A defective flexible loop contributes to the processing and gating defects of the predominant cystic fibrosis-causing mutation.

FASEB J. 2019 Jan 22;:fj201801218RR

Authors: Chen X, Zhu S, Zhenin M, Xu W, Bose SJ, Wong MP, Leung GPH, Senderowitz H, Chen JH

Abstract
People with the genetic disease cystic fibrosis (CF) often carry a deletion mutation ΔF508 on the gene encoding the CF transmembrane conductance regulator (CFTR) Cl- channel. This mutation greatly reduces the CFTR maturation process and slows the channel opening rate. Here, we investigate whether residues near F508 contribute to these defects in ΔF508-CFTR. Most deletion mutations, but not alanine substitutions, of individual residues from positions 503 to 513 impaired CFTR maturation. Interestingly, only protein processing of ΔY512-CFTR, like that of ΔF508-CFTR, was greatly improved by low-temperature culture at 27°C or small-molecule corrector C18. The 2 mutant Cl- channels were equally slow to open, suggesting that they may share common structural flaws. Studies on the H3-H4 loop that links residues F508 and Y512 demonstrate that G509A/V510G mutations, moving G509 1 position backward in the loop, markedly enhanced ΔF508-CFTR maturation and opening rate while promoting protein stability and persistence of the H3 helix in ΔF508 nucleotide-binding domain 1. Moreover, V510A/S511A mutations noticeably increased ΔY512-CFTR maturation at 27°C and its opening rate. Thus, loop abnormalities may contribute to ΔF508- and ΔY512-CFTR defects. Importantly, correcting defects from G509 displacement in ΔF508-CFTR may offer a new avenue for drug discovery and CF treatments.-Chen, X., Zhu, S., Zhenin, M., Xu, W., Bose, S. J., Wong, M.P.-F., Leung, G.P.H., Senderowitz, H., Chen, J.-H. A defective flexible loop contributes to the processing and gating defects of the predominant cystic fibrosis-causing mutation.

PMID: 30668920 [PubMed - as supplied by publisher]

Cost Effectiveness of Inhaled Mannitol (Bronchitol®) in Patients with Cystic Fibrosis.

Pharmacoeconomics. 2019 Jan 22;:

Authors: Warren E, Morgan K, Toward TJ, Schwenkglenks M, Leadbetter J

Abstract
BACKGROUND: Inhaled mannitol (Bronchitol®) is licensed in Australia as a safe and efficacious addition to best supportive care in patients with cystic fibrosis.
OBJECTIVE: The objective of this study was to assess the cost effectiveness of inhaled mannitol (in addition to best supportive care) in the Australian setting from the perspective of a government-funded national healthcare system.
METHODS: A probabilistic patient-level simulation Markov model estimated life-time costs and outcomes of mannitol when added to best supportive care, compared with best supportive care alone in patients aged 6 years and older. We estimated treatment-related inputs (initial change in percentage of predicted forced expiratory volume, relative reduction in severe pulmonary exacerbations, and treatment discontinuations) from two phase III trials. Longer term natural history rates of predicted forced expiratory volume decline over time and severe pulmonary exacerbation rates for best supportive care were taken from Australian CF registries. The utility value for the cystic fibrosis health state was as measured in the trials using the Health Utility Index, whereas the impact of pulmonary exacerbations and lung transplantation on utility was ascertained from the published literature. The underlying cost of managing cystic fibrosis, and the cost associated with pulmonary exacerbations and transplantations was taken from published Australian sources.
RESULTS: The addition of inhaled mannitol to best supportive care resulted in a discounted cost per quality-adjusted life-year of AU$39,165. The result was robust with 77% of probabilistic sensitivity analysis samples below a willingness-to-pay threshold of AU$45,000/quality-adjusted life-year.
CONCLUSION: Benchmarked against an implicit Australian willingness-to-pay threshold for life-threatening diseases, our model suggests inhaled mannitol provides a cost-effective addition to best supportive care in patients with cystic fibrosis, irrespective of concomitant dornase alfa use.

