ECFS standards of care on CFTR-related disorders: Diagnostic criteria of CFTR dysfunction.

The spectrum of disorders involving CFTR (cystic fibrosis transmembrane conductance regulator) dysfunction correlates with a continuous gradient of CFTR function defined by the combination of two allelic CFTR variants. CFTR-related disorders are clinical entities with features of cystic fibrosis (CF) and evidence for presence of CFTR dysfunction but not meeting criteria for diagnosis of CF. Individuals with CFTR-RDs demonstrate a wide range of CFTR activity and are still under-recognized or misclassified. The level of CFTR dysfunction may be measured in vivo (sweat testing, nasal potential difference measurements) and/or by ex vivo tests (intestinal current measurement), or indirectly indicated by CFTR variants, as alteration in sequence of the CFTR gene translates into CFTR dysfunction. CFTR bioassays can aid in the diagnosis of individuals with CF, but we lack parameters to differentiate CF from CFTR-RD. In the era of the CFTR modulators and their potential clinical benefit, it is of utmost importance to diagnose CFTR-RD as unambiguously as possible. We therefore propose the following to define compatible CFTR dysfunction in a person with a suspected diagnosis of CFTR-RD : (1) evidence of CFTR dysfunction in vivo or ex vivo in at least two different CFTR functional test types, or (2) One CFTR variant known to reduce CFTR function and evidence of CFTR dysfunction in vivo or ex vivo in at least two different CFTR functional test types, or (3) Two CFTR variants shown to reduce CFTR function, with at most one CF-causing variant.

[CF Lung Disease - a German S3 Guideline: Module 2: Diagnostics and Treatment in Chronic Infection with Pseudomonas aeruginosa]. / S3-Leitlinie: Lungenerkrankung bei Mukoviszidose ­ Modul 2: Diagnostik und Therapie bei der chronischen Infektion mit Pseudomonas aeruginosa.

Cystic Fibrosis (CF) is the most common autosomal-recessive genetic disease affecting approximately 8000 people in Germany. The disease is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene leading to dysfunction of CFTR, a transmembrane chloride channel. This defect causes insufficient hydration of the epithelial lining fluid which leads to chronic inflammation of the airways. Recurrent infections of the airways as well as pulmonary exacerbations aggravate chronic inflammation, lead to pulmonary fibrosis and tissue destruction up to global respiratory insufficiency, which is responsible for the mortality in over 90 % of patients. The main aim of pulmonary treatment in CF is to reduce pulmonary inflammation and chronic infection. Pseudomonas aeruginosa (Pa) is the most relevant pathogen in the course of CF lung disease. Colonization and chronic infection are leading to additional loss of pulmonary function. There are many possibilities to treat Pa-infection. This is a S3-clinical guideline which implements a definition for chronic Pa-infection and demonstrates evidence-based diagnostic methods and medical treatment for Pa-infection in order to give guidance for individual treatment options.

[Treatment of Cystic Fibrosis with CFTR Modulators]. / Therapie der Mukoviszidose mit CFTR-Modulatoren.

Personalized medicine promises that medical decisions, practices and products are tailored to the individual patient. Cystic fibrosis, an inherited disorder of chloride and bicarbonate transport in exocrine glands, is the first successful example of customized drug development for mutation-specific therapy. There are two classes of CFTR modulators: potentiators that increase the activity of CFTR at the cell surface, and correctors that either promote the read-through of nonsense mutations or facilitate the translation, folding, maturation and trafficking of mutant CFTR to the cell surface. The potentiator ivacaftor and the corrector lumacaftor are approved in Germany for the treatment of people with cystic fibrosis who carry a gating mutation such as p.Gly551Asp or who are homozygous for the most common mutation p.Phe508del, respectively. This report provides an overview of the basic defect in cystic fibrosis, the population genetics of CFTR mutations in Germany and the bioassays to assess CFTR function in humans together with the major achievements of preclinical research and clinical trials to bring CFTR modulators to the clinic. Some practical information on the use of ivacaftor and lumacaftor in daily practice and an update on pitfalls, challenges and novel strategies of bench-to-bedside development of CFTR modulators are also provided.

Friend or foe: genetic and functional characterization of plant endophytic Pseudomonas aeruginosa.

