CFTR Channel Research Articles

Pharmacological rescue of the G85E CFTR variant by preclinical and approved modulators.

Cystic Fibrosis (CF) is a genetic disease due to loss-of-function mutations of the CFTR channel. F508del is the most frequent mutation (70% of alleles in Italy), while other mutations have much lower frequency. Among them, G85E (0.4% frequency globally, 1.13% in Italy) emerges as a mutation characterized by a severe CFTR folding and trafficking defect. To investigate the pharmacological responsiveness of the G85E-CFTR variant, we performed a functional and biochemical characterization in heterologous expression systems and ex vivo models based on patient-derived human nasal epithelial cells (HNEC). Our study demonstrated that treatment of primary airway cells with elexacaftor and tezacaftor causes a significant (although modest) rescue of CFTR function, that reaches 15%-25% of the activity measured in non-CF epithelia. A detrimental effect of chronic treatment with ivacaftor, further limiting G85E rescue, was also observed. A higher rescue of CFTR function, up to 25%-35% of the normal CFTR activity, with no evidence of negative effects upon chronic potentiator treatment, can be achieved by combining elexacaftor with ARN23765, a novel type 1 corrector endowed with very high potency. Importantly, dose-response relationships suggest that G85E might alter the binding of type 1 correctors, possibly affecting their affinity for the target. In conclusion, our studies suggest that novel combinations of modulators, endowed with higher efficacy leading to increased rescue of G85E-CFTR, are needed to improve the clinical benefit in patients for this variant.

The Functional Impact of VX-770 on the Cystic Fibrosis Transmembrane Conductance Regulator Is Enduring and Increases the Constitutive Activity of This Channel in Primary Airway Epithelia Generated from Healthy Donors.

VX-770 is a small-molecule CFTR potentiator that is highly efficacious in individuals with cystic fibrosis caused by mutations in CFTR that result in a defect in channel gating. While studies have reported on the mechanism of action of VX-770, there is still more to learn about the impact that it has on CFTR function in various contexts. The aim of the present study was to examine the longevity and stability of the effect of VX-770 on CFTR function in cultured airway epithelia and to measure the consequences of this interaction. The responses to acute and chronic VX-770 exposure were measured in cultures of expanded and re-differentiated primary human nasal epithelial cells. Acute VX-770 exposure resulted in an increase in CFTR-mediated currents in the absence of exogenous compounds that induce the phosphorylation/activation of CFTR, with acute exposure having the same effect as chronic exposure. The functional impact of VX-770 on CFTR was long-lasting in cultured airway epithelia, as they maintained an electrophysiological profile consistent with the saturation of CFTR with VX-770 over time periods of up to 4 days following a short (0.5 min) or low-dose (100 nM) exposure to VX-770 during an analysis in an Ussing chamber. Rinsing the apical surface prior to VX-770 exposure or exposure during the analysis in the Ussing chamber increased the interaction between VX-770 and the CFTR. Importantly, after short, low-dose exposures to VX-770, the CFTR channels in cultured epithelia appeared to remain saturated with VX-770 for extended periods of time, despite the repetitive rinsing of the apical surface. This finding has implications for patients discontinuing the use of VX-770-containing therapies.

Gene expression responses of CF airway epithelial cells exposed to elexacaftor/tezacaftor/ivacaftor suggest benefits beyond improved CFTR channel function.