PMID: 30666534 [PubMed - as supplied by publisher]

Molecular characterization of the pilS2 gene and its association with the frequency of Pseudomonas aeruginosa plasmid pKLC102 and PAPI-1 pathogenicity island.

Infect Drug Resist. 2019;12:221-227

Authors: Bahramian A, Khoshnood S, Shariati A, Doustdar F, Chirani AS, Heidary M

Abstract
Introduction: Pseudomonas aeruginosa is the most common opportunistic pathogen associated with a broad range of infections, including cystic fibrosis, ocular, otitis media, and burn infections. The aim of this study was to show the frequency of the pilS2 gene, and its association with P. aeruginosa plasmid pKLC102 and PAPI-1 pathogenicity island among P. aeruginosa strains.
Methods: The samples were collected from patients with cystic fibrosis, ocular, otitis media, and burn infections between January 2016 and November 2017. DNA was extracted using the DNA extraction kit and was used for PCR assay. PCR with 4 primer-pairs including 976 F/PAPI-1R, 4542 F/intF, SojR/4541 F, and intF/sojR was performed to identify PAPI-1. pKLC102 was detected using three other primer-pairs including cp10F/cp10R, cp44F/cp44R, and cp97F/cp97R.
Results: A total of 112 P. aeruginosa isolates were collected from patients with cystic fibrosis (36), burn (20), otitis media (26), and ocular (30) infections. The results of PCR showed that pilS2 gene was identified in 96 (85%) strains. PAPI-1-attB integration was detected among 38 (33.9%) isolates and the circular form of PAPI-1 detected among 17 (14%) isolates. In addition, 79 (70.5%) strains were found to be positive for pKLC102.
Conclusion: We found that the majority of the isolates may be susceptible to transfer this significant island and the related element pKLC102 into recipient isolates lacking the island owing to high association of the PilS2 pilus with the islands in the studied strains. It is anticipated that strains isolated from burn and eye with the highest rate of PilS2, PAPI-1, and pKLC102 association have a high level of antibiotic resistance.

PMID: 30666137 [PubMed]

Visceral adipose tissue is associated with poor diet quality and higher fasting glucose in adults with cystic fibrosis.

J Cyst Fibros. 2019 Jan 18;:

Authors: Bellissimo MP, Zhang I, Ivie EA, Tran PH, Tangpricha V, Hunt WR, Stecenko AA, Ziegler TR, Alvarez JA

Abstract
BACKGROUND: Body fat distribution and diet quality influence clinical outcomes in general populations but are understudied in individuals with cystic fibrosis (CF). The aim of this pilot study was to assess body fat distribution and diet quality in relation to fasting glucose and lung function in adults with CF.
METHODS: Subjects were 24 adults (ages 18-50) with CF and 25 age-matched controls. The Healthy Eating Index 2015 (HEI-2015) was calculated from 3-day food records and data were adjusted per 1000 kcal. Whole and regional body composition, including visceral adipose tissue (VAT), was assessed by dual energy X-ray absorptiometry.
RESULTS: Subjects with CF reported more added sugar intake [26.1 (IQR 18.1) vs. 12.9 (12.5) g/1000 kcal, p < 0.001] and had lower HEI-2015 scores [48.3 (IQR 9.9) vs. 63.9 (27.3), p < 0.001] compared to controls. There were no differences in BMI, total body fat, or lean body mass (LBM) between subjects with CF and controls (p > 0.05 for all), although subjects with CF had higher VAT than control subjects [0.3 (IQR 0.3) vs 0.1 (0.3) kg, p = 0.02]. Among subjects with CF, VAT was positively associated with added sugar intake (p < 0.001) and fasting blood glucose (p = 0.04). Lung function was positively associated with BMI (p = 0.005) and LBM (p = 0.03) but not with adiposity indicators.
CONCLUSIONS: These novel data link body fat distribution with diet quality and fasting glucose levels in adults with CF, whereas LBM was associated with lung function. This study highlights the importance of increasing diet quality and assessing body composition and fat distribution in the CF population.