Endophytic Pseudomonas aeruginosa strain BP35 was originally isolated from black pepper grown in the rain forest in Kerala, India. Strain PaBP35 was shown to provide significant protection to black pepper against infections by Phytophthora capsici and Radopholus similis. For registration and implementation in disease management programmes, several traits of PaBP35 were investigated including its endophytic behaviour, biocontrol activity, phylogeny and toxicity to mammals. The results showed that PaBP35 efficiently colonized black pepper shoots and displayed a typical spatiotemporal pattern in its endophytic movement with concomitant suppression of Phytophthora rot. Confocal laser scanning microscopy revealed high populations of PaBP35::gfp2 inside tomato plantlets, supporting its endophytic behaviour in other plant species. Polyphasic approaches to genotype PaBP35, including BOX-PCR, recN sequence analysis, multilocus sequence typing and comparative genome hybridization analysis, revealed its uniqueness among P. aeruginosa strains representing clinical habitats. However, like other P. aeruginosa strains, PaBP35 exhibited resistance to antibiotics, grew at 25-41°C and produced rhamnolipids and phenazines. PaBP35 displayed strong type II secretion effectors-mediated cytotoxicity on mammalian A549 cells. Coupled with pathogenicity in a murine airway infection model, we conclude that this plant endophytic strain is as virulent as clinical P. aeruginosa strains. Safety issues related to the selection of plant endophytic bacteria for crop protection are discussed.

Sinonasal persistence of Pseudomonas aeruginosa after lung transplantation.

UNLABELLED: We report on two CF patients who received double lung transplantation (LTX) due to Pseudomonas aeruginosa related pulmonary destruction. Prior to LTX we detected P. aeruginosa in nasal lavages (NL) and sputum cultures from both patients. Donor lungs of patient 1 became colonized within four weeks with P. aeruginosa identical in genotype with isolates from his pre-transplant sputum cultures and pre- and post-transplant NL. In contrast, patient 2 remained P. aeruginosa free in lower airway samples (bronchial lavage/sputum) for now up to 30 months, despite persistent detection of P. aeruginosa that was identical in genotype with pre-transplant NL and sputum isolates in NL and even in throat swabs. For prevention of pulmonary re-colonization patient 2 has continuously inhaled Colomycin 1 MIU once daily during the preceding more than 36 months with the novel Pari Sinus™ nebulizer, which in scintigraphic studies was shown to deliver vibrating aerosols into paranasal sinuses, additional to bronchial antibiotic inhalation. DISCUSSION: Pulmonary colonization of transplanted donor lungs with identical clones previously colonizing the explanted lungs has been described previously and the upper airways were postulated as reservoir for descending colonization. However, this remained speculative, as upper airway sampling which does not belong to current standards, was not performed in these studies. Our report demonstrates persistence of identical P. aeruginosa genotypes in CF upper airways prior to and after LTX underlining risks of descending colonization of transplanted lungs with P. aeruginosa, which increases risks of graft dysfunction. Therefore, we recommend regular assessment of sinonasal colonization prior to and after LTX. Sinonasal inhalation with antimicrobials should be investigated in prospective trials.

Chronic Pseudomonas aeruginosa infection definition: EuroCareCF Working Group report.

Chronic pulmonary infection with P. aeruginosa develops in most patients with cystic fibrosis (CF); by adulthood 80% of patients are infected and chronic P. aeruginosa infection is the primary cause of increased morbidity and mortality in CF. Chronic infection is preceded by an intermittent stage of infection. The initial stage is characteristically followed by the gradual emergence of mucoid variants of the colonizing strains and a rise in anti-Pseudomonas antibodies. In addition to optimizing existing therapeutic strategies, effective new agents need to be identified. Studies in patients with CF are particularly challenging: the progressive nature of the disease and the wide variation in severity influence considerably the outcome of drug testing. A validated, universally accepted, and clinically useful classification of patients infected with P. aeruginosa, particularly those chronically infected, is needed that can be used as both a criterion for patient selection for clinical trials and as a study endpoint.

Pulmonary exacerbation: towards a definition for use in clinical trials. Report from the EuroCareCF Working Group on outcome parameters in clinical trials.

Pulmonary exacerbations represent a key outcome variable in clinical trials of cystic fibrosis (CF). As there is variation in the trigger for use of intravenous antibiotics compared to the use of oral antibiotics or new nebulised therapy for treatment of exacerbations, the consensus view is that use of intravenous antibiotics cannot be regarded as the key defining character for an exacerbation on its own. The consensus view is that the clinical need for additional treatment as indicated by a recent change in clinical parameters provides the best definition of an exacerbation. Which parameters to include as well as the problems associated with the use of scoring systems and symptom clusters are being discussed.