The combination of elexacaftor/tezacaftor/ivacaftor (ETI, Trikafta) reverses the primary defect in cystic fibrosis (CF) by improving CFTR-mediated Cl- and HCO3- secretion by airway epithelial cells (AECs), leading to improved lung function and less frequent exacerbations and hospitalizations. However, studies have shown that CFTR modulators like ivacaftor, a component of ETI, have numerous effects on CF cells beyond improved CFTR channel function. Because little is known about the effect of ETI on CF AEC gene expression, we exposed primary human AEC to ETI for 48 h and interrogated the transcriptome by RNA-seq and qPCR. ETI increased CFTR Cl- secretion, and defensin gene expression (DEFB1), an observation consistent with reports of decreased bacterial burden in the lungs of people with CF (pwCF). ETI decreased MMP10 and MMP12 gene expression, suggesting that ETI may reduce proteolytic-induced lung destruction in CF. ETI also reduced the expression of the stress response gene heme oxygenase (HMOX1). qPCR analysis confirmed DEFB1, HMOX1, MMP10, and MMP12 gene expression results observed by RNA-seq. Gene pathway analysis revealed that ETI decreased inflammatory signaling, cellular proliferation, and MHC class II antigen presentation. Collectively, these findings suggest that the clinical observation that ETI reduces lung infections in pwCF is related in part to drug-induced increases in DEFB1 and that ETI may reduce lung damage by reducing MMP10 and MMP12 gene expression. Moreover, pathway analysis also identified several other genes responsible for the ETI-induced reduction in inflammation observed in pwCF.NEW & NOTEWORTHY Gene expression responses by CF AECs exposed to ETI suggest that in addition to improving CFTR channel function, ETI is likely to enhance resistance to bacterial infection by increasing levels of beta-defensin 1 (hBD-1). ETI may also reduce lung damage by suppressing MMP10 and MMP12 and reduce airway inflammation by repressing proinflammatory cytokine secretion by CF AECs.

Ribosomal frameshifting selectively modulates the assembly, function, and pharmacological rescue of a misfolded CFTR variant

The cotranslational misfolding of the cystic fibrosis transmembrane conductance regulator chloride channel (CFTR) plays a central role in the molecular basis of CF. The misfolding of the most common CF variant (ΔF508) remodels both the translational regulation and quality control of CFTR. Nevertheless, it is unclear how the misassembly of the nascent polypeptide may directly influence the activity of the translation machinery. In this work, we identify a structural motif within the CFTR transcript that stimulates efficient -1 ribosomal frameshifting and triggers the premature termination of translation. Though this motif does not appear to impact the interactome of wild-type CFTR, silent mutations that disrupt this RNA structure alter the association of nascent ΔF508 CFTR with numerous translation and quality control proteins. Moreover, disrupting this RNA structure enhances the functional gating of the ΔF508 CFTR channel at the plasma membrane and its pharmacological rescue by the CFTR modulators contained in the CF drug Trikafta. The effects of the RNA structure on ΔF508 CFTR appear to be attenuated in the absence of the ER membrane protein complex, which was previously found to modulate ribosome collisions during "preemptive quality control" of a misfolded CFTR homolog. Together, our results reveal that ribosomal frameshifting selectively modulates the assembly, function, and pharmacological rescue of a misfolded CFTR variant. These findings suggest that interactions between the nascent chain, quality control machinery, and ribosome may dynamically modulate ribosomal frameshifting in order to tune the processivity of translation in response to cotranslational misfolding.

Gene expression responses of CF airway epithelial cells exposed to elexacaftor/tezacaftor/ivacaftor (ETI) suggest benefits beyond improved CFTR channel function.

Gene expression responses by CF AEC exposed to ETI suggest that in addition to improving CFTR channel function, ETI is likely to increase resistance to bacterial infection by increasing levels of beta defensin 1 (hBD-1). ETI may also reduce lung damage by suppressing MMP10, and reduce airway inflammation by repressing proinflammatory cytokine secretion by AEC cells.

Ribosomal Frameshifting Selectively Modulates the Assembly, Function, and Pharmacological Rescue of a Misfolded CFTR Variant.

