Terminal glycosylation in cystic fibrosis (CF): a review emphasizing the airway epithelial cell

Altered terminal glycosylation, with increased fucosylation and decreased sialylation is a hallmark of the cystic fibrosis (CF) glycosylation phenotype. Oligosaccharides purified from the surface membrane glycoconjugates of CF airway epithelial cells have the Lewis x, selectin ligand in terminal positions. This review is focused on the investigations of the glycoconjugates of the CF airway epithelial cell surface. Two of the major bacterial pathogens in CF, Pseudomonas aeruginosa and Haemophilus influenzae, have binding proteins which recognize fucose in -1,3 linkage and asialoglycoconjugates. Therefore, consideration has been given to the possibility that the altered terminal glycosylation of airway epithelial glycoproteins in CF contributes to both the chronic infection and the robust, but ineffective, inflammatory response in the CF lung. Since the glycosylation phenotype of CF airway epithelial cells have been modulated by the expression of wtCFTR, the hypotheses which have been proposed to relate altered function of CFTR to the regulation of the glycosyltransferases are discussed. Understanding the effects of mutant CFTR on glycosylation may provide further insight into the regulation of glycoconjugate processing as well as new approaches to the therapy of CF.     

Gene therapy for cystic fibrosis

Cystic fibrosis (CF) is associated with significant morbidity and mortality, despite significant advances in conventional treatment. The field of gene therapy has progressed rapidly since the cystic fibrosis transmembrane conductance regulator (CFTR) gene was cloned. In this review we discuss current knowledge on the underlying molecular defect in CF, and the progress in gene transfer studies from the early in vitro work through to clinical trials, including the development of endpoints to assess efficacy. We highlight the problems encountered, and likely future directions of the field.     

Gene therapy: light is finally in the tunnel

After two decades of ups and downs, gene therapy has recently achieved a milestone in treating patients with Leber’s congenital amaurosis (LCA). LCA is a group of inherited blinding diseases with retinal degeneration and severe vision loss in early infancy. Mutations in several genes, including RPE65, cause the disease. Using adeno-associated virus as a vector, three independent teams of investigators have recently shown that RPE65 can be delivered to retinal pigment epithelial cells of LCA patients by subretinal injections resulting in clinical benefits without side effects. However, considering the whole field of gene therapy, there are still major obstacles to clinical applications for other diseases. These obstacles include innate and immune barriers to vector delivery, toxicity of vectors and the lack of sustained therapeutic gene expression. Therefore, new strategies are needed to overcome these hurdles for achieving safe and effective gene therapy. In this article, we shall review the major advancements over the past two decades and, using lung gene therapy as an example, discuss the current obstacles and possible solutions to provide a roadmap for future gene therapy research.     

Gene targeting in livestock: a preview

Until recently genetically modified livestock could only be generated by pronuclear injection. The discovery that animals can be cloned by nuclear transfer from cultured somatic cells means that it will now be possible to achieve gene targeting in these species. We discuss current developments in NT, the prospects and technical challenges for introducing targeted changes into the germline by this route, and the types of application for which this new technology will be used. This revised version was published online in August 2006 with corrections to the Cover Date.     

Cystic fibrosis: A look into the future of prenatal screening and therapy

Despite recent guidelines suggesting prenatal screening for carriers of cystic fibrosis (CF) mutations, many physicians do not offer patients this service or even counseling. Some argue that the risks of miscarriage associated with prenatal diagnostic techniques outweigh the benefit of added insight, but with the advent of newer, noninvasive techniques, risks of miscarriage may be significantly lowered. Prenatal diagnosis provides parents the time to prepare for raising a child with CF, and soon, could provide treatment options in utero that could improve quality of life. Here, we describe two of the most promising gene therapy approaches: lentivirus and adenoassociated virus (AAV)‐mediated gene transduction. Thus, prenatal detection and treatment is in a most crucial stage for care of patients with CF. Birth Defects Research (Part C) 105:73–80, 2015. © 2015 Wiley Periodicals, Inc.     