Characterization of the porcine transferrin gene (TF) and its association with disease severity following an experimental Actinobacillus pleuropneumoniae infection.

Transferrin (TF)-mediated provision of iron is essential for a productive infection by many bacterial pathogens, and iron-depletion of TF is a first line defence against bacterial infections. Therefore, the transferrin (TF) gene can be considered a candidate gene for disease resistance. We obtained the complete DNA sequence of the porcine TF gene, which spans 40 kb and contains 17 exons. We identified polymorphisms on a panel of 10 different pig breeds. Comparative intra- and interbreed sequence analysis revealed 62 polymorphisms in the TF gene including one microsatellite. Ten polymorphisms were located in the coding sequence of the TF gene. Four SNPs (c.902A>T, c.980G>A, c.1417A>G, c.1810A>C) were predicted to cause amino acid exchanges (p.Lys301Ile, p.Arg327Lys, p.Lys473Glu, p.Asn604His). We performed association analyses using six selected TF markers and 116 pigs experimentally infected with Actinobacillus pleuropneumoniae serotype 7. The analysis showed breed-specific TF allele frequencies. In German Landrace, we found evidence for a possible association of the severity of A. pleuropneumoniae infection with TF genotypes.

Concordant genotype of upper and lower airways P aeruginosa and S aureus isolates in cystic fibrosis.

RATIONALE: Lower airway (LAW) infection with Pseudomonas aeruginosa and Staphylococcus aureus is the leading cause of morbidity in cystic fibrosis (CF). The upper airways (UAW) were shown to be a gateway for acquisition of opportunistic bacteria and to act as a reservoir for them. Therefore, tools for UAW assessment within CF routine care require evaluation. OBJECTIVES: The aims of the study were non-invasive assessment of UAW and LAW microbial colonisation, and genotyping of P aeruginosa and S aureus strains from both segments. METHODS: 182 patients with CF were evaluated (age 0.4-68 years, median 17 years). LAW specimens were preferably sampled as expectorated sputum and UAW specimens by nasal lavage. P aeruginosa and S aureus isolates were typed by informative single nucleotide polymorphisms (SNPs) or by spa typing, respectively. RESULTS: Of the typable S aureus and P aeruginosa isolates from concomitant UAW- and LAW-positive specimens, 31 of 36 patients were carrying identical S aureus spa types and 23 of 24 patients identical P aeruginosa SNP genotypes in both compartments. Detection of S aureus or P aeruginosa in LAW specimens was associated with a 15- or 88-fold higher likelihood also to identify S aureus or P aeruginosa in a UAW specimen from the same patient. CONCLUSIONS: The presence of identical genotypes in UAW and LAW suggests that the UAW play a role as a reservoir of S aureus and P aeruginosa in CF. Nasal lavage appears to be suitable for non-invasive UAW sampling, but further longitudinal analyses and comparison with invasive methods are required. While UAW bacterial colonisation is typically not assessed in regular CF care, the data challenge the need to discuss diagnostic and therapeutic standards for this airway compartment. TRIAL REGISTRATION NUMBER: NCT00266474.

Transient correction of the basic defect in sweat glands in an individual with cystic fibrosis carrying the complex CFTR allele F508del-R553Q.

The molecular pathology of mutant F508del CFTR is partially corrected in vitro by the secondary amino acid substitution R553Q in the ABC signature motif. An individual with the CFTR genotype R553X/F508del-R553Q showed the typical symptoms and electrophysiological anomalies of cystic fibrosis in the airways and intestine. Sweat chloride concentrations were normal early in life, but were later raised into the range that is diagnostic for cystic fibrosis, suggesting that R553Q could temporarily correct the basic defect in sweat glands. R553Q caused a delay in diagnosis because of false negative sweat tests but was not a disease reverting suppressor mutation as had been inferred from cellular models.

Expression levels of FAS are regulated through an evolutionary conserved element in intron 2, which modulates cystic fibrosis disease severity.