PMID: 30665857 [PubMed - as supplied by publisher]

Lack of GAS2L2 Causes PCD by Impairing Cilia Orientation and Mucociliary Clearance.

Am J Hum Genet. 2019 Jan 18;:

Authors: Bustamante-Marin XM, Yin WN, Sears PR, Werner ME, Brotslaw EJ, Mitchell BJ, Jania CM, Zeman KL, Rogers TD, Herring LE, Refabért L, Thomas L, Amselem S, Escudier E, Legendre M, Grubb BR, Knowles MR, Zariwala MA, Ostrowski LE

Abstract
Primary ciliary dyskinesia (PCD) is a genetic disorder in which impaired ciliary function leads to chronic airway disease. Exome sequencing of a PCD subject identified an apparent homozygous frameshift variant, c.887_890delTAAG (p.Val296Glyfs∗13), in exon 5; this frameshift introduces a stop codon in amino acid 308 of the growth arrest-specific protein 2-like 2 (GAS2L2). Further genetic screening of unrelated PCD subjects identified a second proband with a compound heterozygous variant carrying the identical frameshift variant and a large deletion (c.867_∗343+1207del; p.?) starting in exon 5. Both individuals had clinical features of PCD but normal ciliary axoneme structure. In this research, using human nasal cells, mouse models, and X.laevis embryos, we show that GAS2L2 is abundant at the apical surface of ciliated cells, where it localizes with basal bodies, basal feet, rootlets, and actin filaments. Cultured GAS2L2-deficient nasal epithelial cells from one of the affected individuals showed defects in ciliary orientation and had an asynchronous and hyperkinetic (GAS2L2-deficient = 19.8 Hz versus control = 15.8 Hz) ciliary-beat pattern. These results were recapitulated in Gas2l2-/- mouse tracheal epithelial cell (mTEC) cultures and in X. laevis embryos treated with Gas2l2 morpholinos. In mice, the absence of Gas2l2 caused neonatal death, and the conditional deletion of Gas2l2 impaired mucociliary clearance (MCC) and led to mucus accumulation. These results show that a pathogenic variant in GAS2L2 causes a genetic defect in ciliary orientation and impairs MCC and results in PCD.

PMID: 30665704 [PubMed - as supplied by publisher]

Take it personally: how personal we reach when we are so different from each other?

J Cyst Fibros. 2019 Jan;18(1):6-7

Authors: Kerem E, Oren YS, Kerem B

PMID: 30665545 [PubMed - in process]

JCF - progress in 2018.

J Cyst Fibros. 2019 Jan;18(1):1-5

Authors: Bell SC, Castellani C, Flume PA

PMID: 30665544 [PubMed - in process]

An evaluation of methods for the isolation of nontuberculous mycobacteria from patients with cystic fibrosis, bronchiectasis and patients assessed for lung transplantation.

BMC Pulm Med. 2019 Jan 21;19(1):19

Authors: Stephenson D, Perry A, Appleby MR, Lee D, Davison J, Johnston A, Jones AL, Nelson A, Bourke SJ, Thomas MF, De Soyza A, Lordan JL, Lumb J, Robb AE, Samuel JR, Walton KE, Perry JD