The cotranslational misfolding of the cystic fibrosis transmembrane conductance regulator chloride channel (CFTR) plays a central role in the molecular basis of cystic fibrosis (CF). The misfolding of the most common CF variant (ΔF508) remodels both the translational regulation and quality control of CFTR. Nevertheless, it is unclear how the misassembly of the nascent polypeptide may directly influence the activity of the translation machinery. In this work, we identify a structural motif within the CFTR transcript that stimulates efficient -1 ribosomal frameshifting and triggers the premature termination of translation. Though this motif does not appear to impact the interactome of wild-type CFTR, silent mutations that disrupt this RNA structure alter the association of nascent ΔF508 CFTR with numerous translation and quality control proteins. Moreover, disrupting this RNA structure enhances the functional gating of the ΔF508 CFTR channel at the plasma membrane and its pharmacological rescue by the CFTR modulators contained in the CF drug Trikafta. The effects of the RNA structure on ΔF508 CFTR appear to be attenuated in the absence of the ER membrane protein complex (EMC), which was previously found to modulate ribosome collisions during "preemptive quality control" of a misfolded CFTR homolog. Together, our results reveal that ribosomal frameshifting selectively modulates the assembly, function, and pharmacological rescue of a misfolded CFTR variant. These findings suggest interactions between the nascent chain, quality control machinery, and ribosome may dynamically modulate ribosomal frameshifting in order to tune the processivity of translation in response to cotranslational misfolding.

Inflammation in the COVID-19 airway is due to inhibition of CFTR signaling by the SARS-CoV-2 spike protein

SARS-CoV-2-contributes to sickness and death in COVID-19 patients partly by inducing a hyper-proinflammatory immune response in the host airway. This hyper-proinflammatory state involves activation of signaling by NFκB, and unexpectedly, ENaC, the epithelial sodium channel. Post-infection inflammation may also contribute to "Long COVID"/PASC. Enhanced signaling by NFκB and ENaC also marks the airway of patients suffering from cystic fibrosis, a life-limiting proinflammatory genetic disease due to inactivating mutations in the CFTR gene. We therefore hypothesized that inflammation in the COVID-19 airway might similarly be due to inhibition of CFTR signaling by SARS-CoV-2 spike protein, and therefore activation of both NFκB and ENaC signaling. We used western blot and electrophysiological techniques, and an organoid model of normal airway epithelia, differentiated on an air–liquid-interface (ALI). We found that CFTR protein expression and CFTR cAMP-activated chloride channel activity were lost when the model epithelium was exposed to SARS-CoV-2 spike proteins. As hypothesized, the absence of CFTR led to activation of both TNFα/NFκB signaling and α and γ ENaC. We had previously shown that the cardiac glycoside drugs digoxin, digitoxin and ouabain blocked interaction of spike protein and ACE2. Consistently, addition of 30 nM concentrations of the cardiac glycoside drugs, prevented loss of both CFTR protein and CFTR channel activity. ACE2 and CFTR were found to co-immunoprecipitate in both basal cells and differentiated epithelia. Thus spike-dependent CFTR loss might involve ACE2 as a bridge between Spike and CFTR. In addition, spike exposure to the epithelia resulted in failure of endosomal recycling to return CFTR to the plasma membrane. Thus, failure of CFTR recovery from endosomal recycling might be a mechanism for spike-dependent loss of CFTR. Finally, we found that authentic SARS-CoV-2 virus infection induced loss of CFTR protein, which was rescued by the cardiac glycoside drugs digitoxin and ouabain. Based on experiments with this organoid model of small airway epithelia, and comparisons with 16HBE14o- and other cell types expressing normal CFTR, we predict that inflammation in the COVID-19 airway may be mediated by inhibition of CFTR signaling by the SARS-CoV-2 spike protein, thus inducing a cystic fibrosis-like clinical phenotype. To our knowledge this is the first time COVID-19 airway inflammation has been experimentally traced in normal subjects to a contribution from SARS-CoV-2 spike-dependent inhibition of CFTR signaling.