Gene therapy of cystic fibrosis: the glycofection approach

Many cells express surface membrane lectins that selectively bind and carry glycoconjugates into intracellular endosomes; in addition, various intracellular membrane and soluble lectins act as shuttles between different compartments. On this basis, we developed glycosylated polycations, now called glycofectins (glycosylated polylysine and polyethyleneimine). Recently, we set up a simple way to transform oligosaccharides into glycosynthons suitable to substitute proteins or polymers. Glycofectins bind plasmid DNA leading to compact glycoplexes. Glycoplexes prepared with glycofectins were found to be much more active than naked plasmid to transfer genes to various types of cells including human airway epithelial and serous cells. The gene transfer efficiency was found to depend on the nature of the sugars borne by glycofectins. It appeared that the sugar-dependent efficiency was not only related to the uptake but also to the intracellular traffic of glycoplexes.     

Successful targeting of the mouse cystic fibrosis transmembrane conductance regulator gene in embryonal stem cells

We wish to construct a mouse model for the human inherited disease cystic fibrosis. We describe here the successful targeting in embryonal stem cells of the murine homologue (Cftr) of the cystic fibrosis transmembrane conductance regulator gene, as the first critical step towards this end. The targeting event precisely disrupts exon 10, the site of the major mutation in patients with cystic fibrosis. The targeted cells are pluripotent and competent to form chimaeras.     

microRNA调控的靶向性病毒载体在基因治疗中的应用

安全、有效、具有靶向性的病毒载体是基因治疗药物在临床上得以应用的关键。microRNA是一类单链、内源性的转录后调控小分子,它的发现为开发具有靶向性调控能力的病毒载体提供了新的研究方法。以下在介绍microRNA调节病毒载体靶向性原理的基础上,着重介绍microRNA在清除复制能力病毒的污染、消除转基因特异性免疫、增强肿瘤靶向性基因治疗、开发活体疫苗等领域的应用。     

Human airway mucin glycosylation: a combinatory of carbohydrate determinants which vary in cystic fibrosis

Human airway mucins represent a very broad family of polydisperse high molecular mass glycoproteins, which are part of the airway innate immunity. Apomucins, which correspond to their peptide part, are encoded by at least 6 different mucin genes (MUC1, MUC2, MUC4, MUC5B, MUC5AC and MUC7). The expression of some of these genes (at least MUC2 and MUC5AC) is induced by bacterial products, tobacco smoke and different cytokines.Human airway mucins are highly glycosylated (70–80% per weight). They contain from one single to several hundred carbohydrate chains. The carbohydrate chains that cover the apomucins are extremely diverse, adding to the complexity of these molecules. Structural information is available for more than 150 different O-glycan chains corresponding to the shortest chains (less than 12 sugars).The biosynthesis of these carbohydrate chains is a stepwise process involving many glycosyl- or sulfo-transferases. The only structural element shared by all mucin O-glycan chains is a GalNAc residue linked to a serine or threonine residue of the apomucin. There is growing evidence that the apomucin sequences influence the first glycosylation reactions. The elongation of the chains leads to various linear or branched extensions. Their non-reducing end, which corresponds to the termination of the chains, may bear different carbohydrate structures, such as histo-blood groups A or B determinants, H and sulfated H determinants, Lewis a, Lewis b, Lewis x or Lewis y epitopes, as well as sialyl- or sulfo- (sometimes sialyl- and sulfo-) Lewis a or Lewis x determinants. The synthesis of these different terminal determinants involves three different pathways with a whole set of glycosyl- and sulfo-transferases.Due to their wide structural diversity forming a combinatory of carbohydrate determinants as well as their location at the surface of the airways, mucins are involved in multiple interactions with microorganisms and are very important in the protection of the underlying airway mucosa.Airway mucins are oversulfated in cystic fibrosis and this feature has been considered as being linked to a primary defect of the disease. However, a similar pattern is observed in mucins from patients suffering from chronic bronchitis when they are severely infected. Airway mucins from severely infected patients suffering either from cystic fibrosis or from chronic bronchitis are also highly sialylated, and highly express sialylated and sulfated Lewis x determinants, a feature which may reflect severe mucosal inflammation or infection.These determinants are potential sites of attachment for Pseudomonas aeruginosa, the pathogen responsible for most of the morbidity and mortality in cystic fibrosis, and the expression of the sulfo- and glycosyl-transferases involved in their biosynthesis is increased by TNF.In summary, airway inflammation may simultaneously induce the expression of mucin genes (MUC2 and MUC5AC) and the expression of several glycosyl- and sulfo-transferases, therefore modifying the combinatory glycosylation of these molecules.     