Abstract
BACKGROUND: RGM medium is an agar-based, selective culture medium designed for the isolation of nontuberculous mycobacteria (NTM) from the sputum of patients with cystic fibrosis (CF). We evaluated RGM medium for the detection of NTM in patients with CF (405 samples), bronchiectasis (323 samples) and other lung diseases necessitating lung transplantation (274 samples).
METHODS: In total, 1002 respiratory samples from 676 patients were included in the study. Direct culture on RGM medium, with incubation at two temperatures (30 °C and 37 °C), was compared with conventional culture of decontaminated samples for acid-fast bacilli (AFB) using both a solid medium (Löwenstein-Jensen medium) and a liquid medium (the Mycobacterial Growth Indicator Tube; MGIT).
RESULTS: For all three patient groups, significantly more isolates of NTM were recovered using RGM medium incubated at 30 °C than by any other method (sensitivity: 94.6% vs. 22.4% for conventional AFB culture; P < 0.0001). Significantly more isolates of Mycobacterium abscessus complex were isolated on RGM at 30 °C than by AFB culture (sensitivity: 96.1% vs. 58.8%; P < 0.0001). The recovery of Mycobacterium avium complex was also greater using RGM medium at 30 °C compared to AFB culture (sensitivity: 83% vs. 70.2%), although this difference was not statistically significant and a combination of methods was necessary for optimal recovery (P = 0.21).
CONCLUSIONS: In the largest study of RGM medium to date, we reaffirm its utility for isolation of NTM from patients with CF. Furthermore; we show that it also provides an effective tool for culture of respiratory samples from patients with bronchiectasis and other lung diseases.

PMID: 30665395 [PubMed - in process]

Penicillin binding protein 3 is a common adaptive target among Pseudomonas aeruginosa isolates from adult cystic fibrosis patients treated with β-lactams.

Int J Antimicrob Agents. 2019 Jan 18;:

Authors: Clark ST, Sinha U, Zhang Y, Wang PW, Donaldson SL, Coburn B, Waters VJ, Yau YCW, Tullis DE, Guttman DS, Hwang DM

Abstract
Determining the mechanisms that modulate β-lactam resistance in clinical P. aeruginosa isolates can be challenging, as the molecular profiles identified in mutation- or expression-based resistance determinant screens may not correlate with in vitro phenotypes. One of the lesser studied resistance mechanisms in P. aeruginosa is the modification of penicillin binding protein 3 (pbpB/ftsI). Here, we report that nonsynonymous polymorphisms within pbpB frequently occur among β-lactam resistant sputum isolates, and are associated with unique antibiotic susceptibility patterns. Longitudinally collected isolates (n=126) from cystic fibrosis (CF) patients with or without recent β-lactam therapy or of non-clinical origin were tested for susceptibility to six β-lactams (aztreonam, ceftazidime, cefsulodin, cefepime, meropenem and piperacillin). Known β-lactam resistance mechanisms were characterized by PCR-based methods, and polymorphisms in the transpeptidase-encoding domain of pbpB identified by sequencing. Twelve nonsynonymous polymorphisms were detected among 86 isolates (67%) from five CF patients with a history of β-lactam therapy, compared with only one polymorphism in 30 (3.3%) from three patients who had not received β-lactam treatments. No nonsynonymous polymorphisms were found in ten environmental isolates. Multiple pbpB alleles, often with different combinations of polymorphisms, were detected within the population of strains from each CF patient for up to 2.6 years. Traditional patterns of ampC or mexA de-repression, reduced expression of oprD or the presence of extended spectrum β-lactamases were not observed in resistant isolates with nonsynonymous polymorphisms in pbpB. Our findings suggest that pbpB is a common adaptive target, and may contribute to the development of β-lactam resistance in P. aeruginosa.

PMID: 30664925 [PubMed - as supplied by publisher]

Lung Allocation Score Thresholds Prioritize Survival after Lung Transplantation.