Comparison of a novel potentiator of CFTR channel activity to ivacaftor in ameliorating mucostasis caused by cigarette smoke in primary human bronchial airway epithelial cells

BackgroundCystic Fibrosis causing mutations in the gene CFTR, reduce the activity of the CFTR channel protein, and leads to mucus aggregation, airway obstruction and poor lung function. A role for CFTR in the pathogenesis of other muco-obstructive airway diseases such as Chronic Obstructive Pulmonary Disease (COPD) has been well established. The CFTR modulatory compound, Ivacaftor (VX-770), potentiates channel activity of CFTR and certain CF-causing mutations and has been shown to ameliorate mucus obstruction and improve lung function in people harbouring these CF-causing mutations. A pilot trial of Ivacaftor supported its potential efficacy for the treatment of mucus obstruction in COPD. These findings prompted the search for CFTR potentiators that are more effective in ameliorating cigarette-smoke (CS) induced mucostasis.MethodsSmall molecule potentiators, previously identified in CFTR binding studies, were tested for activity in augmenting CFTR channel activity using patch clamp electrophysiology in HEK-293 cells, a fluorescence-based assay of membrane potential in Calu-3 cells and in Ussing chamber studies of primary bronchial epithelial cultures. Addition of cigarette smoke extract (CSE) to the solutions bathing the apical surface of Calu-3 cells and primary bronchial airway cultures was used to model COPD. Confocal studies of the velocity of fluorescent microsphere movement on the apical surface of CSE exposed airway epithelial cultures, were used to assess the effect of potentiators on CFTR-mediated mucociliary movement.ResultsWe showed that SK-POT1, like VX-770, was effective in augmenting the cyclic AMP-dependent channel activity of CFTR. SK-POT-1 enhanced CFTR channel activity in airway epithelial cells previously exposed to CSE and ameliorated mucostasis on the surface of primary airway cultures.ConclusionTogether, this evidence supports the further development of SK-POT1 as an intervention in the treatment of COPD.

Personalized assessment of the effectiveness of targeted drugs for the treatment of cystic fibrosis in a patient with c.264_268delATATT and c.3139+1G>C rare variants of the <i>CFTR</i> gene

BACKGROUND: When new, most often rare CFTR gene variants, are detected in patients with cystic fibrosis, intestinal organoids are used, which allows for the assessment of the residual functional activity of the CFTR channel and the effect of targeted drugs to determine the possibility of further pathogenetic therapy. Clinical trials of the effectiveness of the targeted drugs in patients with rare CFTR variants are expensive, and patient groups are extremely small. A forskolin-induced swelling assay on a patient’s intestinal organoids allows for a personalized approach when studying rare or even single CFTR variants. AIM: To examine the effect of the CFTR potentiator ivacaftor and the combination of ivacaftor with the CFTR correctors lumacaftor, tezacaftor, and elexacaftor on the restoration of CFTR channel functions on a culture of intestinal organoids obtained from a patient with two rare CFTR variants c.264_268delATATT and c.3139+1GC. MATERIALS AND METHODS: To assess the activity of the CFTR channel, the forskolin-induced swelling assay on organoids and intestinal current measurements on rectal biopsy samples method were used. The clinical picture of a patient with the c.264_268delATATT/c.3139+1GC genotype was described. RESULTS: In vitro, the genetic variants c.264_268delATATT and c.3139+1GC lead to a complete loss of the functional CFTR protein, whereas CFTR modulators do not positively affect and do not lead to the restoration of CFTR function, in contrast from F508del/F508del control. The disease severity in a child is consistent with the results of functional tests. CONCLUSION: Both CFTR variants are classified as “severe” and cause a complete loss of chloride channel activity. No effective CFTR modulators were found for the c.264_268delATATT and c.3139+1GC variants; thus, targeted therapy cannot be recommended to the patient.