We have analyzed frequent naturally occurring variants in the autogene FAS in two independent cystic fibrosis (CF) patient populations. Analysis of FAS expression levels from intestinal epithelial biopsies from 16 unrelated F508del-CFTR homozygotes showed a correlation between FAS intron 2 SNP rs7901656 and signals for Affymetrix GeneChip U133 Plus 2.0 probeset 204781_s_at consistent with a dominant model (P=0.0009). Genotype and haplotype analysis at six informative SNPs spanning the FAS gene locus was carried out on 37 nuclear families representing extreme clinical phenotypes that were selected from the European CF Twin and Sibling Study population of more than 300 affected sibling pairs. Case-control comparison of the haplotype composed of rs2296603-rs7901656-rs1571019 encompassing intron 2 of FAS reached significance (P=0.0246). Comparative phylogenetic analysis and functional annotation of the FAS intron 2 sequence revealed a conserved non-coding sequence surrounding rs7901656 and predicted binding sites for four transcription factors whereby the binding site of c-Rel is altered by rs7901656. Taken together, these findings from two independent CF patient cohorts indicate that allelic variants within FAS intron 2 alter FAS gene expression and that these functional variants modulate the manifestation of CF disease.

Diversity of the basic defect of homozygous CFTR mutation genotypes in humans.

BACKGROUND: Knowledge of how CFTR mutations other than F508del translate into the basic defect in cystic fibrosis (CF) is scarce due to the low incidence of homozygous index cases. METHODS: 17 individuals who are homozygous for deletions, missense, stop or splice site mutations in the CFTR gene were investigated for clinical symptoms of CF and assessed in CFTR function by sweat test, nasal potential difference and intestinal current measurement. RESULTS: CFTR activity in sweat gland, upper airways and distal intestine was normal for homozygous carriers of G314E or L997F and in the range of F508del homozygotes for homozygous carriers of E92K, W1098L, R553X, R1162X, CFTRdele2(ins186) or CFTRdele2,3(21 kb). Homozygotes for M1101K, 1898+3 A-G or 3849+10 kb C-T were not consistent CF or non-CF in the three bioassays. 14 individuals exhibited some chloride conductance in the airways and/or in the intestine which was identified by the differential response to cAMP and DIDS as being caused by CFTR or at least two other chloride conductances. DISCUSSION: CFTR mutations may lead to unusual electrophysiological or clinical manifestations. In vivo and ex vivo functional assessment of CFTR function and in-depth clinical examination of the index cases are indicated to classify yet uncharacterised CFTR mutations as either disease-causing lesions, risk factors, modifiers or neutral variants.

[Molecular genetics principles in cystic fibrosis. An example of genetic illness in pneumology]. / Molekulargenetische Grundlagen der zystischen Fibrose. Ein Beispiel genetischer Erkrankungen in der Pneumologie.

The generalized exocrinopathy cystic fibrosis (CF) is caused by molecular lesions in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The basic defect of this autosomal-recessive disorder manifests in decreased permeability for chloride ions across the apical epithelial membrane. Of the more than 1,000 known CFTR mutations the most frequent mutation F508del occurs on about 70% of North- and Mideuropean CF chromosomes. CFTR mutations are also causatively involved in male infertility, pancreatitis and several airway diseases like disseminated bronchiectasis. The differential diagnosis between CF, other CFTR-opathies and diseases of unrelated etiology can be achieved by the assessment of clinical symptoms, CFTR mutation analysis and electrophysiological bioassays (sweat test, nasal potential difference, intestinal current measurements).

Fatal outcome of lung transplantation in cystic fibrosis patients due to small-colony variants of the Burkholderia cepacia complex.

The aim of this study was to investigate the possible role of small-colony variant morphotypes of Burkholderia cepacia-like organisms in infectious complications in cystic fibrosis patients following lung transplantation. Respiratory tract specimens from 470 cystic fibrosis patients were screened over a 22-month period for Burkholderia cepacia-like organisms. Nineteen patients were positive for these organisms, eight of whom harboured small-colony-variant morphotypes. Three patients underwent bilateral lung transplantation during the study, two of whom harboured small-colony variants in addition to clonally identical wildtypes of Burkholderia multivorans and Burkholderia cepacia genomovar III prior to lung transplantation. Both patients developed fatal systemic infections post transplantation due to small-colony variants. In vitro testing revealed that small-colony variants exhibited increased serum resistance in comparison to wildtypes. The results of this study indicate that diagnostic efforts should be undertaken to carefully identify small-colony variants of Burkholderia cepacia complex, since they might be an indicator of poor post-transplantation outcome in patients with cystic fibrosis.