Chest. 2019 Jan 18;:

Authors: Li SS, Miller R, Tumin D, Stewart WCL, Tobias JD, Hayes D

Abstract
BACKGROUND: The Lung Allocation Score (LAS) prioritizes lung transplant (LTx) candidates with poor transplant-free survival and expected survival benefit from LTx. Although patients with the highest LAS have the shortest waiting time, mortality benefit is unclear in this group, raising criticism that LAS inappropriately prioritizes critically ill candidates. We aim to identify a threshold above which increasing LAS values do not predict increasing survival benefit.
METHODS: The United Network for Organ Sharing Registry was queried for first-time adult LTx candidates with LAS ≥30 between May 2005 and December 2016. Survival was tracked from the time of listing through the post-transplant period, and compared to survival while remaining on the waitlist using proportional hazards regression. The survival benefit of LTx was modeled as a piecewise-constant time-dependent covariate, moderated by candidate LAS.
RESULTS: Of the overall cohort (N=21,157), LTx was particularly protective for 365 patients with initial LAS of 70-79 (hazard ratio [HR] of death after undergoing LTx relative to remaining on the waitlist=0.2; 95% confidence interval [CI]: 0.1, 0.3). However, the survival benefit of LTx benefit did not meaningfully increase for 1,042 patients listed with even higher LAS. Among cystic fibrosis (CF) patients, the survival benefit of LTx was constant above LAS of approximately 50.
CONCLUSIONS: Consistent survival benefit of LTx was observed among patients with initial LAS of 70 and greater. This result supports equalizing priority for donor lung allocation for patients with LAS ≥70. A lower LAS threshold for maximum priority is indicated in patients with CF.

PMID: 30664859 [PubMed - as supplied by publisher]

The Effect of Pulmonary Rehabilitation on the Physical Activity Level and General Clinical Status of Patients with Bronchiectasis.

Turk Thorac J. 2019 Jan 01;20(1):30-35

Authors: Pehlivan E, Niksarlıoğlu EY, Balcı A, Kılıç L

Abstract
OBJECTIVES: We aimed to investigate the effects of pulmonary rehabilitation (PR) on the level of physical activity (PA) and the general clinical status in adult patients with non-cystic fibrosis bronchiectasis.
MATERIALS AND METHODS: The patients were included prospectively in the study and followed a home-based PR program for 2 months comprising breathing exercises, training in chest hygiene techniques, peripheral muscle strengthening training, and self-walking. The outcomes measurements were the following: 6-minute walking distance, pulmonary function test, peripheral and respiratory muscle strength measurements, International Physical Activity Questionnaire (IPAQ), Saint George Respiratory Questionnaire, and modified Medical Research Council dyspnea scores.
RESULTS: Of the total 25 patients included in the study, six were excluded due to follow-up and adherence problems. A comparison of the outcome measures recorded before and after PR showed statistically significant improvements in the IPAQ total (p=0.015) and walking scores (p=0.011). While the proportion of patients in the low PA category was 73% (n=14) prior to PR, this rate decreased to 42% (n=8) post-PR. The proportion of patients in the moderate PA category was 26% (n=5) prior to PR and increased to 52% (n=10) post-PR. While positive improvements were seen in all clinically monitored parameters, aside from spirometric values, these changes did not reach a statistically significant level.
CONCLUSION: The majority of patients with bronchiectasis have a low level of PA. PR ensures positive improvements in the level of PA and general physical clinical status of such patients.

PMID: 30664424 [PubMed]

Anaerobes in cystic fibrosis patients' airways.

Crit Rev Microbiol. 2019 Jan 21;:1-15

Authors: Lamoureux C, Guilloux CA, Beauruelle C, Jolivet-Gougeon A, Héry-Arnaud G

Abstract
Anaerobes are known to constitute an important part of the airway microbiota in both healthy subjects and cystic fibrosis (CF) patients. Studies on the potential role of anaerobic bacteria in CF and thus their involvement in CF pathophysiology have reported contradictory results, and the question is still not elucidated. The aim of this study was to summarize anaerobe diversity in the airway microbiota and its potential role in CF, to provide an overview of the state of knowledge on anaerobe antibiotic resistances (resistome), and to investigate the detectable metabolites produced by anaerobes in CF airways (metabolome). This review emphasizes key metabolites produced by strict anaerobic bacteria (sphingolipids, fermentation-induced metabolites and metabolites involved in quorum-sensing), which may be essential for the better understanding of lung disease pathophysiology in CF.

