Efficacy of lumacaftor-ivacaftor for the treatment of cystic fibrosis patients homozygous for the F508del-CFTR mutation

Cystic fibrosis (CF) results from mutations in the CF transmembrane conductance regulator (CFTR) gene, which codes for the CFTR channel protein. The most common mutation in CF is F508del, which produces a misfolded protein with diminished channel activity. The development of small-molecule CFTR-modulator compounds offers an exciting and novel approach for pharmacological treatment of CF. The corrector lumacaftor helps rescue F508del-CFTR to the cell surface, and potentiator ivacaftor increases F508del-CFTR channel activity. The combination of lumacaftor-ivacaftor (Vertex Pharmaceuticals Incorporated) represents the first FDA-approved therapy for CF patients with two copies of the F508del mutation. Although this combination therapy is the first treatment to directly target the F508del-CFTR mutation, patients taking this drug displayed only modest improvements in lung function. This article summarizes recent data from clinical trials and research discoveries relating to the lumacaftor-ivacaftor treatment, and considers options for identifying future therapies that will be most efficacious for all CF patients

Bioactive Thymosin Alpha-1 Does Not Influence F508del-CFTR Maturation and Activity.

Deletion of phenylalanine 508 (F508del) in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is the most frequent mutation causing cystic fibrosis (CF). F508del-CFTR is misfolded and prematurely degraded. Recently thymosin a-1 (Tα-1) was proposed as a single molecule-based therapy for CF, improving both F508del-CFTR maturation and function by restoring defective autophagy. However, three independent laboratories failed to reproduce these results. Lack of reproducibility has been ascribed by the authors of the original paper to the use of DMSO and to improper handling. Here, we address these potential issues by demonstrating that Tα-1 changes induced by DMSO are fully reversible and that Tα-1 peptides prepared from different stock solutions have equivalent biological activity. Considering the negative results here reported, six independent laboratories failed to demonstrate F508del-CFTR correction by Tα-1. This study also calls into question the autophagy modulator cysteamine, since no rescue of mutant CFTR function was detected following treatment with cysteamine, while deleterious effects were observed when bronchial epithelia were exposed to cysteamine plus the antioxidant food supplement EGCG. Although these studies do not exclude the possibility of beneficial immunomodulatory effects of thymosin α-1, they do not support its utility as a corrector of F508del-CFTR

Streptomyces coelicolor strains lacking polyprenol phosphate mannose synthase and protein O-mannosyl transferase are hyper-susceptible to multiple antibiotics

Polyprenol phosphate mannose (PPM) is a lipid-linked sugar donor used by extra-cytoplasmic glycosyl tranferases in bacteria. PPM is synthesiszed by polyprenol phosphate mannose synthase, Ppm1, and in most Actinobacteria is used as the sugar donor for protein O-mannosyl transferase, Pmt, in protein glycosylation. Ppm1 and Pmt have homologues in yeasts and humans, where they are required for protein O-mannosylation. Actinobacteria also use PPM for lipoglycan biosynthesis. Here we show that ppm1 mutants of Streptomyces coelicolor have increased susceptibility to a number of antibiotics that target cell wall biosynthesis. The pmt mutants also have mildly increased antibiotic susceptibilities, in particular to β-lactams and vancomycin. Despite normal induction of the vancomycin gene cluster, vanSRJKHAX, the pmt and ppm1 mutants remained highly vancomycin sensitive indicating that the mechanism of resistance is blocked post-transcriptionally. Differential RNA expression analysis indicated that catabolic pathways were downregulated and anabolic ones upregulated in the ppm1 mutant compared to the parent or complemented strains. Of note was the increase in expression of fatty acid biosynthetic genes in the ppm1-mutant. A change in lipid composition was confirmed using Raman spectroscopy, which showed that the ppm1-mutant had a greater relative proportion of unsaturated fatty acids compared to the parent or the complemented mutant. Taken together, these data suggest that an inability to synthesize PPM (ppm1) and loss of the glycoproteome (pmt-mutant) can detrimentally affect membrane or cell envelope functions leading to loss of intrinsic and, in the case of vancomycin, acquired antibiotic resistance

The N terminus of α-ENaC mediates ENaC cleavage and activation by furin

Epithelial Na+ channels comprise three homologous subunits (α, β, and γ) that are regulated by alternative splicing and proteolytic cleavage. Here, we determine the basis of the reduced Na+ current (INa) that results from expression of a previously identified, naturally occurring splice variant of the a subunit (α-ENaC), in which residues 34-82 are deleted (αΔ34-82). αΔ34-82-ENaC expression with WT β and γ subunits in Xenopus oocytes produces reduced basal INa, which can largely be recovered by exogenous trypsin. With this αΔ34-82-containing ENaC, both α and γ subunits display decreased cleavage fragments, consistent with reduced processing by furin or furin-like convertases. Data using MTS ET modification of a cysteine, introduced into the degenerin locus in β-ENaC, suggest that the reduced INa of αΔ34-82-ENaC arises from an increased population of uncleaved, near-silent ENaC, rather than from a reduced open probability spread uniformly across all channels. After treatment with brefeldin A to disrupt anterograde trafficking of channel subunits, INa in oocytes expressing αΔ34-82-ENaC is reestablished more slowly than that in oocytes expressing WT ENaC. Overnight or acute incubation of oocytes expressing WT ENaC in the pore blocker amiloride increases basal ENaC proteolytic stimulation, consistent with relief of Na+ feedback inhibition. These responses are reduced in oocytes expressing αΔ34-82-ENaC. We conclude that the α-ENaC N terminus mediates interactions that govern the delivery of cleaved and uncleaved ENaC populations to the oocyte membrane