The anion exchanger slc26a3 regulates colonic mucus expansion during steady state and in response to prostaglandin E2, while Cftr regulates de novo mucus release in response to carbamylcholine

SummaryThe intestinal epithelium is covered by mucus that protects the tissue from the luminal content. Studies have shown that anion secretion via the cystic fibrosis conductance regulator (Cftr) regulates mucus formation in the small intestine. However, mechanisms regulating mucus formation in the colon are less understood. The aim of this study was to explore the role of anion transport in the regulation of mucus formation during steady state and in response to carbamylcholine (CCh) and prostaglandin E2 (PGE2). The broad-spectrum anion transport inhibitor 4,4′-diisothiocyanatostilbene-2,2′-disulfonate (DIDS), CftrdF508 (CF) mice, and the slc26a3 inhibitor SLC26A3-IN-2 were used to inhibit anion transport. In the distal colon, steady-state mucus expansion was reduced by SLC26A3-IN-2 and normal in CF mice. PGE2 stimulated mucus expansion without de novo mucus release in wild type (WT) and CF colon via slc26a3 sensitive mechanisms, while CCh induced de novo mucus secretion in WT but not in CF colon. However, when added simultaneously, CCh and PGE2 stimulated de novo mucus secretion in the CF colon via DIDS-sensitive pathways. A similar response was observed in CF ileum that responded to CCh and PGE2 with DIDS-sensitive de novo mucus secretion. In conclusion, this study suggests that slc26a3 regulates colonic mucus expansion, while Cftr regulates CCh-induced de novo mucus secretion from ileal and distal colon crypts. Furthermore, these findings demonstrate that in the absence of a functional Cftr channel, parallel stimulation with CCh and PGE2 activates additional anion transport processes that help release mucus from intestinal goblet cells.

Heterogeneity in response to Elexacaftor/Tezacaftor/Ivacaftor in people with cystic fibrosis

BackgroundHighly effective modulators of the CFTR channel have been demonstrated to dramatically impact disease progression and outcome. However, real-world data indicates that the magnitude of the clinical benefit is not equal among all patients receiving the treatment. We aimed to assess the variability in treatment response (as defined by the 6-month change in sweat chloride concentration, forced expiratory volume in one second [ppFEV1], body mass index [BMI], and CF Questionnaire-Revised [CFQ-R] respiratory domain score) and identify potential predictors in a group of patients receiving Elexacaftor-Tezacaftor-Ivacaftor (ETI) triple combination therapy. MethodsThis was a single-center, prospective cohort study enrolling adults with CF at a major center in Italy. We used linear regression models to identify a set of potential predictors (including CFTR genotype, sex, age, and baseline clinical characteristics) and estimate the variability in treatment response. ResultsThe study included 211 patients (median age: 29 years, range: 12–58). Median changes (10–90th percentile) from baseline were: - 56 mEq/L (–76; –27) for sweat chloride concentration, +14.5 points (2.5; 32.0) for ppFEV1, +0.33 standard deviation scores (–0.13; 1.05) for BMI and +17 points (0; 39) for the CFQ-R respiratory domain score. The selected predictors explained 23 % of the variability in sweat chloride concentration changes, 18 % of the variability in ppFEV1 changes, 39 % of the variability in BMI changes, and 65 % of the variability in CFQ-R changes. ConclusionsThis study highlights a high level of heterogeneity in treatment response to ETI, which can only be partially explained by the baseline characteristics of the disease.

Contribution of the intestinal current measurement method to assessment of the efficacy of CFTR modulators in cystic fibrosis

Cystic fibrosis (CF) is a disease caused by pathogenic variants of the CFTR gene. In the last decade, the treatment algorithm has entered a new era as several drugs have become available that restore the function of the CFTR chloride channel and are called CFTR modulators. The efficacy and safety of targeted drugs in cystic fibrosis needs to be further investigated using additional assessment methods.The aim of this study was to investigate the role of intestinal current measurement (ICM) in assessing the efficacy of targeted therapy for cystic fibrosis.Methods. The efficacy of CFTR modulator therapy was evaluated in 15 patients, of which 10 were children and 5 were adults. In addition to the ICM method, patients’ clinical parameters, sweat test, and pulmonary function were also evaluated according to clinical guidelines.Results. Patients with genotypes 2143delT/7121G>T and G542X/R785X had no restoration of chloride channel function with elexacaftor + tezacaftor + ivacaftor therapy, and patients with the L467F;F508del genotype with lumacaftor + ivacaftor therapy. In patients with the F508del/F508del, N1303K/G461E, N1303K/3321delG genotype, improvements were noted in terms of the restoration of CFTR channel function during therapy with elexacaftor + tezacaftor + ivacaftor therapy, and in patients with the F508del/F508del genotype during therapy with tezacaftor + ivacaftor therapy and lumacaftor + ivacaftor.Conclusion. Restoring the function of the epithelial chloride channel (CFTR) is the basis for increasing life expectancy in CF. The crucial role of the ICM method in determining the efficacy of CFTR modulators is shown.

