CFTR function is impaired in a subset of patients with pancreatitis carrying rare CFTR variants.

BACKGROUND: Many affected by pancreatitis harbor rare variants of the cystic fibrosis (CF) gene, CFTR, which encodes an epithelial chloride/bicarbonate channel. We investigated CFTR function and the effect of CFTR modulator drugs in pancreatitis patients carrying CFTR variants. METHODS: Next-generation sequencing was performed to identify CFTR variants. Sweat tests and nasal potential difference (NPD) assays were performed to assess CFTR function in vivo. Intestinal current measurement (ICM) was performed on rectal biopsies. Patient-derived intestinal epithelial monolayers were used to evaluate chloride and bicarbonate transport and the effects of a CFTR modulator combination: elexacaftor, tezacaftor and ivacaftor (ETI). RESULTS: Of 32 pancreatitis patients carrying CFTR variants, three had CF-causing mutations on both alleles and yielded CF-typical sweat test, NPD and ICM results. Fourteen subjects showed a more modest elevation in sweat chloride levels, including three that were provisionally diagnosed with CF. ICM indicated impaired CFTR function in nine out of 17 non-CF subjects tested. This group of nine included five carrying a wild type CFTR allele. In epithelial monolayers, a reduction in CFTR-dependent chloride transport was found in six out of 14 subjects tested, whereas bicarbonate secretion was reduced in only one individual. In epithelial monolayers of four of these six subjects, ETI improved CFTR function. CONCLUSIONS: CFTR function is impaired in a subset of pancreatitis patients carrying CFTR variants. Mutations outside the CFTR locus may contribute to the anion transport defect. Bioassays on patient-derived intestinal tissue and organoids can be used to detect such defects and to assess the effect of CFTR modulators.

Theratyping of the Rare CFTR Genotype A559T in Rectal Organoids and Nasal Cells Reveals a Relevant Response to Elexacaftor (VX-445) and Tezacaftor (VX-661) Combination.

Despite the promising results of new CFTR targeting drugs designed for the recovery of F508del- and class III variants activity, none of them have been approved for individuals with selected rare mutations, because uncharacterized CFTR variants lack information associated with the ability of these compounds in recovering their molecular defects. Here we used both rectal organoids (colonoids) and primary nasal brushed cells (hNEC) derived from a CF patient homozygous for A559T (c.1675G>A) variant to evaluate the responsiveness of this pathogenic variant to available CFTR targeted drugs that include VX-770, VX-809, VX-661 and VX-661 combined with VX-445. A559T is a rare mutation, found in African-Americans people with CF (PwCF) with only 85 patients registered in the CFTR2 database. At present, there is no treatment approved by FDA (U.S. Food and Drug Administration) for this genotype. Short-circuit current (Isc) measurements indicate that A559T-CFTR presents a minimal function. The acute addition of VX-770 following CFTR activation by forskolin had no significant increment of baseline level of anion transport in both colonoids and nasal cells. However, the combined treatment, VX-661-VX-445, significantly increases the chloride secretion in A559T-colonoids monolayers and hNEC, reaching approximately 10% of WT-CFTR function. These results were confirmed by forskolin-induced swelling assay and by western blotting in rectal organoids. Overall, our data show a relevant response to VX-661-VX-445 in rectal organoids and hNEC with CFTR genotype A559T/A559T. This could provide a strong rationale for treating patients carrying this variant with VX-661-VX-445-VX-770 combination.

In Vivo Inflammation Caused by Achromobacter spp. Cystic Fibrosis Clinical Isolates Exhibiting Different Pathogenic Characteristics.