Terminal glycosylation of cystic fibrosis airway epithelial cells

Cystic fibrosis (CF) has a characteristic glycosylation phenotype usually expressed as a decreased ratio of sialic acid to fucose. The glycosylation phenotype was found in CF/T1 airway epithelial cells (F508/F508). When these cells were transfected and were expressing high amounts of wtCFTR, as detected by Western blot analysis and in situ hybridization, the cell membrane glycoconjugates had an increased sialic acid content and decreased fucosyl residues in 1,3/4 linkage to antennary N[emsp4 ]-acetyl glucosamine (Fuc1,3/4GlcNAc). After the expression of wtCFTR decreased, the amount of sialic acid and Fuc1,3/4GlcNAc returned to levels shown by the parent CF cells. Sialic acid was measured by chemical analysis and Fuc1,3/4GlcNAc was detected with a specific 1,3/4 fucosidase. CF and non-CF airway cells in primary culture also had a similar reciprocal relationship between fucosylation and sialylation. It is possible that the glycosylation phenotype is involved in the pathogenesis of CF lung disease by facilitating bacterial colonization and leukocyte recruitment.     

Development of a Gram-negative selective agar (GNSA) for the detection of Gram-negative microflora in sputa in patients with cystic fibrosis

AIMS: To develop a selective agar medium to help detect and quantify Gram-negative flora in the sputum of patients with cystic fibrosis (CF). METHODS AND RESULTS: A novel Gram-negative Selective Agar (GNSA) medium was developed consisting of tryptone soya broth (30 g), bacteriological agar no.1 (10 g), yeast extract (5 g), crystal violet (2 mg), nisin (48 mg), novobiocin (5 mg), cycloheximide (100 mg), amphotericin (2 mg) and double distilled water (1 l), for the selective culture of all Gram-negative flora from the sputum of patients with CF. GNSA was able to support the proliferation of all 34 Gram-negative organisms examined, including 23 species most commonly associated with CF, but was unable to support the growth of the 12 Gram-positive or seven fungal organisms examined. Sensitivity studies demonstrated that the GNSA medium was able to detect not less than 1.50 x 102 CFU ml-1 sputum Pseudomonas aeruginosa, 2.38 x 102 CFU ml-1 sputum Burkholderia cepacia genomovar IIIb and 6.70 x 103 CFU ml-1 sputum Stenotrophomonas maltophilia. A comparison of the microbial flora detected in the sputa of 12 adult CF patients by employment of routine bacteriological agar media and GNSA, demonstrated that GNSA was able to detect all Gram-negative organisms cultured by routine media, but had the advantage of detecting Alcaligenes xylosoxidans in two CF patients, whom had no previous history of Gram-negative infection. CONCLUSIONS: GNSA was unable to support the proliferation of any Gram-positive organism or yeast/fungi, but was successful in supporting the growth of all Gram-negative organisms challenged. SIGNIFICANCE AND IMPACT OF THE STUDY: Employment of this medium coupled with semi-automated technology may aid in helping to efficiently determine Gram-negative loading of respiratory secretions, particularly in response to antibiotic intervention.     