BEST4+ cells in the intestinal epithelium.

The recent development of single-cell transcriptomics highlighted the existence of a new lineage of mature absorptive cells in the human intestinal epithelium. This subpopulation is characterized by the specific expression of Bestrophin 4 (BEST4) and of other marker genes including OTOP2, CA7, GUCA2A, GUCA2B, and SPIB. BEST4+ cells appear early in development and are present in all regions of the small and large intestine at a low abundance (<5% of all epithelial cells). Location-specific gene expression profiles in BEST4+ cells suggest their functional specialization in each gut region, as exemplified by the small intestine-specific expression of the ion channel CFTR. The putative roles of BEST4+ cells include sensing and regulation of luminal pH, tuning of guanylyl cyclase-C signaling, transport of electrolytes, hydration of mucus, and secretion of antimicrobial peptides. However, most of these hypotheses lack functional validation, notably because BEST4+ cells are absent in mice. The presence of BEST4+ cells in human intestinal organoids indicates that this in vitro model should be suitable to study their role. Recent studies showed that BEST4+ cells are also present in the intestinal epithelium of macaque, pig, and zebrafish and, here, we report their presence in rabbits, which suggests that these species could be appropriate animal models to study BEST4+ cells during the development of diseases and their interactions with environmental factors such as diet or the microbiota. In this review, we summarize the existing literature regarding BEST4+ cells and emphasize the description of their predicted roles in the intestinal epithelium in health and disease.NEW & NOTEWORTHY BEST4+ cells are a novel subtype of mature absorptive cells in the human intestinal epithelium highlighted by single-cell transcriptomics. The gene expression profile of BEST4+ cells suggests their role in pH regulation, electrolyte secretion, mucus hydration, and innate immune defense. The absence of BEST4+ cells in mice requires the use of alternative animal models or organoids to decipher the role of this novel type of intestinal epithelial cells.

The first described pathogenic variant c.2617G&gt;T of the CFTR gene

Cystic fibrosis (CF) is a disease caused by pathogenic variants in the CFTR gene. The greatest difficulty at the moment is assessing the contribution to the development of the disease of rare and previously unidentified genetic variants (mutations), mutations of uncertain clinical significance, as well as determining the genotype-phenotype relationship. Additional diagnostic methods are needed due to the large number of patients with borderline sweat test values. Purpose of the study: to study the pathogenicity of a newly identified variant of the nucleotide sequence of the CFTR gene based on assessing the functional activity of epithelial ion channels (ENaC, CFTR, CaCCs). Materials and methods: medical history data, method intestinal current measurement (ICM). Results: the patient had a severe course of the disease. Positive sweat test results, low fecal pancreatic elastase. The ICM method showed the absence of chlorine channel function. Conclusion: For the first time, the clinical features of a patient with the c.2617G&gt;T genetic variant in the genotype have been described. The results of the examination and the clinical course of the disease correspond to the severe course of the disease. Additional use of the ICM method confirmed the absence of CFTR channel function in a patient carrier of this variant of the CFTR gene.