Achromobacter spp. lung infection in cystic fibrosis has been associated with inflammation, increased frequency of exacerbations, and decline of respiratory function. We aimed to evaluate in vivo the inflammatory effects of clinical isolates exhibiting different pathogenic characteristics. Eight clinical isolates were selected based on different pathogenic characteristics previously assessed: virulence in Galleria mellonella larvae, cytotoxicity in human bronchial epithelial cells, and biofilm formation. Acute lung infection was established by intratracheal instillation with 10.5 × 108 bacterial cells in wild-type and CFTR-knockout (KO) mice expressing a luciferase gene under control of interleukin-8 promoter. Lung inflammation was monitored by in vivo bioluminescence imaging up to 48 h after infection, and mortality was recorded up to 96 h. Lung bacterial load was evaluated by CFU count. Virulent isolates caused higher lung inflammation and mice mortality, especially in KO animals. Isolates both virulent and cytotoxic showed higher persistence in mice lungs, while biofilm formation was not associated with lung inflammation, mice mortality, or bacterial persistence. A positive correlation between virulence and lung inflammation was observed. These results indicate that Achromobacter spp. pathogenic characteristics such as virulence and cytotoxicity may be associated with clinically relevant effects and highlight the importance of elucidating their mechanisms.

CFTR Modulators Rescue the Activity of CFTR in Colonoids Expressing the Complex Allele p.[R74W;V201M;D1270N]/dele22_24.

An Italian, 46-year-old female patient carrying the complex allele p.[R74W;V201M;D1270N] in trans with CFTR dele22_24 was diagnosed at the Cystic Fibrosis (CF) Center of Verona as being affected by CF-pancreatic sufficient (CF-PS) in 2021. The variant V201M has unknown significance, while both of the other variants of this complex allele have variable clinical consequences, according to the CFTR2 database, with reported clinical benefits for treatment with ivacaftor + tezacaftor and ivacaftor + tezacaftor + elexacaftor in patients carrying the R74W-D1270N complex allele, which are currently approved (in USA, not yet in Italy). She was previously followed up by pneumologists in northern Italy because of frequent bronchitis, hemoptysis, recurrent rhinitis, Pseudomonas aeruginosa lung colonization, bronchiectasis/atelectasis, bronchial arterial embolization and moderately compromised lung function (FEV1: 62%). Following a sweat test with borderline results, she was referred to the Verona CF Center where she presented abnormal values in both optical beta-adrenergic sweat tests and intestinal current measurement (ICM). These results were consistent with a diagnosis of CF. CFTR function analyses were also performed in vitro by forskolin-induced swelling (FIS) assay and short-circuit currents (Isc) in the monolayers of the rectal organoids. Both of these assays showed significantly increased CFTR activity following treatment with the CFTR modulators. Western-blot analysis revealed increased fully glycosylated CFTR protein after treatment with correctors, in line with the functional analysis. Interestingly, tezacaftor, together with elexacaftor, rescued the total organoid area under steady-state conditions, even in the absence of the CFTR agonist forskolin. In conclusion, in ex vivo and in vitro assays, we measured a residual function that was significantly enhanced by in vitro incubation with CFTR modulators, especially by ivacaftor + tezacaftor + elexacaftor, suggesting this combination as a potentially optimal treatment for this case.

Achromobacter spp. Adaptation in Cystic Fibrosis Infection and Candidate Biomarkers of Antimicrobial Resistance.

Achromobacter spp. can establish occasional or chronic lung infections in patients with cystic fibrosis (CF). Chronic colonization has been associated with worse prognosis highlighting the need to identify markers of bacterial persistence. To this purpose, we analyzed phenotypic features of 95 Achromobacter spp. isolates from 38 patients presenting chronic or occasional infection. Virulence was tested in Galleria mellonella larvae, cytotoxicity was tested in human bronchial epithelial cells, biofilm production in static conditions was measured by crystal violet staining and susceptibility to selected antibiotics was tested by the disk diffusion method. The presence of genetic loci associated to the analyzed phenotypic features was evaluated by a genome-wide association study. Isolates from occasional infection induced significantly higher mortality of G. mellonella larvae and showed a trend for lower cytotoxicity than chronic infection isolates. No significant difference was observed in biofilm production among the two groups. Additionally, antibiotic susceptibility testing showed that isolates from chronically-infected patients were significantly more resistant to sulfonamides and meropenem than occasional isolates. Candidate genetic biomarkers associated with antibiotic resistance or sensitivity were identified. Achromobacter spp. strains isolated from people with chronic and occasional lung infection exhibit different virulence and antibiotic susceptibility features, which could be linked to persistence in CF lungs. This underlines the possibility of identifying predictive biomarkers of persistence that could be useful for clinical purposes.