Increased plasma membrane cholesterol in cystic fibrosis cells correlates with CFTR genotype and depends on de novo cholesterol synthesis

<p>Abstract</p> <p>Background</p> <p>Previous observations demonstrate that <it>Cftr</it>-null cells and tissues exhibit alterations in cholesterol processing including perinuclear cholesterol accumulation, increased <it>de novo </it>synthesis, and an increase in plasma membrane cholesterol accessibility compared to wild type controls. The hypothesis of this study is that membrane cholesterol accessibility correlates with CFTR genotype and is in part influenced by <it>de novo </it>cholesterol synthesis.</p> <p>Methods</p> <p>Electrochemical detection of cholesterol at the plasma membrane is achieved with capillary microelectrodes with a modified platinum coil that accepts covalent attachment of cholesterol oxidase. Modified electrodes absent cholesterol oxidase serves as a baseline control. Cholesterol synthesis is determined by deuterium incorporation into lipids over time. Incorporation into cholesterol specifically is determined by mass spectrometry analysis. All mice used in the study are on a C57Bl/6 background and are between 6 and 8 weeks of age.</p> <p>Results</p> <p>Membrane cholesterol measurements are elevated in both R117H and ΔF508 mouse nasal epithelium compared to age-matched sibling wt controls demonstrating a genotype correlation to membrane cholesterol detection. Expression of wt CFTR in CF epithelial cells reverts membrane cholesterol to WT levels further demonstrating the impact of CFTR on these processes. In wt epithelial cell, the addition of the CFTR inhibitors, Gly H101 or CFTR_inh-172, for 24 h surprisingly results in an initial drop in membrane cholesterol measurement followed by a rebound at 72 h suggesting a feedback mechanism may be driving the increase in membrane cholesterol. <it>De novo </it>cholesterol synthesis contributes to membrane cholesterol accessibility.</p> <p>Conclusions</p> <p>The data in this study suggest that CFTR influences cholesterol trafficking to the plasma membrane, which when depleted, leads to an increase in <it>de novo </it>cholesterol synthesis to restore membrane content.</p

Delayed enzymatic debridement in severe burns: Proof of concept

Introduction Enzymatic debridement (ED) is a novel powerful therapy for debridement of severe burns. Standard ED is usually performed within 72 h after injury following a presoaking phase. Little evidence exists on the effectiveness of ED later than 72 h after trauma. In this retrospective study, we compared outcomes of burn patients treated within versus later than 72 h after injury. Patients and Methods 110 patients with severe burns treated with ED between 2016 and 2020 were evaluated. Patients treated later than 72 h after trauma were identified and matched to a control group treated within 72 h. Matching criteria included age, area treated with ED, and localization of ED. Exclusion criteria were abbreviated burn severity index (ABSI) greater than 12 and death within the first 10 days after burn injury. Primary outcomes were time to full epithelialization and number of secondary surgical interventions. Results 16 patients (11 female, 5 male) matched the inclusion criteria and were assigned to the late treatment group. Mean age was 54.0 ± 19.0 years, the = and mean ABSI score 6.3 ± 3.2. 16 matched patients were assigned to the early ED group. Secondary surgical procedures were performed in 62.5% of cases in both groups with a mean of 1.7 (late treatment) vs. 2.2 (control; p = 0.29) secondary procedures in each group, respectively. No significant difference between groups regarding time to complete epithelialization (28.2 days vs. 27.3 days, p = 0.45) was observed. Infection rate was higher (18.8% vs. 6.3%, p = 0.28) in the delayed group. Conclusion Delayed ED is a feasible procedure as part of personalized care in burn surgery. In our retrospective study, we could not identify r safety issues except a slightly higher infection rate. This may however be attributed to delayed initiation of burn treatment itself

Accumulation and persistence of ivacaftor in airway epithelia with prolonged treatment