PMID: 30663924 [PubMed - as supplied by publisher]

CF derived scoring systems do not fully describe the range of structural changes seen on CT scans in PCD.

Pediatr Pulmonol. 2019 Jan 21;:

Authors: Tadd K, Morgan L, Rosenow T, Schultz A, Susanto C, Murray C, Robinson P

Abstract
BACKGROUND: Structural lung changes seen on computed tomography (CT) scans in Cystic Fibrosis (CF) and Primary Ciliary Dyskinesia (PCD) are currently described using scoring systems derived from CF populations. This practise assumes lung damage in the two conditions is identical, potentially resulting in a failure to identify PCD-specific changes. Our study addresses this assumption.
METHODS: A total of 58 CT scans from 41 PCD patients (age 2-48 years) were examined and the presence and extent of abnormalities common in CF; bronchiectasis, bronchial wall thickening, atelectasis, mucous plugging, and air trapping noted. Further assessment of the PCD scans by an experienced chest radiologist identified several unique PCD specific changes.
RESULTS: Bronchial wall thickening was the commonest abnormality seen in PCD. All abnormalities were present more often in middle and lower lobes than in upper lobes (P < 0.001). Bronchiectasis, mucus plugging, atelectasis, and air trapping were present more often in PCD than in the historic CF cohorts which formed the basis of two CF scoring systems (P < 0.05). Extensive tree-in-bud pattern of mucus plugging, thickening of interlobar, and interlobular septa, and whole lobe atelectasis were seen significantly more frequently in PCD than CF.
CONCLUSIONS: Structural changes identified on CT scans in PCD are not identical to those previously described in CF patients and suggest assessment of PCD structural changes on CT should not use CF derived scoring systems.

PMID: 30663844 [PubMed - as supplied by publisher]

Lung disease and vitamin D levels in cystic fibrosis infants and preschoolers.

Pediatr Pulmonol. 2019 Jan 20;:

Authors: Oliveira MS, Matsunaga NY, Rodrigues MLE, Morcillo AM, de Oliveira Ribeiro MAG, Ribeiro AF, de Fátima C P Servidoni M, Nogueira RJN, Pereira MC, Ribeiro JD, Toro AADC

Abstract
INTRODUCTION: Vitamin D acts on the immune system and lung response. Patients with cystic fibrosis (CF) may be deficient in this vitamin. The aims of the study were to evaluate vitamin D levels and severity of lung disease in infants and preschoolers diagnosed with CF, and to compare them to a group of children without pancreatic insufficiency (PI).
METHODS: Patients with CF up to 4 years old were included, and compared to an age-matched group of children without diagnosis of CF. CF group had medical records and High Resolution Thorax Computed Tomography (HRCCT) evaluated in order to verify the severity of lung disease. Information on demographic data, sun exposure habits, supplemental vitamin D therapy, and on the season at the time of vitamin D sampling were collected for both groups.
RESULTS: This study included 45 patients in the CF group and 102 in the non-CF group, with no differences in age (P = 0.327) between them. There was no association between vitamin D levels and markers of lung disease in the CF group. The non-CF group had lower daily sun exposure (P = 0.034), and lower supplementation than the CF group (P < 0.001). Supplementation and seasonality were the determinant variables for vitamin D levels, which were lower for non-supplemented children and for assessments during fall/winter.
CONCLUSION: There was no association between lung disease severity and vitamin D levels in CF group. Supplementation and seasonality were associated to higher vitamin levels.

PMID: 30663283 [PubMed - as supplied by publisher]

Animal Models in the Pathophysiology of Cystic Fibrosis.

