithelium. Hence, it is not recommended to use those cell lines to study CFTR-dependent epithelial transport

Familial hypercholesterolaemia in children and adolescents from 48 countries: a cross-sectional study

Background: Approximately 450 000 children are born with familial hypercholesterolaemia worldwide every year, yet only 2·1% of adults with familial hypercholesterolaemia were diagnosed before age 18 years via current diagnostic approaches, which are derived from observations in adults. We aimed to characterise children and adolescents with heterozygous familial hypercholesterolaemia (HeFH) and understand current approaches to the identification and management of familial hypercholesterolaemia to inform future public health strategies. Methods: For this cross-sectional study, we assessed children and adolescents younger than 18 years with a clinical or genetic diagnosis of HeFH at the time of entry into the Familial Hypercholesterolaemia Studies Collaboration (FHSC) registry between Oct 1, 2015, and Jan 31, 2021. Data in the registry were collected from 55 regional or national registries in 48 countries. Diagnoses relying on self-reported history of familial hypercholesterolaemia and suspected secondary hypercholesterolaemia were excluded from the registry; people with untreated LDL cholesterol (LDL-C) of at least 13·0 mmol/L were excluded from this study. Data were assessed overall and by WHO region, World Bank country income status, age, diagnostic criteria, and index-case status. The main outcome of this study was to assess current identification and management of children and adolescents with familial hypercholesterolaemia. Findings: Of 63 093 individuals in the FHSC registry, 11 848 (18·8%) were children or adolescents younger than 18 years with HeFH and were included in this study; 5756 (50·2%) of 11 476 included individuals were female and 5720 (49·8%) were male. Sex data were missing for 372 (3·1%) of 11 848 individuals. Median age at registry entry was 9·6 years (IQR 5·8-13·2). 10 099 (89·9%) of 11 235 included individuals had a final genetically confirmed diagnosis of familial hypercholesterolaemia and 1136 (10·1%) had a clinical diagnosis. Genetically confirmed diagnosis data or clinical diagnosis data were missing for 613 (5·2%) of 11 848 individuals. Genetic diagnosis was more common in children and adolescents from high-income countries (9427 [92·4%] of 10 202) than in children and adolescents from non-high-income countries (199 [48·0%] of 415). 3414 (31·6%) of 10 804 children or adolescents were index cases. Familial-hypercholesterolaemia-related physical signs, cardiovascular risk factors, and cardiovascular disease were uncommon, but were more common in non-high-income countries. 7557 (72·4%) of 10 428 included children or adolescents were not taking lipid-lowering medication (LLM) and had a median LDL-C of 5·00 mmol/L (IQR 4·05-6·08). Compared with genetic diagnosis, the use of unadapted clinical criteria intended for use in adults and reliant on more extreme phenotypes could result in 50-75% of children and adolescents with familial hypercholesterolaemia not being identified. Interpretation: Clinical characteristics observed in adults with familial hypercholesterolaemia are uncommon in children and adolescents with familial hypercholesterolaemia, hence detection in this age group relies on measurement of LDL-C and genetic confirmation. Where genetic testing is unavailable, increased availability and use of LDL-C measurements in the first few years of life could help reduce the current gap between prevalence and detection, enabling increased use of combination LLM to reach recommended LDL-C targets early in life

Paracellular transport through healthy and cystic fibrosis bronchial epithelial cell lines--do we have a proper model?

It has been reported recently that the cystic fibrosis transmembrane conductance regulator (CFTR) besides transcellular chloride transport, also controls the paracellular permeability of bronchial epithelium. The aim of this study was to test whether overexpressing wtCFTR solely regulates paracellular permeability of cell monolayers. To answer this question we used a CFBE41o- cell line transfected with wtCFTR or mutant F508del-CFTR and compered them with parental line and healthy 16HBE14o- cells. Transepithelial electrical resistance (TER) and paracellular fluorescein flux were measured under control and CFTR-stimulating conditions. CFTR stimulation significant decreased TER in 16HBE14o- and also in CFBE41o- cells transfected with wtCFTR. In contrast, TER increased upon stimulation in CFBE41o- cells and CFBE41o- cells transfected with F508del-CFTR. Under non-stimulated conditions, all four cell lines had similar paracellular fluorescein flux. Stimulation increased only the paracellular permeability of the 16HBE14o- cell monolayers. We observed that 16HBE14o- cells were significantly smaller and showed a different structure of cell-cell contacts than CFBE41o- and its overexpressing clones. Consequently, 16HBE14o- cells have about 80% more cell-cell contacts through which electrical current and solutes can leak. Also tight junction protein composition is different in 'healthy' 16HBE14o- cells compared to 'cystic fibrosis' CFBE41o- cells. We found that claudin-3 expression was considerably stronger in 16HBE14o- cells than in the three CFBE41o- cell clones and thus independent of the presence of functional CFTR. Together, CFBE41o- cell line transfection with wtCFTR modifies transcellular conductance, but not the paracellular permeability. We conclude that CFTR overexpression is not sufficient to fully reconstitute transport in CF bronchial epithelium. Hence, it is not recommended to use those cell lines to study CFTR-dependent epithelial transport

Differences in TJ length.