Structural basis of typhoid: Salmonella typhi type IVb pilin (PilS) and cystic fibrosis transmembrane conductance regulator interaction

The type IVb pilus of the enteropathogenic bacteria Salmonella typhi is a major adhesion factor during the entry of this pathogen into gastrointestinal epithelial cells. Its target of adhesion is a stretch of 10 residues from the first extracellular domain of cystic fibrosis transmembrane conductance regulator (CFTR). The crystal structure of the N‐terminal 25 amino acid deleted S. typhi native PilS protein (ΔPilS), which makes the pilus, was determined at 1.9 Å resolution by the multiwavelength anomalous dispersion method. Also, the structure of the complex of ΔPilS and a target CFTR peptide, determined at 1.8 Å, confirms that residues 113–117 (NKEER) of CFTR are involved in binding with the pilin protein and gives us insight on the amino acids that are essential for binding. Furthermore, we have also explored the role of a conserved disulfide bridge in pilus formation. The subunit structure and assembly architecture are crucial for understanding pilus functions and designing suitable therapeutics against typhoid. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Terminal glycosylation and disease: Influence on cancer and cystic fibrosis

Terminal glycosylation has been a recurring theme of the laboratory. In cystic fibrosis (CF), decreased sialic acid and increased fucosyl residues in 1,3 position to antennary N -acetyl glucosamine is the CF glycosylation phenotype. The glycosylation phenotype is reversed by transfection of CF airway cells with wtCFTR. In neuronal cells, polymers of 2,8sialyl residues are prominent in oligodendrocytes and human neuroblastoma. These findings are discussed in relationship to early studies in our laboratories and those of other investigators. The potential extension of these concepts to future clinical therapeutics is presented.     

A survey of detergents for the purification of stable,active human cystic fibrosis transmembrane conductance regulator (CFTR)

Structural knowledge of the cystic fibrosis transmembrane conductance regulator (CFTR) requires developing methods to purify and stabilize this aggregation-prone membrane protein above 1 mg/ml. Starting with green fluorescent protein- and epitope-tagged human CFTR produced in mammalian cells known to properly fold and process CFTR, we devised a rapid tandem affinity purification scheme to minimize CFTR exposure to detergent in order to preserve its ATPase function. We compared a panel of detergents, including widely used detergents (maltosides, neopentyl glycols (MNG), C₁₂E₈, lysolipids, Chaps) and innovative detergents (branched alkylmaltosides, facial amphiphiles) for CFTR purification, function, monodispersity and stability. ATPase activity after reconstitution into proteoliposomes was 2–3 times higher when CFTR was purified using facial amphiphiles. ATPase activity was also demonstrated in purified CFTR samples without detergent removal using a novel lipid supplementation assay. By electron microscopy, negatively stained CFTR samples were monodisperse at low concentration, and size exclusion chromatography showed a predominance of monomer even after CFTR concentration above 1 mg/ml. Rates of CFTR aggregation quantified in an electrophoretic mobility shift assay showed that detergents which best preserved reconstituted ATPase activity also supported the greatest stability, with CFTR monomer half-lives of 6–9 days in MNG or Chaps, and 12–17 days in facial amphiphile. Cryoelectron microscopy of concentrated CFTR in MNG or facial amphiphile confirmed mostly monomeric protein, producing low resolution reconstructions in conformity with similar proteins. These protocols can be used to generate samples of pure, functional, stable CFTR at concentrations amenable to biophysical characterization.     

Exopolysaccharides produced by Inquilinus limosus, a new pathogen of cystic fibrosis patients: novel structures with usual components