Front Pharmacol. 2018;9:1475

Authors: Semaniakou A, Croll RP, Chappe V

Abstract
Our understanding of the multiorgan pathology of cystic fibrosis (CF) has improved impressively during the last decades, but we still lack a full comprehension of the disease progression. Animal models have greatly contributed to the elucidation of specific mechanisms involved in CF pathophysiology and the development of new therapies. Soon after the cloning of the CF transmembrane conductance regulator (CFTR) gene in 1989, the first mouse model was generated and this model has dominated in vivo CF research ever since. Nonetheless, the failure of murine models to mirror human disease severity in the pancreas and lung has led to the generation of larger animal models such as pigs and ferrets. The following review presents and discusses data from the current animal models used in CF research.

PMID: 30662403 [PubMed]

The nutritional status in CF: Being certain about the uncertainties.

Clin Nutr ESPEN. 2019 Feb;29:15-21

Authors: Declercq D, Van Meerhaeghe S, Marchand S, Van Braeckel E, van Daele S, De Baets F, Van Biervliet S

Abstract
BACKGROUND: Nutritional therapy is one of the cornerstones in cystic fibrosis (CF) therapy. There is a strong association between nutritional status and pulmonary function and thus longevity. Therefore nutritional therapy should be continuously adapted to preserve or improve the nutritional status. This narrative review was written to reconsider nutritional therapy in CF based on the latest evidence available since the publication of the ESPEN - ESPGHAN - ECFS guidelines on nutrition care for infants, children and adults with CF.
METHODS: A literature search in Pubmed, Scopus and Web of Science was conducted to identify new research focusing on the use of growth charts, body composition, protein intake and pancreatic enzyme therapy (PERT) in CF between June 2014 and June 2017.
RESULTS: The search strategy resulted in a total of 1810 hits across the databases. After reviewing title and abstract only 17 studies were included of which 2 animal studies. The use of growth charts was discussed in 3 studies, body composition in 6, protein intake and digestion in 4 and PERT in 4.
CONCLUSION: According to the current guidelines and the available evidence of the discussed topics, it is important that the nutritional therapy in CF is redefined according to age, pancreatic function and disease stage. Macronutrients balances are of importance and change over lifetime. As a consequence an accurate PERT intake is required and thus further research on timing and dosage is necessary. To improve the nutritional assessment a proper use of the growth charts and a consensus on body composition measurements, references and thresholds is advised.

PMID: 30661680 [PubMed - in process]

Heterogeneous production of proteases from Brazilian clinical isolates of Pseudomonas aeruginosa.

Enferm Infecc Microbiol Clin. 2017 Dec;35(10):630-637

Authors: Galdino ACM, Viganor L, Ziccardi M, Nunes APF, Dos Santos KRN, Branquinha MH, Santos ALS

Abstract
BACKGROUND: Pseudomonas aeruginosa is an important human pathogen that causes severe infections in a wide range of immunosuppressed patients. Herein, we evaluated the proteolytic profiles of 96 Brazilian clinical isolates of P. aeruginosa recovered from diverse anatomical sites.
METHODS: Cell-associated and extracellular proteases were evidenced by gelatin-SDS-PAGE and by the cleavage of soluble gelatin. Elastase was measured by using the peptide substrate N-succinyl-Ala-Ala-Ala-p-nitroanilide. The prevalence of elastase genes (lasA and lasB) was evaluated by PCR.
RESULTS: Bacterial extracts were initially applied on gelatin-SDS-PAGE and the results revealed four distinct zymographic profiles as follows: profile I (composed by bands of 145, 118 and 50kDa), profile II (118 and 50kDa), profile III (145kDa) and profile IV (118kDa). All the proteolytic enzymes were inhibited by EDTA, identifying them as metalloproteases. The profile I was the most detected in both cellular (79.2%) and extracellular (84.4%) extracts. Overall, gelatinase and elastase activities measured in the spent culture media were significantly higher (around 2-fold) compared to the cellular extracts and the production level varied according to the site of bacterial isolation. For instance, tracheal secretion isolates produced elevated amount of gelatinase and elastase measured in both cellular and extracellular extracts. The prevalence of elastase genes revealed that 100% isolates were lasB-positive and 85.42% lasA-positive. Some positive/negative correlations were showed concerning the production of gelatinase, elastase, isolation site and antimicrobial susceptibility.
CONCLUSION: The protease production was highly heterogeneous in Brazilian clinical isolates of P. aeruginosa, which corroborates the genomic/metabolic versatility of this pathogen.

