Proteomics investigation of human platelets in healthy donors and cystic fibrosis patients by shotgun nUPLC-MSE and 2DE: a comparative study

Platelets are of pathophysiological relevance in haemostasis, wound repair, inflammation and cardiovascular disease. We have shown that human platelets express a biologically active Cystic Fibrosis Transmembrane Conductance Regulator, which is dysfunctional in Cystic Fibrosis (CF) patients, and regulate platelet responses related to inflammation and its resolution. In order to further elucidate platelet involvement in CF inflammation, we pursued a comparative proteomic analysis of cells from healthy donors and CF patients, in association with a non-supervised comparative analysis of the Gene Ontology. Our results, showing changes in the integrin signalling in CF, support a pro-inflammatory profile of CF platelets.     

Impact of MIF Gene Promoter Polymorphism on F508del Cystic Fibrosis Patients

Macrophage migration Inhibitory Factor (MIF) is a pro-inflammatory cytokine sustaining the acute response to gram–negative bacteria and a regulatory role for MIF in Cystic Fibrosis has been suggested by the presence of a functional, polymorphic, four-nucleotide repeat in this gene''s promoter at position −794, with the 5-repeat allele displaying lower promoter activity. We aimed at assessing the association of this polymorphism with disease severity in a group of Cystic Fibrosis patients homozygous for F508del CFTR gene mutation. Genotype frequencies were determined in 189 Cystic Fibrosis and 134 control subjects; key clinical features of patients were recorded and compared among homozygous 5-allele patients and the other MIF genotypes. Patients homozygous for the 5-repeat allele of MIF promoter displayed a slower rate of lung function decline (p = 0.027) at multivariate survival analysis. Multiple regression analysis on age-normalized respiratory volume showed no association of the homozygous 5-repeat genotype with lung function under stable conditions and no correlation with P.aeruginosa chronic colonization. Therefore, only the Homozygous 5-repeat genotype at MIF −794 is associated with milder disease in F508del Cystic Fibrosis patients.     

Cultured epithelial cells from patients with cystic fibrosis have an increased expression of the 14 kDa Ca2(+)-binding protein CFA

The Cystic Fibrosis antigen (CFA) is a 14 kDa. Ca2(+)-binding protein known to be expressed in cells of myeloid origin during normal cell differentiation. CFA serum levels are elevated in Cystic Fibrosis (CF) patients and heterozygotes. We examined the expression of CFA in different cultured epithelial cells from controls and patients with CF. The steady state level of CFA was in general higher in epithelial cells from CF patients compared to control cells and was found to increase during cell aging. The latter difference could be attributed to an increased rate of CFA synthesis rather than to an impairment of CFA degradation or secretion, as shown by pulse chase experiments.     

Anaerobic bacteria infection in cystic fibrosis airway disease

Depletion of the periciliary liquid in "Cystic Fibrosis" airway disease results in reduced mucociliary transport, persistent mucus hypersecretion and consequently increased height of the luminal mucus layer, so hypoxic gradients in the mucus plugs are developed. Because of anaerobic lung zones, it is highly probable that anaerobic bacteria not detected by routine bacteriologic culture methods also reside within the mucus. Notwithstanding this evidence, microbiology laboratories working in the cystic fibrosis field do not generally use strict anaerobic bacteriologic cultures to determine the presence of anaerobic bacteria in the Cystic Fibrosis lung. The aim of this review is to focus on the published data regarding the finding of anaerobic bacteria in cystic fibrosis airway disease. Therefore, microbiology, diagnosis, antimicrobial susceptibility and possible impact on clinical management of anaerobic bacteria lung infection in cystic fibrosis are described.     