The major cause of morbidity and mortality in patients with cystic fibrosis, an autosomal recessive disorder, is chronic microbial colonisation of the major airways that leads to exacerbation of pulmonary infection. Several different microbes colonise cystic fibrosis lungs, and Pseudomonas aeruginosa is one of the most threatening, since the establishment of mucoid (alginate producing) strains is ultimately associated with the patient's death. Very recently a new bacterium, named Inquilinus limosus, was repeatedly found infecting the respiratory tract of cystic fibrosis patients. Its multi-resistance characteristic to antibiotics might result in the spreading of I. limosus infection among the cystic fibrosis community, as recently happened with strains of the Burkholderia cepacia complex. Since exopolysaccharides are recognised as important virulence factors in lung infections, the primary structure of the polysaccharide produced by I. limosus strain LMG 20952(T) was investigated as the first step in understanding its role in pathogenesis. The structure was determined by means of methylation analysis, acid degradations, mass spectrometry and NMR spectroscopy. The results showed that the bacterium produced a mixture constituted of the following polymers: [3)-[4,6-O-(1-carboxyethylidene)]-beta-D-Glcp(1-->]n; [2)-[4,6-O-(1-carboxyethylidene)]-alpha-D-Manp(1-->]n. Both polymers were completely substituted with pyruvyl ketal groups, a novel structural characteristic not previously found in bacterial polysaccharides. The absolute configuration of all pyruvyl groups was S. Inspection of possible local conformations assumed by the two polysaccharide chains showed features, which might provide interesting clues for understanding structure-function relationships.     

Gene therapy with cytokine-transfected xenogenic cells (Vero-IL-2) in patients with metastatic solid tumors: mechanism(s) of elimination of the transgene-carrying cells

Eleven patients with advanced cancer were treated in a clinical gene therapy trial by repeated intra- tumoral injections with different doses of xenogenic fibroblasts secreting high amounts of human interleukin-2 (Vero-IL2). Treatments in a total of 14 courses were well tolerated and resulted in clinical responses and measurable biological effects. Together with increases in serum interleukin-2 (IL-2), modifications of the V-β T cell receptor repertoire and induction of intratumoral T-cell infiltration were observed. When the intratumoral expression of endogenous cytokine genes and the persistence of the IL-2 transgene at the application site and in peripheral blood were investigated, rapid disappearance of the transgene at the application site appeared to be the most prominent biological effect. Tests detecting a single Vero-IL2 cell against a background of 10⁵ non-transfected cells were not able to demonstrate significant expression of exogenous IL-2 (i.e. the transgene or transgene-carrying cells) in tumor biopsies or blood at different times. Therefore, further studies were performed to evaluate the mechanism(s) involved in the rapid disappearance of xenogenic carrier cells in more detail. We show here that significant in vitro cytotoxicity against transgene-carrying Vero cells can be observed in peripheral blood of all the patients before treatment as well as in healthy controls. “Cold” target inhibition shows that significant killing of Vero-IL2 cells is mediated by natural killer (NK) cells. This was confirmed by showing that established CD3⁻/CD16 ⁺ /CD56 ⁺ peripheral blood NK cell clones kill both K562 and Vero-IL2 target cells. The failure of other mechanisms (complement, antibody-dependent cell cytotoxicity or cytotoxic T lymphocytes) to destroy xenogenic, histoincompatible Vero cells in vitro suggests that NK cells also might be responsible for the killing of Vero-IL2 in vivo and for the failure to detect the transgene at the application site. These results might also be of importance for some aspects of the current discussion of xenotransplantation. Received: 9 April 1999 / Accepted: 14 June 1999     

Inhibition of corneal neovascularization with endostatin delivered by adeno-associated viral (AAV) vector in a mouse corneal injury model

The use of a recombinant adeno-associated viral (rAAV) vector carrying endostatin gene as an anti-angiogenesis strategy to treat corneal neovascularization in a mouse model was evaluated. Subconjunctival injection of recombinant endostatin-AAV was used to examine the inhibition of corneal neovascularization induced by silver nitrate cauterization in mice. The results showed that gene expression in corneal tissue was observed as early as 4 days after gene transfer and stably lasted for over 8 months with minimal immune reaction. Subconjunctival injection of a high-titer rAAV-endostatin successfully inhibited neovascularization. Immunohistchemistry staining of CD 31 and endostatin showed that the treatment significantly inhibits angiogenesis in cornea. We concluded that the rAAV was capable of directly delivering genes to the ocular surface epithelium by way of subconjunctival injection and was able to deliver sustained, high levels of gene expression in vivo to inhibit angiogenesis.