Identification of Potential Leukocyte Biomarkers Related to Drug Recovery of CFTR: Clinical Applications in Cystic Fibrosis.

The aim of this study was the identification of specific proteomic profiles, related to a restored cystic fibrosis transmembrane conductance regulator (CFTR) activity in cystic fibrosis (CF) leukocytes before and after ex vivo treatment with the potentiator VX770. We used leukocytes, isolated from CF patients carrying residual function mutations and eligible for Ivacaftor therapy, and performed CFTR activity together with proteomic analyses through micro-LC-MS. Bioinformatic analyses of the results obtained revealed the downregulation of proteins belonging to the leukocyte transendothelial migration and regulation of actin cytoskeleton pathways when CFTR activity was rescued by VX770 treatment. In particular, we focused our attention on matrix metalloproteinase 9 (MMP9), because the high expression of this protease potentially contributes to parenchyma lung destruction and dysfunction in CF. Thus, the downregulation of MMP9 could represent one of the possible positive effects of VX770 in decreasing the disease progression, and a potential biomarker for the prediction of the efficacy of therapies targeting the defect of Cl- transport in CF.

Mutations of Cystic Fibrosis Transmembrane Conductance Regulator Gene Cause a Monocyte-Selective Adhesion Deficiency.

RATIONALE: Cystic fibrosis (CF) is a common genetic disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Persistent lung inflammation, characterized by increasing polymorphonuclear leukocyte recruitment, is a major cause of the decline in respiratory function in patients with CF and is a leading cause of morbidity and mortality. CFTR is expressed in various cell types, including leukocytes, but its involvement in the regulation of leukocyte recruitment is unknown. OBJECTIVES: We evaluated whether CF leukocytes might present with alterations in cell adhesion and migration, a key process governing innate and acquired immune responses. METHODS: We used ex vivo adhesion and chemotaxis assays, flow cytometry, immunofluorescence, and GTPase activity assays in this study. MEASUREMENTS AND MAIN RESULTS: We found that chemoattractant-induced activation of ß1 and ß2 integrins and of chemotaxis is defective in mononuclear cells isolated from patients with CF. In contrast, polymorphonuclear leukocyte adhesion and chemotaxis were normal. The functionality of ß1 and ß2 integrins was restored by treatment of CF monocytes with the CFTR-correcting drugs VRT325 and VX809. Moreover, treatment of healthy monocytes with the CFTR inhibitor CFTR(inh)-172 blocked integrin activation by chemoattractants. In a murine model of lung inflammation, we found that integrin-independent migration of CF monocytes into the lung parenchyma was normal, whereas, in contrast, integrin-dependent transmigration into the alveolar space was impaired. Finally, signal transduction analysis showed that, in CF monocytes, chemoattractant-triggered activation of RhoA and CDC42 Rho small GTPases (controlling integrin activation and chemotaxis, respectively) was strongly deficient. CONCLUSIONS: Altogether, these data highlight the critical regulatory role of CFTR in integrin activation by chemoattractants in monocytes and identify CF as a new, cell type-selective leukocyte adhesion deficiency disease, providing new insights into CF pathogenesis.

In vivo monitoring of lung inflammation in CFTR-deficient mice.