Building an understanding of cystic fibrosis on the foundation of ABC transporter structures

Cystic fibrosis (CF) is a fatal disease affecting the lungs and digestive system by impairment of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). While over 1000 mutations in CFTR have been associated with CF, the majority of cases are linked to the deletion of phenylalanine 508 (ΔF508). F508 is located in the first nucleotide binding domain (NBD1) of CFTR. This mutation is sufficient to impair the trafficking of CFTR to the plasma membrane and, thus, its function. As an ABC transporter, recent structural data from the family provide a framework on which to consider the effect of the ΔF508 mutation on CFTR. There are fifty-seven known structures of ABC transporters and domains thereof. Only six of these structures are of the intact transporters. In addition, modern bioinformatic tools provide a wealth of sequence and structural information on the family. We will review the structural information from the RCSB structure repository and sequence databases of the ABC transporters. The available structural information was used to construct a model for CFTR based on the ABC transporter homologue, Sav1866, and provide a context for understanding the molecular pathology of Cystic Fibrosis.     

Microsatellite haplotypes associations with 5 CFTR mutations in "Grande Brière", an isolate located in southern Brittany

The variability at three microsatellites in the Cystic Fibrosis Transmembrane Conductance Regulator Gene (CFTR) locus has been studied for frequent mutations encountered in an isolated population of "Grande Brière", a small region located in Southern Brittany. Fluorescent multiplex PCR of these microsatellites were assayed in 16 Cystic Fibrosis (CF) families carrying 5 different mutations. The four most frequent haplotypes on df508 chromosomes were the same as those found in Northern France and Europe but the distribution of these haplotypes provides new enlightenment on the population origin of this insular community.     

Effect of genotype on selected clinical features of Polish cystic fibrosis adults

Cystic fibrosis (CF), the most common autosomal recessive disorder of Caucasians, is caused by the mutations in the gene encoding CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) protein. Until now, approximately 1000 mutations of the CFTR gene have been described. The genotype-phenotype relationships in CF are still not completely understood. This study was undertaken in an attempt to characterise the distribution of CFTR mutations and their effect on selected clinical parameters in a group of Polish CF adults. A total number of 38 adult CF patients (mean age 21.6 +/- 6.8); 18 females & 20 males were enrolled in the study. The CFTR gene identification was conducted with the use of PCR & InnoLipa-CF set. The assessed clinical parameters included: age at diagnosis, age, lung function test, X-ray scored in Brasfield score, weight & height. We found that: (1) the genotypes of the studied population were unevenly distributed (65.8%- genotype deltaF508/M), (2) a high percentage of 3849+10kbC-->T was noted, (3) patients homozygous for the deltaF508 mutation were diagnosed significantly earlier and had a lower body mass index, (4) no differences were observed in the patients' length of life or the progression of lung disease. Conclusions: 1. In comparison to other populations, Polish adult CF patients display a relatively higher frequency of mild mutations. 2. Late diagnosis of CF in the studied group may be partially caused by a high percentage of CFTR mutations connected with the mild course of the disease that are difficult to identify. 3. Cystic fibrosis should be more commonly taken into consideration in the differential diagnosis in adult patients with milder symptoms.     

Current concepts of immune dysregulation in cystic fibrosis

Cystic fibrosis (CF) lung disease is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene and is characterized by a perpetuated feedback loop of bacterial infection and inflammation. Both intrinsic (CFTR-dependent) and extrinsic (CFTR-independent) mechanisms contribute to the inflammatory phenotype of CF lung disease. Innate immune cells, initially recruited to combat bacterial pathogens, are acting in a dysregulated and non-resolving fashion in CF airways and cause harm to the host by releasing proteases and oxidants. Targeting harmful immune pathways, while preserving protective ones, remains the challenge for the future. This review highlights current concepts of innate immune dysregulation in CF lung disease.This article is part of a Directed Issue entitled: Cystic Fibrosis: From o-mics to cell biology, physiology, and therapeutic advances.     