<p><b>A)</b> Upper image show immunostaining of ZO-1 (green) and nuclei staining (blue). Lower image shows magnifications of representative cell borders. <b>B)</b> Ratio of persistent length to contour length of all tested cell monolayers. 16HBE14o^– shows a cell-cell contact enlargement by 40% while CFBE41o^– cells and its transfected clones exhibit only a tiny enlargement by 10%. <b>C)</b> TJ lengths per area in µm per µm². 16HBE14o^– cells show 80% longer TJ lengths per area than CFBE41o^– cells. Furthermore, there is no difference in TJ lengths per area between CFBE41o^– cells and its transfected clones. Results are presented as mean ± SD (n = 7–8, p<0.05).</p

Expression of CFTR mRNA.

<p>The relative quantity of CFTR gene expression was calculated by the 2^−ΔΔCt method, using GAPDH as the internal reference. CFTR mRNA expression value of 16HBE14o^– cells was defined as 1 and expression of all other cells were normalized to this value. CFTR mRNA levels of the other cell lines were displayed as a fold change relative to16HBE14o^–. Results are presented as mean ± SEM (n = 9–12, p<0.001).</p

Changes in transepithelial electrical resistance (cTER) upon cAMP.

<p><b>A)</b> 16HBE14o^– (triangles) and CFBE41o^– (circles) monolayer were grown on ThinCert supports. After 8–11 days in culture they obtained 800 and 500 Ω*cm² resistance respectively. After 5 min 8cpt-cAMP was added, causing dramatic TER decrease in 16HBE14o^– cells (red triangles) and an increase of TER for CFBE41o^– (green circles). Addition of the same amount of medium to control cells (open circles and triangles) did not show any effect. <b>B)</b> CFBE-WT monolayer (blue triangles) and CFBE-delF monolayer (brown circles) were grown on ThinCert supports. After 8–11 days in culture both clones obtained 500–600 Ω*cm² resistance. Stimulation with 8cpt-cAMP caused a decrease of TER in CFBE-WT cells (blue triangles) and an increase for CFBE-delF (brown circles). Addition of the same amount of medium to control cells (open circles and triangles) did not show any effect. Results are presented as mean ± SD (n = 3–6, p<0.05).</p

Scheme of the continuous transepithelial resistance measurement device (cTER).

<p>The ThinCert culture plate contains eight filter inserts with cell monolayers. The upper plate (lid) has six titanium electrodes for each insert, four electrodes to inject the current and two to measures the voltage. Electrodes are arranged in a way that resulted in a fairly homogenous electrical field.</p

Detection of tight junction proteins.

<p>Presence of tight junction proteins known to be expressed in alveolar epithelial cells (claudin-3, -4, -5, -7) and their junctional localization in 16HBE14o^–, CFBE41o^– cells and its transfected clones was verified by Western blot (A) and confocal laser scanning microscopy (B). For densitometric evaluation of Western blots (C), all signals were normalized to β-actin. All values are expressed relative to the respective value detected in 16HBE14o^– cell layers. One-way Anova analysis revealed that claudin-3 expression differed (p<0.05) in 16HBE14o^– and CFBE41o^– clones, whereas claudin-4, -5 and -7 expression was not significantly different (n = 4). No claudin-18 expression was detected (not shown).</p

Fluorescein flux per TJ length.

<p><b>A)</b> Calculation of flux per TJ length revealed a significant lower value for 16HBE14o^– cells under resting conditions (red box) compared to CFBE41o^– cells (green box). Stimulation with cAMP caused a 3 fold increase for 16HBE14o^– cells (red hatched box) while CFBE41o^– cells do not respond to stimulation (green hatched box). <b>B)</b> CFBE-WT cells (blue) and CFBE-delF (brown) do not show statistically significant differences in fluorescein flux per TJ length neither under resting conditions nor upon stimulation with cAMP (hatched boxes). Data are presented as a box-plot showing raw data (circles), median (horizontal line) 25 and 75 percentile (box) and SD (whiskers (n = 7–13, p<0.05).</p