BACKGROUND: Experimentally, lung inflammation in laboratory animals is usually detected by the presence of inflammatory markers, such as immune cells and cytokines, in the bronchoalveolar lavage fluid (BALF) of sacrificed animals. This method, although extensively used, is time, money and animal life consuming, especially when applied to genetically modified animals. Thus a new and more convenient approach, based on in vivo imaging analysis, has been set up to evaluate the inflammatory response in the lung of CFTR-deficient (CF) mice, a murine model of cystic fibrosis. METHODS: Wild type (WT) and CF mice were stimulated with P. aeruginosa LPS, TNF-alpha and culture supernatant derived from P. aeruginosa (strain VR1). Lung inflammation was detected by measuring bioluminescence in vivo in mice transiently transgenized with a luciferase reporter gene under the control of a bovine IL-8 gene promoter. RESULTS: Differences in bioluminescence (BLI) signal were revealed by comparing the two types of mice after intratracheal challenge with pro-inflammatory stimuli. BLI increased at 4 h after stimulation with TNF-alpha and at 24 h after administration of LPS and VR1 supernatant in CF mice with respect to untreated animals. The BLI signal was significantly more intense and lasted for longer times in CF animals when compared to WT mice. Analysis of BALF markers: leukocytes, cytokines and histology revealed no significant differences between CF and WT mice. CONCLUSIONS: In vivo gene delivery technology and non-invasive bioluminescent imaging has been successfully adapted to CFTR-deficient mice. Activation of bIL-8 transgene promoter can be monitored by non-invasive BLI imaging in the lung of the same animal and compared longitudinally in both CF or WT mice, after challenge with pro-inflammatory stimuli. The combination of these technologies and the use of CF mice offer the unique opportunity of evaluating the impact of therapies aimed to control inflammation in a CF background.

Genotypic and phenotypic relatedness of Pseudomonas aeruginosa isolates among the major cystic fibrosis patient cohort in Italy.

BACKGROUND: Pseudomonas aeruginosa is the predominant pathogen associated with the decline of pulmonary function in cystic fibrosis (CF) patients. Both environment-to-host acquisition and patient-to-patient transmission have been described for P. aeruginosa infection. Epidemic clones and bacterial phenotypic adaptation to the CF lung have been recognised as independent risk factors for disease progression. So far, there is no established link between genotypic prevalence and phenotypic traits. Here, we look at the major CF patient cohort in Italy to identify shared P. aeruginosa clones and associated common phenotypic traits. RESULTS: A comprehensive analysis of P. aeruginosa genotypes to determine the presence of high-risk shared clones and their association to specific phenotypic traits has been performed in a major Italian CF centre. Pulsed-field gel electrophoresis (PFGE) of P. aeruginosa isolates from 338 CF subjects identified 43 profiles shared by two or more patients and 214 profiles exclusive to individual patients. There was no evidence of a P. aeruginosa outbreak, but four most prevalent pulsotypes were detected. Common phenotypic traits were recorded intra-pulsotypes, but we detected heterogeneity inter-pulsotypes. Two of the four major pulsotypes included P. aeruginosa isolates with hallmarks of adaptation to the CF airways, including loss of motility, low production of siderophore, pyocyanin and proteases, and antibiotic resistance. One of these pulsotypes grouped a high percentage of hypermutable isolates. No clear correlation between epidemiological and clinical data was found. CONCLUSIONS: We conclude that CF patients of this cohort shared common pulsotypes, but their phenotypic heterogeneity indicates an absence of specific traits associated to P. aeruginosa genotypic prevalence.

Whole-gene CFTR sequencing combined with digital RT-PCR improves genetic diagnosis of cystic fibrosis.

Despite extensive screening, 1-5% of cystic fibrosis (CF) patients lack a definite molecular diagnosis. Next-generation sequencing (NGS) is making affordable genetic testing based on the identification of variants in extended genomic regions. In this frame, we analyzed 23 CF patients and one carrier by whole-gene CFTR resequencing: 4 were previously characterized and served as controls; 17 were cases lacking a complete diagnosis after a full conventional CFTR screening; 3 were consecutive subjects referring to our centers, not previously submitted to any screening. We also included in the custom NGS design the coding portions of the SCNN1A, SCNN1B and SCNN1G genes, encoding the subunits of the sodium channel ENaC, which were found to be mutated in CF-like patients. Besides 2 novel SCNN1B missense mutations, we identified 22 previously-known CFTR mutations, including 2 large deletions (whose breakpoints were precisely mapped), and novel deep-intronic variants, whose role on splicing was excluded by ex-vivo analyses. Finally, for 2 patients, compound heterozygotes for a CFTR mutation and the intron-9c.1210-34TG[11-12]T5 allele-known to be associated with decreased CFTR mRNA levels-the molecular diagnosis was implemented by measuring the residual level of wild-type transcript by digital reverse transcription polymerase chain reaction performed on RNA extracted from nasal brushing.