超声与CT 在肝脏囊型包虫病与非寄生虫性囊肿鉴别中的比较

目的:分析囊型包虫病的CT以及超声的影像学特征以及联合诊断的效能,以减少对于CL及Ⅰ型包虫囊肿误诊率。方法:回顾性分析自2008年1月～2010年9月在我院行CT和超声检查的包虫病患者22例,肝脏单纯囊肿25例,分析其表现并进行比较。结果:CT检出36个病灶,超声检测出34个病灶,单纯囊肿组和包虫组病灶分布及形态等无明显差别,22例超声显示位于包虫囊肿底部可以随体位运动棘球呦砂样改变,显示囊肿壁不规则增厚3例,病灶周围胆管扩张2例,CT显示包虫囊肿壁钙化5例,病灶周围胆管扩张3例。结论:联合CT和超声学检查可以帮助提高CEI及CL型包虫囊肿的诊断率。     

Cystic fibrosis: Demonstration of an abnormality in mucopolysaccharides in cultured lymphoid lines

Cultured lymphoid cells of both homozygotes and heterozygotes for cystic fibrosis could be distinguished from those of normals by (1) growth pattern, gross clumping, and (2) a relative increase in dermatan sulfate, with a normal total mucopolysaccharide content. Lines derived from the genetic mucopolysaccharidoses also had these characteristics, but their total mucopolysaccharide content was markedly increased. These observations support the hypothesis that the cellular disturbance in cystic fibrosis resides in those cellular regions whose functions would be altered by mucopolysaccharide composition.This research was made possible through a grant from The National Foundation-March of Dimes and, in part, supported by The Cystic Fibrosis Children's Fund and The National Cystic Fibrosis Research Foundation.Career Scientist of the Health Research Council of New York City (I-797).     

The spectrum of cystic fibrosis mutations.

Although the major mutation causing cystic fibrosis accounts for almost 70% of mutant chromosomes screened, almost 300 sequence alterations have been identified in the gene during the past two and a half years. At least 230 of these mutations are probably associated with disease. This rapid accumulation of data is in part due to the highly coordinated effort by members of the Cystic Fibrosis Genetic Analysis Consortium. The information is not only essential to genetic diagnosis, but also will aid in understanding the structure and function of the protein, and possibly in correlating genotype with phenotype.     

TPL2 signalling: From Toll-like receptors-mediated ERK1/ERK2 activation to Cystic Fibrosis lung disease

Cystic Fibrosis (CF) is the most common lethal genetic recessive disorder, with a carrier frequency of 1 in 27 among North American Caucasians. Mitogen-activated protein kinases (MAPKs) and pro-inflammatory cytokines have crucial functions in the innate immune response of epithelial cells. They determine the inflammation status and the host response to pathogenic infections. However, in CF, bacterial-driven inflammation leads to tissue destruction, reduction in lung function and mortality. Recognition of invading pathogens is mediated in part by Toll-like receptors (TLR) activation of intracellular signalling cascade leading to cytokines’ synthesis. The protein kinase Tumour Progression Locus 2 (TPL2) is a key molecule in relaying inflammatory stimuli to ERK1/ERK2 MAPKs. In this review, we summarized the recent findings on TPL2 signalling and how TPL2 can contribute to the excessive inflammation found in CF. Pharmacologically targeting this kinase could have a significant benefit for CF patients dealing with chronic bacterial infections such as Pseudomonas aeruginosa.This article is part of a Directed Issue entitled: Cystic Fibrosis: From o-mics to cell biology, physiology, and therapeutic advances.     

Cytoskeleton and CFTR

Cystic Fibrosis Transmembrane conductance Regulator, CFTR, is a membrane protein expressed in epithelia. A protein kinase A (PKA)-regulated Cl⁻ channel, it is a rate-limiting factor in fluid transport. Mutations in CFTR are responsible for cystic fibrosis, CF, an autosomal recessive disease. The most frequent mutation is deletion of phenylalanine at position 508, ΔF508. The regulation of trafficking and degradation of CFTR/ΔF508CFTR as well as its function(s) is a complex process which involves a number of proteins including chaperones and adaptors. It is now known that cytoskeletal proteins, previously considered only as structural proteins, are also important factors in the regulation of cellular processes and functions. The aim of the present review is to focus on how microfilaments, microtubules and intermediary filaments form a dynamic interactome with CFTR to participate in the regulation of CFTR-dependent transepithelial ion transport, CFTR trafficking and degradation.This article is part of a Directed Issue entitled: Cystic Fibrosis: From o-mics to cell biology, physiology, and therapeutic advances.     