Background: Current dosing strategies of CFTR modulators are based on serum pharmacokinetics, but drug concentrations in target tissues such as airway epithelia are not known. Previous data suggest that CFTR modulators may accumulate in airway epithelia, and serum pharmacokinetics may not accurately predict effects of chronic treatment. Methods: CF (F508del homozygous) primary human bronchial epithelial (HBE) cells grown at air-liquid interface were treated for 14 days with ivacaftor plus lumacaftor or ivacaftor plus tezacaftor, followed by a 14-day washout period. At various intervals during treatment and washout phases, drug concentrations were measured via mass spectrometry, electrophysiological function was assessed in Ussing chambers, and mature CFTR protein was quantified by Western blotting. Results: During treatment, ivacaftor accumulated in CF-HBEs to a much greater extent than either lumacaftor or tezacaftor and remained persistently elevated even after 14 days of washout. CFTR activity peaked at 7 days of treatment but diminished with further ivacaftor accumulation, though remained above baseline even after washout. Conclusions: Intracellular accrual and persistence of CFTR modulators during and after chronic treatment suggest complex pharmacokinetic and pharmacodynamic properties within airway epithelia that are not predicted by serum pharmacokinetics. Direct measurement of drugs in target tissues may be needed to optimize dosing strategies, and the persistence of CFTR modulators after treatment cessation has implications for personalized medicine approaches

ScotGrid: Providing an Effective Distributed Tier-2 in the LHC Era

ScotGrid is a distributed Tier-2 centre in the UK with sites in Durham, Edinburgh and Glasgow. ScotGrid has undergone a huge expansion in hardware in anticipation of the LHC and now provides more than 4MSI2K and 500TB to the LHC VOs. Scaling up to this level of provision has brought many challenges to the Tier-2 and we show in this paper how we have adopted new methods of organising the centres, from fabric management and monitoring to remote management of sites to management and operational procedures, to meet these challenges. We describe how we have coped with different operational models at the sites, where Glagsow and Durham sites are managed "in house" but resources at Edinburgh are managed as a central university resource. This required the adoption of a different fabric management model at Edinburgh and a special engagement with the cluster managers. Challenges arose from the different job models of local and grid submission that required special attention to resolve. We show how ScotGrid has successfully provided an infrastructure for ATLAS and LHCb Monte Carlo production. Special attention has been paid to ensuring that user analysis functions efficiently, which has required optimisation of local storage and networking to cope with the demands of user analysis. Finally, although these Tier-2 resources are pledged to the whole VO, we have established close links with our local physics user communities as being the best way to ensure that the Tier-2 functions effectively as a part of the LHC grid computing framework..Comment: Preprint for 17th International Conference on Computing in High Energy and Nuclear Physics, 7 pages, 1 figur

Restoration of R117H CFTR folding and function in human airway cells through combination treatment with VX-809 and VX-770

Cystic fibrosis (CF) is a lethal recessive genetic disease caused primarily by the F508del mutation in the CF transmembrane conductance regulator (CFTR). The potentiator VX-770 was the first CFTR modulator approved by the FDA for treatment of CF patients with the gating mutation G551D. Orkambi is a drug containing VX-770 and corrector VX809 and is approved for treatment of CF patients homozygous for F508del, which has folding and gating defects. At least 30% of CF patients are heterozygous for the F508del mutation with the other allele encoding for one of many different rare CFTR mutations. Treatment of heterozygous F508del patients with VX-809 and VX-770 has had limited success, so it is important to identify heterozygous patients that respond to CFTR modulator therapy. R117H is a more prevalent rare mutation found in over 2,000 CF patients. In this study we investigated the effectiveness of VX-809/VX-770 therapy on restoring CFTR function in human bronchial epithelial (HBE) cells from R117H/F508del CF patients. We found that VX-809 stimulated more CFTR activity in R117H/F508del HBEs than in F508del/F508del HBEs. R117H expressed exclusively in immortalized HBEs exhibited a folding defect, was retained in the ER, and degraded prematurely. VX-809 corrected the R117H folding defect and restored channel function. Because R117 is involved in ion conductance, VX-770 acted additively with VX-809 to restore CFTR function in chronically treated R117H/F508del cells. Although treatment of R117H patients with VX-770 has been approved, our studies indicate that Orkambi may be more beneficial for rescue of CFTR function in these patients

Regulation of the epithelial Na+ channel and airway surface liquid volume by serine proteases

Mammalian airways are protected from infection by a thin film of airway surface liquid (ASL) which covers airway epithelial surfaces and acts as a lubricant to keep mucus from adhering to the epithelial surface. Precise regulation of ASL volume is essential for efficient mucus clearance and too great a reduction in ASL volume causes mucus dehydration and mucus stasis which contributes to chronic airway infection. The epithelial Na+ channel (ENaC) is the rate-limiting step that governs Na+ absorption in the airways. Recent in vitro and in vivo data have demonstrated that ENaC is a critical determinant of ASL volume and hence mucus clearance. ENaC must be cleaved by either intracellular furin-type proteases or extracellular serine proteases to be active and conduct Na+, and this process can be inhibited by protease inhibitors. ENaC can be regulated by multiple pathways, and once proteolytically cleaved ENaC may then be inhibited by intracellular second messengers such as cAMP and PIP2. In the airways, however, regulation of ENaC by proteases seems to be the predominant mode of regulation since knockdown of either endogenous serine proteases such as prostasin, or inhibitors of ENaC proteolysis such as SPLUNC1, has large effects on ENaC activity in airway epithelia. In this review, we shall discuss how ENaC is proteolytically cleaved, how this process can regulate ASL volume, and how its failure to operate correctly may contribute to chronic airway disease