Electrophysiological evaluation of cystic fibrosis conductance transmembrane regulator (CFTR) expression in human monocytes.

BACKGROUND: Cystic fibrosis is caused by mutations of CFTR gene, a protein kinase A-activated anion channel, and is associated to a persistent and excessive chronic lung inflammation, suggesting functional alterations of immune cells. Leukocytes express detectable levels of CFTR but the molecule has not been fully characterized in these cells. METHODS: Freshly isolated monocytes from healthy individuals and CF patients were assessed by protein expression, single cell electrophysiological and membrane depolarization assays. RESULTS: We recorded chloride currents by patch clamp in healthy monocytes, after the administration of a CFTR stimulus. Currents were sensitive to a specific blocker of the CFTR channel, CFTRinh-172 and were absent in CF monocytes. Next, we evaluated the effects of ex vivo exposure of monocytes from cystic fibrosis patients carrying the F508del mutation to a chemical corrector, Vertex-325. We found an increase in CFTR expression by confocal microscopy and a recovery of CFTR function by both patch clamp and single cell fluorescence analysis. CONCLUSIONS: We confirm the expression of functional CFTR in human monocytes and demonstrate that blood monocytes can represent an adequate source of primary cells to assess new therapies and define diagnosis of CF. GENERAL SIGNIFICANCE: Tests to evaluate CFTR functional abnormalities in CF disease might greatly benefit from the availability of a convenient source of primary cells. This electrophysiological study promotes the use of monocytes as a minimally invasive tool to study and monitor CFTR function in individual patients.

In vivo imaging of the lung inflammatory response to Pseudomonas aeruginosa and its modulation by azithromycin.

BACKGROUND: Chronic inflammation of the airways is a central component in lung diseases and is frequently associated with bacterial infections. Monitoring the pro-inflammatory capability of bacterial virulence factors in vivo is challenging and usually requires invasive methods. METHODS: Lung inflammation was induced using the culture supernatants from two Pseudomonas aeruginosa clinical strains, VR1 and VR2, isolated from patients affected by cystic fibrosis and showing different phenotypes in terms of motility, colony characteristics and biofilm production as well as pyoverdine and pyocyanine release. More interesting, the strains differ also for the presence in supernatants of metalloproteases, a family of virulence factors with known pro-inflammatory activity. We have evaluated the benefit of using a mouse model, transiently expressing the luciferase reporter gene under the control of an heterologous IL-8 bovine promoter, to detect and monitoring lung inflammation. RESULTS: In vivo imaging indicated that VR1 strain, releasing in its culture supernatant metalloproteases and other virulence factors, induced lung inflammation while the VR2 strain presented with a severely reduced pro-inflammatory activity. The bioluminescence signal was detectable from 4 to 48 h after supernatant instillation. The animal model was also used to test the anti-inflammatory activity of azithromycin (AZM), an antibiotic with demonstrated inhibitory effect on the synthesis of bacterial exoproducts. The inflammation signal in mice was in fact significantly reduced when bacteria grew in the presence of a sub-lethal dose of AZM causing inhibition of the synthesis of metalloproteases and other bacterial elements. The in vivo data were further supported by quantification of immune cells and cytokine expression in mouse broncho-alveolar lavage samples. CONCLUSIONS: This experimental animal model is based on the transient transduction of the bovine IL-8 promoter, a gene representing a major player during inflammation, essential for leukocytes recruitment to the inflamed tissue. It appears to be an appropriate molecular read-out for monitoring the activation of inflammatory pathways caused by bacterial virulence factors. The data presented indicate that the model is suitable to functionally monitor in real time the lung inflammatory response facilitating the identification of bacterial factors with pro-inflammatory activity and the evaluation of the anti-inflammatory activity of old and new molecules for therapeutic use.

Detection of CFTR protein in human leukocytes by flow cytometry.