MiR-101 and miR-144 Regulate the Expression of the CFTR Chloride Channel in the Lung

The Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is a chloride channel that plays a critical role in the lung by maintaining fluid homeostasis. Absence or malfunction of CFTR leads to Cystic Fibrosis, a disease characterized by chronic infection and inflammation. We recently reported that air pollutants such as cigarette smoke and cadmium negatively regulate the expression of CFTR by affecting several steps in the biogenesis of CFTR protein. MicroRNAs (miRNAs) have recently received a great deal of attention as both biomarkers and therapeutics due to their ability to regulate multiple genes. Here, we show that cigarette smoke and cadmium up-regulate the expression of two miRNAs (miR-101 and miR-144) that are predicted to target CFTR in human bronchial epithelial cells. When premature miR-101 and miR-144 were transfected in human airway epithelial cells, they directly targeted the CFTR 3′UTR and suppressed the expression of the CFTR protein. Since miR-101 was highly up-regulated by cigarette smoke in vitro, we investigated whether such increase also occurred in vivo. Mice exposed to cigarette smoke for 4 weeks demonstrated an up-regulation of miR-101 and suppression of CFTR protein in their lungs. Finally, we show that miR-101 is highly expressed in lung samples from patients with severe chronic obstructive pulmonary disease (COPD) when compared to control patients. Taken together, these results suggest that chronic cigarette smoking up-regulates miR-101 and that this miRNA could contribute to suppression of CFTR in the lungs of COPD patients.     

Conditioned media from cultured cystic fibrosis fibroblasts inhibits Na/K ATPase activity

Conditioned culture media taken from fibroblast cell lines derived from skin biopsies of control or of patients with Cystic Fibrosis (CF) were incubated with membranes of rat submandibular glands. The Na/K - ATPase activity of these membranes was inhibited when treated with CF-media, including both ouabain sensitive and insensitive activities. However, the membrane associated Mg-ATPase, Ca-ATPase, and both basal and hormone-stimulated adenylate cyclase activities were relatively unaffected. Thus, a factor or factors produced by CF-fibroblasts was shown to be active in a cell-free system derived from an exocrine gland.     

Laboratory Diagnosis and Characterization of Fungal Disease in Patients with Cystic Fibrosis (CF): A Survey of Current UK Practice in a Cohort of Clinical Microbiology Laboratories

There is much uncertainty as to how fungal disease is diagnosed and characterized in patients with cystic fibrosis (CF). A 19-question anonymous electronic questionnaire was developed and distributed to ascertain current practice in clinical microbiology laboratories providing a fungal laboratory service to CF centres in the UK. Analyses of responses identified the following: (1) current UK laboratory practice, in general, follows the current guidelines, but the scope and diversity of what is currently being delivered by laboratories far exceeds what is detailed in the guidelines; (2) there is a lack of standardization of fungal tests amongst laboratories, outside of the current guidelines; (3) both the UK CF Trust Laboratory Standards for Processing Microbiological Samples from People with Cystic Fibrosis and the US Cumulative Techniques and Procedures in Clinical Microbiology (Cumitech) Guidelines 43 Cystic Fibrosis Microbiology need to be updated to reflect both new methodological innovations, as well as better knowledge of fungal disease pathophysiology in CF; (4) there is a need for clinical medicine to decide upon a stratification strategy for the provision of new fungal assays that will add value to the physician in the optimal management of CF patients; (5) there is also a need to rationale what assays should be performed at local laboratory level and those which are best served at National Mycology Reference Laboratory level; and (6) further research is required in developing laboratory assays, which will help ascertain the clinical importance of ‘old’ fungal pathogens, as well as ‘emerging’ fungal pathogens.     