PMID: 27480954 [PubMed - indexed for MEDLINE]

Secondhand smoke alters arachidonic acid metabolism and inflammation in infants and children with cystic fibrosis.

Thorax. 2019 Jan 19;:

Authors: Kopp BT, Thompson R, Kim J, Konstan R, Diaz A, Smith B, Shrestha C, Rogers LK, Hayes D, Tumin D, Woodley FW, Ramilo O, Sanders DB, Groner JA, Mejias A

Abstract
BACKGROUND: Mechanisms that facilitate early infection and inflammation in cystic fibrosis (CF) are unclear. We previously demonstrated that children with CF and parental-reported secondhand smoke exposure (SHSe) have increased susceptibility to bacterial infections. SHSe hinders arachidonic acid (AA) metabolites that mediate immune function in patients without CF, and may influence CF immune dysfunction. We aimed to define SHSe's impact on inflammation mediators and infection in children with CF.
METHODS: Seventy-seven children with CF <10 years of age (35 infants <1 year; 42 children 1-10 years) were enrolled and hair nicotine concentrations measured as an objective surrogate of SHSe. AA signalling by serum and macrophage lipidomics, inflammation using blood transcriptional profiles and in vitro macrophage responses to bacterial infection after SHSe were assessed.
RESULTS: Hair nicotine concentrations were elevated in 63% of patients. Of the AA metabolites measured by plasma lipidomics, prostaglandin D2 (PGD2) concentrations were decreased in children with CF exposed to SHSe, and associated with more frequent hospitalisations (p=0.007) and worsened weight z scores (p=0.008). Children with CF exposed to SHSe demonstrated decreased expression of the prostaglandin genes PTGES3 and PTGR2 and overexpression of inflammatory pathways. These findings were confirmed using an in vitro model, where SHSe was associated with a dose-dependent decrease in PGD2 and increased methicillin-resistant Staphylococcus aureus survival in human CF macrophages.
CONCLUSIONS: Infants and young children with CF and SHSe have altered AA metabolism and dysregulated inflammatory gene expression resulting in impaired bacterial clearance. Our findings identified potential therapeutic targets to halt early disease progression associated with SHSe in the young population with CF.

PMID: 30661024 [PubMed - as supplied by publisher]

Cystic fibrosis: a call for papers for ECFS 2019.

Lancet Respir Med. 2019 Jan 16;:

Authors: Grainger E

PMID: 30660510 [PubMed - as supplied by publisher]

Folding-function relationship of the most common cystic fibrosis-causing CFTR conductance mutants.

Life Sci Alliance. 2019 Feb;2(1):

Authors: van Willigen M, Vonk AM, Yeoh HY, Kruisselbrink E, Kleizen B, van der Ent CK, Egmond MR, de Jonge HR, Braakman I, Beekman JM, van der Sluijs P

Abstract
Cystic fibrosis is caused by mutations in the CFTR gene, which are subdivided into six classes. Mutants of classes III and IV reach the cell surface but have limited function. Most class-III and class-IV mutants respond well to the recently approved potentiator VX-770, which opens the channel. We here revisited function and folding of some class-IV mutants and discovered that R347P is the only one that leads to major defects in folding. By this criterion and by its functional response to corrector drug VX-809, R347P qualifies also as a class-II mutation. Other class-IV mutants folded like wild-type CFTR and responded similarly to VX-809, demonstrating how function and folding are connected. Studies on both types of defects complement each other in understanding how compounds improve mutant CFTR function. This provides an attractive unbiased approach for characterizing mode of action of novel therapeutic compounds and helps address which drugs are efficacious for each cystic fibrosis disease variant.

PMID: 30659068 [PubMed]