Leukocytes have previously been shown to express detectable levels of the protein cystic fibrosis transmembrane conductance regulator (CFTR). This study aims to evaluate the application of flow cytometric (FC) analysis to detect CFTR expression, and changes thereof, in these cells. Aliquots (200 µL) of peripheral whole blood from 12 healthy control volunteers (CTRLs), 12 carriers of a CFTR mutation (CFC), and 40 patients with cystic fibrosis (CF) carrying various combinations of CFTR mutations were incubated with specific fluorescent probes recognizing CFTR protein expressed on the plasma membrane of leukocytes. FC was applied to analyze CFTR expression in monocytes, lymphocytes, and polymorphonuclear (PMN) cells. CFTR protein was detected in monocytes and lymphocytes, whereas inconclusive results were obtained from the analysis of PMN cells. Mean fluorescence intensity (MFI) ratio value and %CFTR-positive cells above a selected threshold were the two parameters selected to quantify CFTR expression in cells. Lowest variability and the highest reproducibility were obtained when analyzing monocytes. ANOVA results indicated that both parameters were able to discriminate monocytes of healthy controls and CF individuals according to CFTR mutation classes with high accuracy. Significantly increased MFI ratio values were recorded in CFTR-defective cells that were also able to improve CFTR function after ex vivo treatment with PTC124 (Ataluren), an investigative drug designed to permit the ribosome to read through nonsense CFTR mutations. The method described is minimally invasive and may be used in the monitoring of responses to drugs whose efficacy can depend on increased CFTR protein expression levels. © 2014 International Society for Advancement of Cytometry.

Challenging the diagnosis of cystic fibrosis in a patient carrying the 186-8T/C allelic variant in the CF transmembrane conductance regulator gene.

BACKGROUND: This report describe for the first time a clinical case with a CFTR allelic variant 186-8T/C (c.54-8 T/C) in intron 1 of CFTR and underline the importance of applying a combination of genetic and functional tests to establish or exclude a diagnosis of Cystic Fibrosis. In this case the diagnostic algorithm proposed for CF has been successfully applied at our Center and previous CF diagnosis assigned in a different Center was not confirmed. CASE PRESENTATION: A 38 year-old Italian woman had been treated as affected by CF since 2010, following diagnosis based on sweat tests (reported values of 73 and 57 mEq/L) and a clinical history consistent with CF. No mutations were identified by first level of genetic analysis. Afterwards the patient referred to our center for assessing the relevance of these findings. The genetic variant 186-8T/C (c.54-8 T/C) in intron 1 of the CFTR gene was detected by sequencing. Low-level interstitial-alveolar infiltration was recorded by high-resolution computerized tomography. Lung function was normal and sputum and Broncho Alveolar Lavage cultures resulted bacteriologically negative. Sweat chloride levels was re-assessed and resulted with values of 57 and 35 mEq/L, with a borderline range between 40 and 60 mEq/L. Nasal Potential Difference measurements resulted in three reliable measurements consistent with a non-CF phenotype. Differential diagnosis with ciliary dyskinesia was excluded, as was exon 2 skipping of CFTR gene that might have caused a CFTR functional defect. Furthermore, single cell fluorescence analysis in response to cAMP agonists performed in patient's monocytes overlapped those obtained in healthy donors. CONCLUSION: We concluded that this patient was not affected by CF. This case highlights the need for referrals to highly specialized centers and the importance of combined functional and genetic tests in making a correct diagnosis. Moreover, we confirmed a correlation between NPD tracings and cell depolarization in monocytes providing a rationale for proposing the use of leukocytes as a potential support for CF diagnosis.

Functional rescue of CFTR in rectal organoids from patients carrying R334W variant by CFTR modulators and PDE4 inhibitor Roflumilast.