Molecular dynamics analysis of the wild type and dF508 mutant structures of the human CFTR–nucleotide binding domain 1

Mutations of CFTR (Cystic Fibrosis transmembrane Conductance Regulator), a membrane protein expressed in the epithelium that forms a chloride channel, cause a chronic, developmental and hereditary disease, known as Cystic Fibrosis. The most common mutation is the deletion of F508, a residue present in the first nucleotide binding domain (NBD1). We studied the thermodynamic properties of NBD1 wild type (WT) and mutant (dF508), starting from the crystallographic structures in the Protein Data Bank using the techniques of Molecular Dynamics. The two structures were similarly stable at room temperature, showed no change enthalpy or entropy, maintaining the same dimensions and the same order of magnitude of atomic fluctuations; the only difference was the energy of interaction with the solvent, in which the mutant appears slightly disadvantaged; these differences between the two models are at microscopic level and relate to local variations (in residues at 8 Å from F508) of the surface exposed to the solvent. We also found a decrease in the mutant of about 30 times of affinity for ATP compared to WT.     

Lessons from the on-site quality audit of data transmitted to the French cystic fibrosis registry

Background

The French Cystic Fibrosis Registry takes a census of the population of patients and records their annual data transmitted by Cystic Fibrosis Centers (CFCs). Quality of patient data has been a focus in the past years, with the implementation of automated controls before data integration. The objective was to assess, at the 14 CFCs trained in the quality improvement named Hospital Program to Improve Outcomes and Expertise in Cystic Fibrosis (PHARE-M), the quality of the 2012 and 2013 data transmitted to the French Registry with respect to the rules established to obtain forced expiratory volume in 1 second (FEV1%) and anthropometric data.

Methods

The clinical researcher selected 20 patients at each CFC from age ranges corresponding to different visit frequencies and measurement procedures in order to reach saturation of error causes. The control consisted in comparing source data, pulmonary function tests (PFTs), patient records, and data in the Registry.

Results

The audit focused on 242 patients, 2455 consultations and 1855 PFTs. Less than 5% of data concerning weight, height, or FEV1 (L) in the patient records files had discrepancies with source data. Discrepancies on patient height between patient records and PFT files were found in 11% of cases. For one hundred and ten patients (45%), anomalies were found between the patient record and the Registry for the FEV1% and the associated anthropometric measurements mainly related to the interpretation of the selection rule of the venue corresponding to the “best spirometry in the year” and the reference standard used (local standards versus Knudson reference equations). For the 33 children in the age range of 6–17 years old (27% out of 120 children records controlled), the FEV1% value in the Registry presented an average deviation of +4.25% (min. = ?9.3%; max. = +16.9%; median?=?4%) with the value from the Patient record.

Conclusions

This first on-site quality audit of the data transmitted to the Registry pointed out variability in the measurement process at the CFCs. The rule for selecting the data for the Registry was applied differently at some CFCs, and various local References for the FEV1% calculation were used. Avenues for improvement have been identified.

     

"Bronchial Artery Delivery of Viral Vectors for Gene delivery in Cystic Fibrosis; Superior to Airway Delivery?"

Background

Attempts at gene therapy for the pulmonary manifestations of Cystic Fibrosis have relied mainly on airway delivery. However the efficiency of gene transfer and expression in the airway epithelia has not reached therapeutic levels. Access to epithelial cells is not homogenous for a number of reasons and the submucosal glands cannot be reached via the airways.

Presentation

We propose to inject gene delivery vectors directly into bronchial arteries combined with pre-delivery of vascular endothelial growth factor to increase vascular endothelial permeability and post-delivery flow reduction by balloon occlusion. Thus it may be possible to reach mucous secreting cells of the bronchial luminal epithelium and the submucosal glands in an increased and homogenous fashion.

Testing

This combination of techniques to the best of our knowledge has not previously been investigated, and may enable us to overcome some of the current limitations to gene therapy for Cystic Fibrosis.