Functional activity of the CFTR channel in a patient with the [L467F;F508del]/W1310X genotype

An important factor influencing the severity of cystic fibrosis (CF) and the effectiveness of treatment is the presence of complex alleles in the CFTR gene ≥ 2 variants in one allele. The influence of such alleles on the manifestations of CF has not been sufficiently studied.The aim of this study was to investigate the effect of the complex allele [L467F;F508del] on the phenotypic manifestations of CF and the efficacy of targeted therapy in an intestinal organoid (IO) model in a patient with the [L467F;F508del]/W1310X genotype.Methods. Methods for determining the difference in intestinal potentials (IDP), the KO method, and the forskolin test are presented using the medical history of a patient with the [L467F;F508del]/ W1310X genotype as an example.Results. The patient was diagnosed with progressive disease with obvious deterioration of pulmonary function. The ORKP method showed the absence of chlorine channel function. An assay with a KO culture from intestinal tissue showed a complete loss of functional activity of the chloride channel. In addition, the complex allele [L467F;F508del] is not sensitive to the effect of all tested CFTR modulators.Conclusion. The complex allele [L467F;F508del] causes a complete loss of functional CFTR protein and is not sensitive to the effect of any of the registered targeted drugs.

Impact of hypoxia in Cystic Fibrosis bronchial epithelial cells: Focus on CFTR and TRPA1 channels

Impact of hypoxia in Cystic Fibrosis bronchial epithelial cells: Focus on CFTR and TRPA1 channels

Cystic Fibrosis Transmembrane Conductance Regulator Protein Modulators in Children and Adolescents with Different CF Genotypes - Systematic Review and Meta-Analysis.

To determine the efficacy of the first triple CFTR protein modulators in children and adolescents with cystic fibrosis. Systematic review and meta-analysis were conducted, following PRISMA guidelines. The following databases were searched extensively: PubMed/Medline, Clinical trials.gov, Google Scholar, Scopus, Embase, and Europe PMC using the keywords: "Ivacaftor", "Elexacaftor", "Tezacaftor", VX_661", VX_770", "VX_445", "cystic fibrosis". A total of ten randomized clinical trials were included in our analysis. Primary outcomes included: Absolute change in predicted FEV1 from baseline, Absolute change in sweat chloride test from baseline, Absolute change in BMI from baseline, Absolute change in CF-QR from baseline, and Adverse Events. Among primary findings, significant absolute change in predictive FEV1 from baseline through 4 weeks favoured the triple CFTR protein modulators. (MD = 11.80, 95% CI = 8.47_15.12, p value = <0.00001); as well as CF_QR score (MD = 0.00, 95% CI = -2.50_2.50, p value= 1.00), and BMI kg/m² change (MD = 16.90, 95% CI = 12.73_21.06, p value= <0.00001). No significant change was noted for CFTR channels activity in the treatment group when compared to placebo or VX_770/VX_661 (MD = -12.57, 95% CI = -94.46_69.32, p value= 0.76). In children aged ≥ 6 y old and adolescents with F508del_CFTR mutation, Elexacaftor- Tezacaftor-Ivacaftor tend to be more effective than first-generation therapy, demonstrating promising results by exhibiting significant improvement in lung function, body weight, and respiratory-related quality of life.

Abnormal functional lymphoid tolerance and enhanced myeloid exocytosis are characteristics of resting and stimulated PBMCs in cystic fibrosis patients.