BACKGROUND: Many disease-causing variants in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene remain uncharacterized and untreated. Restoring the function of the impaired CFTR protein is the goal of personalized medicine, particularly in patients carrying rare CFTR variants. In this study, functional defects related to the rare R334W variant were evaluated after treatment with CFTR modulators or Roflumilast, a phosphodiesterase-4 inhibitor (PDE4i). METHODS: Rectal organoids from subjects with R334W/2184insA and R334W/2183AA > G genotypes were used to perform the Forskolin-induced swelling (FIS) assay. Organoids were left drug-untreated or treated with modulators VX-770 (I), VX-445 (E), and VX-661 (T) mixed, and their combination (ETI). Roflumilast (R) was used alone or as a combination of I + R. RESULTS: Our data show a significant increase in FIS rate following treatment with I alone. The combined use of modulators, such as ETI, did not increase further swelling than I alone, nor in protein maturation. Treatment with R shows an increase in FIS response similar to those of I, and the combination R + I significantly increases the rescue of CFTR activity. CONCLUSIONS: Equivalent I and ETI treatment efficacy was observed for both genotypes. Furthermore, significant organoid swelling was observed with combined I + R used that supports the recently published data describing a potentiating effect of only I in patients carrying the variant R334W and, at the same time, corroborating the role of strategies that include PDE4 inhibitors further to potentiate the effect of I for this variant.

Influence of perfusate temperature on nasal potential difference.

Nasal potential difference (NPD) quantifies abnormal ion transport in cystic fibrosis. It has gained acceptance as an outcome measure for the investigation of new therapies. To quantify the effect of solution temperature on NPD, we first examined the effect of switching from room temperature (20-25°C) to warmed (32-37°C) solutions and vice versa during each perfusion step. Secondly, standard protocols were repeated at both temperatures in the same subjects. Changing solution temperature did not alter NPD during perfusion with Ringer's solution (<1 mV) (p>0.1). During perfusion with zero chloride solution, changing from room temperature to warmed solutions tended to decrease absolute NPD (i.e. it became less negative) by 0.9 mV (p>0.1); changing from warmed to room temperature increased NPD by 2.1 mV (p<0.05). During isoprenaline perfusion, changing from room temperature to warmed solutions increased NPD by 1.5 mV (p<0.01) and from warmed to room temperature decreased NPD by 1.4 mV (p<0.05). For full protocols at room temperature or warmed in the same subjects, mean values were similar (n = 24). During warmed perfusion, group results for total chloride response had a larger standard deviation. As this increased variability will probably decrease the power of trials, this study suggests that solutions at room temperature should be recommended for the measurement of NPD.

In silico analysis and theratyping of an ultra-rare CFTR genotype (W57G/A234D) in primary human rectal and nasal epithelial cells.

Mutation targeted therapy in cystic fibrosis (CF) is still not eligible for all CF subjects, especially for cases carrying rare variants such as the CFTR genotype W57G/A234D (c.169T>G/c.701C>A). We performed in silico analysis of the effects of these variants on protein stability, which we functionally characterized using colonoids and reprogrammed nasal epithelial cells. The effect of mutations on cystic fibrosis transmembrane conductance regulator (CFTR) protein was analyzed by western blotting, forskolin-induced swelling (FIS), and Ussing chamber analysis. We detected a residual CFTR function that increases following treatment with the CFTR modulators VX661±VX445±VX770, correlates among models, and is associated with increased CFTR protein levels following treatment with CFTR correctors. In vivo treatment with VX770 reduced sweat chloride concentration to non-CF levels, increased the number of CFTR-dependent sweat droplets, and induced a 6% absolute increase in predicted FEV1% after 27 weeks of treatment indicating the relevance of theratyping with patient-derived cells in CF.

Organoid Technology and Its Role for Theratyping Applications in Cystic Fibrosis.

Cystic fibrosis (CF) is a autosomal recessive, multisystemic disease caused by different mutations in the CFTR gene encoding CF transmembrane conductance regulator. Although symptom management is important to avoid complications, the approval of CFTR modulator drugs in the clinic has demonstrated significant improvements by targeting the primary molecular defect of CF and thereby preventing problems related to CFTR deficiency or dysfunction. CFTR modulator therapies have positively changed the patients' quality of life, especially for those who start their use at the onset of the disease. Due to early diagnosis with the implementation of newborn screening programs and considerable progress in the treatment options, nowadays pediatric mortality was dramatically reduced. In any case, the main obstacle to treat CF is to predict the drug response of patients due to genetic complexity and heterogeneity. Advances in 3D culture systems have led to the extrapolation of disease modeling and individual drug response in vitro by producing mini organs called "organoids" easily obtained from nasal and rectal mucosa biopsies. In this review, we focus primarily on patient-derived intestinal organoids used as in vitro model for CF disease. Organoids combine high-validity of outcomes with a high throughput, thus enabling CF disease classification, drug development and treatment optimization in a personalized manner.