Pulmonary microbial infection in mice: comparison of different application methods and correlation of bacterial numbers and histopathology.

Many investigations have been performed in characterising experimental bacterial infections in the lung of mice using several pathogens. Robust experimental pulmonary infection models require a reproducible method of application with defined numbers of pathogens to the respiratory tract without contaminating extrapulmonary tissues. At the same time trauma due to the experimental procedure should be kept to a minimum. So far several routes of administration have been used but a systematic comparison of these methods is still missing. Here we provide a comprehensive evaluation of view controlled i.t. instillation, tracheotomy, intranasal application, blind instillation and aerosol infection. An infection dose of up to 5 x 10(4) bacteria (L. monocytogenes) was applied to a group of ten mice by each technique and the animals were killed after 1 h or 24h. The number of viable bacteria was estimated by plating homogenates of the lungs and spleens. In addition, pathological effects on lung tissue were examined by histology 24h after infection. The highest reproducibility was achieved after applying Listeria directly in the trachea under view or by tracheotomy. However, mice were severely affected in their vitality after tracheotomy. Thus, for topical application of bacterial suspension into the lung the view controlled i.t. instillation is most appropriate.

Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440.

Pseudomonas putida is a metabolically versatile saprophytic soil bacterium that has been certified as a biosafety host for the cloning of foreign genes. The bacterium also has considerable potential for biotechnological applications. Sequence analysis of the 6.18 Mb genome of strain KT2440 reveals diverse transport and metabolic systems. Although there is a high level of genome conservation with the pathogenic Pseudomonad Pseudomonas aeruginosa (85% of the predicted coding regions are shared), key virulence factors including exotoxin A and type III secretion systems are absent. Analysis of the genome gives insight into the non-pathogenic nature of P. putida and points to potential new applications in agriculture, biocatalysis, bioremediation and bioplastic production.

Chloride conductance and genetic background modulate the cystic fibrosis phenotype of Delta F508 homozygous twins and siblings.

To investigate the impact of chloride (Cl(-)) permeability, mediated by residual activity of the cystic fibrosis transmembrane conductance regulator (CFTR) or by other Cl(-) channels, on the manifestations of cystic fibrosis (CF), we determined Cl(-) transport properties of the respiratory and intestinal tracts in Delta F508 homozygous twins and siblings. In the majority of patients, cAMP and/or Ca(2+)-regulated Cl(-) conductance was detected in the airways and intestine. Our finding of cAMP-mediated Cl(-) conductance suggests that, in vivo, at least some Delta F508 CFTR can reach the plasma membrane and affect Cl(-) permeability. In respiratory tissue, the expression of basal CFTR-mediated Cl(-) conductance, demonstrated by 30% of Delta F508 homozygotes, was identified as a positive predictor of milder CF disease. In intestinal tissue, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid-insensitive (DIDS-insensitive) Cl(-) secretion, which is indicative of functional CFTR channels, correlated with a milder phenotype, whereas DIDS-sensitive Cl(-) secretion was observed mainly in more severely affected patients. The more concordant Cl(-) secretory patterns within monozygous twins compared with dizygous pairs imply that genes other than CFTR significantly influence the manifestation of the basic defect.

General method of rapid Smith/Birnstiel mapping adds for gap closure in shotgun microbial genome sequencing projects: application to Pseudomonas putida KT2440.

A physical mapping strategy has been developed to verify and accelerate the assembly and gap closure phase of a microbial genome shotgun-sequencing project. The protocol was worked out during the ongoing Pseudomonas putida KT2440 genome project. A macro-restriction map was constructed by linking probe hybridisation of SwaI- or I-CeuI-restricted chromosomes to serve as a backbone for the quick quality control of sequence and contig assemblies. The library of PCR-generated SwaI linking probes was derived from the sequence assembly after 3- and 6-fold genome coverage. In order to support gap closure in regions with ambiguous assemblies such as the repetitive sequence of the seven ribosomal operons, high-resolution Smith/Birnstiel maps were generated by Southern hybridisation of pulsed-field gel electrophoresis-separated rare-cutter complete/frequent-cutter partial digestions with rare-cutter fragment end probes. Overall 1.5 Mb of the 6.1 Mb P.putida KT2440 genome has been subjected to high-resolution physical mapping in order to align assemblies generated from shotgun sequencing.