Cystic Fibrosis (CF) is the commonest genetically inherited disease (1 in 4,500 newborns) and 70% of people with CF (pwCF) harbour the F508Del mutation, resulting in misfolding and incorrect addressing of the channel CFTR to the epithelial membrane and subsequent dysregulation of fluid homeostasis. Although studies have underscored the importance and over-activation of myeloid cells, and in particular neutrophils in the lungs of people with CF (pwCF), relatively less emphasis has been put on the potential immunological bias in CF blood cells, at homeostasis or following stimulation/infection. Here, we revisited, in an exhaustive fashion, in pwCF with mild disease (median age of 15, median % FEV1 predicted = 87), whether their PBMCs, unprimed or primed with a 'non specific' stimulus (PMA+ionomycin mix) and a 'specific' one (live P.a =PAO1 strain), were differentially activated, compared to healthy controls (HC) PBMCs. 1) we analysed the lymphocytic and myeloid populations present in CF and Control PBMCs (T cells, NKT, Tgd, ILCs) and their production of the signature cytokines IFN-g, IL-13, IL-17, IL-22. 2) By q-PCR, ELISA and Luminex analysis we showed that CF PBMCs have increased background cytokines and mediators production and a partial functional tolerance phenotype, when restimulated. 3) we showed that CF PBMCs low-density neutrophils release higher levels of granule components (S100A8/A9, lactoferrin, MMP-3, MMP-7, MMP-8, MMP-9, NE), demonstrating enhanced exocytosis of potentially harmful mediators. In conclusion, we demonstrated that functional lymphoid tolerance and enhanced myeloid protease activity are key features of cystic fibrosis PBMCs.

Effects of Dietary Supplementation with Tea Residue on Growth Performance, Digestibility, and Diarrhea in Piglets.

Thirty-six healthy 21-day-old weaned ternary piglets (Duroc × Landrace × Yorkshire) were randomly divided into two treatments with 18 replicates per treatment and one pig per replicate. The control group was fed with a basal diet and the test group was fed with diets supplemented with 1 kg/t tea residue. The test period was 28 days. The results are as follows: The addition of tea residue in the diet had no significant effect on the growth performance of weaned piglets (p > 0.05), but it could significantly reduce the diarrhea rate of piglets from 1 to 7 days and 1 to 28 days (p < 0.05). Compared with the control group, the dietary supplementation of tea residue had no significant effect on nutrient apparent digestibility, plasma biochemical indexes and plasma immune indexes (p > 0.05) but increased the content of glutathione in plasma (p < 0.05). Tea residue had no significant effect on the morphology of the jejunum and ileum of piglets (p > 0.05), but it could significantly reduce the content of chloride ions in feces (p < 0.05). Compared with the basal diet group, there was no significant difference in the relative expression of TMEM16A and CFTR mRNA in the colon of weaned piglets (p > 0.05). The whole-cell patch clamp recording showed that the TMEM16A and CFTR ion channels could be activated by ionomycin and forskolin, respectively. However, when HT-29 cells transfected with TMEM16A and CFTR channels were treated with tea residue extract, it could significantly inhibit the chloride current of the TMEM16A and CFTR ion channels (p < 0.05).

Benchmarking AlphaMissense pathogenicity predictions against cystic fibrosis variants.

Variants in the cystic fibrosis transmembrane conductance regulator gene (CFTR) result in cystic fibrosis-a lethal autosomal recessive disorder. Missense variants that alter a single amino acid in the CFTR protein are among the most common cystic fibrosis variants, yet tools for accurately predicting molecular consequences of missense variants have been limited to date. AlphaMissense (AM) is a new technology that predicts the pathogenicity of missense variants based on dual learned protein structure and evolutionary features. Here, we evaluated the ability of AM to predict the pathogenicity of CFTR missense variants. AM predicted a high pathogenicity for CFTR residues overall, resulting in a high false positive rate and fair classification performance on CF variants from the CFTR2.org database. AM pathogenicity score correlated modestly with pathogenicity metrics from persons with CF including sweat chloride level, pancreatic insufficiency rate, and Pseudomonas aeruginosa infection rate. Correlation was also modest with CFTR trafficking and folding competency in vitro. By contrast, the AM score correlated well with CFTR channel function in vitro-demonstrating the dual structure and evolutionary training approach learns important functional information despite lacking such data during training. Different performance across metrics indicated AM may determine if polymorphisms in CFTR are recessive CF variants yet cannot differentiate mechanistic effects or the nature of pathophysiology. Finally, AM predictions offered limited utility to inform on the pharmacological response of CF variants i.e., theratype. Development of new approaches to differentiate the biochemical and pharmacological properties of CFTR variants is therefore still needed to refine the targeting of emerging precision CF therapeutics.