Theratyping of the Rare CFTR Variants E193K and R334W in Rectal Organoid-Derived Epithelial Monolayers.

BACKGROUND: The effect of presently available CFTR modulator combinations, such as elexacaftor (ELX), tezacaftor (TEZ), and ivacaftor (IVA), on rare CFTR alleles is often unknown. Several assays have been developed, such as forskolin-induced swelling (FIS), to evaluate the rescue of such uncommon CFTR alleles both by established and novel modulators in patient-derived primary cell cultures (organoids). Presently, we assessed the CFTR-mediated electrical current across rectal organoid-derived epithelial monolayers. This technique, which allows separate measurement of CFTR-dependent chloride or bicarbonate transport, was used to assess the effect of ELX/TEZ/IVA on two rare CFTR variants. METHODS: Intestinal organoid cultures were established from rectal biopsies of CF patients carrying the rare missense mutations E193K or R334W paired with F508del. The effect of the CFTR modulator combination ELX/TEZ/IVA on CFTR-mediated Cl- and HCO3- secretion was assessed in organoid-derived intestinal epithelial monolayers. Non-CF organoids were used for comparison. Clinical biomarkers (sweat chloride, FEV1) were monitored in patients receiving modulator therapy. RESULTS: ELX/TEZ/IVA markedly enhanced CFTR-mediated bicarbonate and chloride transport across intestinal epithelium of both patients. Consistent with the rescue of CFTR function in cultured intestinal cells, ELX/TEZ/IVA therapy improved biomarkers of CFTR function in the R334W/F508del patient. CONCLUSIONS: Current measurements in organoid-derived intestinal monolayers can readily be used to monitor CFTR-dependent epithelial Cl- and HCO3- transport. This technique can be explored to assess the functional consequences of rare CFTR mutations and the efficacy of CFTR modulators. We propose that this functional CFTR assay may guide personalized medicine in patients with CF-like clinical manifestations as well as in those carrying rare CFTR mutations.

A PI3Kγ mimetic peptide triggers CFTR gating, bronchodilation, and reduced inflammation in obstructive airway diseases.

Cyclic adenosine 3',5'-monophosphate (cAMP)-elevating agents, such as ß2-adrenergic receptor (ß2-AR) agonists and phosphodiesterase (PDE) inhibitors, remain a mainstay in the treatment of obstructive respiratory diseases, conditions characterized by airway constriction, inflammation, and mucus hypersecretion. However, their clinical use is limited by unwanted side effects because of unrestricted cAMP elevation in the airways and in distant organs. Here, we identified the A-kinase anchoring protein phosphoinositide 3-kinase γ (PI3Kγ) as a critical regulator of a discrete cAMP signaling microdomain activated by ß2-ARs in airway structural and inflammatory cells. Displacement of the PI3Kγ-anchored pool of protein kinase A (PKA) by an inhaled, cell-permeable, PI3Kγ mimetic peptide (PI3Kγ MP) inhibited a pool of subcortical PDE4B and PDE4D and safely increased cAMP in the lungs, leading to airway smooth muscle relaxation and reduced neutrophil infiltration in a murine model of asthma. In human bronchial epithelial cells, PI3Kγ MP induced unexpected cAMP and PKA elevations restricted to the vicinity of the cystic fibrosis transmembrane conductance regulator (CFTR), the ion channel controlling mucus hydration that is mutated in cystic fibrosis (CF). PI3Kγ MP promoted the phosphorylation of wild-type CFTR on serine-737, triggering channel gating, and rescued the function of F508del-CFTR, the most prevalent CF mutant, by enhancing the effects of existing CFTR modulators. These results unveil PI3Kγ as the regulator of a ß2-AR/cAMP microdomain central to smooth muscle contraction, immune cell activation, and epithelial fluid secretion in the airways, suggesting the use of a PI3Kγ MP for compartment-restricted, therapeutic cAMP elevation in chronic obstructive respiratory diseases.