bromochloroacetic-acid and Cystic-Fibrosis

Respiratory epithelium is the target of therapies, such as gene therapy, for cystic fibrosis (CF) lung disease. To determine the usefulness of the nasal epithelium as a pre-screen for lung-directed therapies, we profiled gene expression in CF and non-CF nasal and bronchial epithelium samples using Illumina HumanRef-8 Expression BeadChips. 863 genes were differentially expressed between CF and non-CF bronchial epithelium but only 15 were differentially expressed between CF and non-CF nasal epithelium (≥1.5-fold, P≤0.05). The most enriched pathway in CF bronchial epithelium was inflammatory response, whereas in CF nasal epithelium it was amino acid metabolism. We also compared nasal and bronchial epithelium in each group and identified differential expression of cellular movement genes in CF patients and cellular growth genes in non-CF subjects. We conclude that CF and non-CF nasal and bronchial epithelium are transcriptionally distinct and CF nasal epithelium is not a good surrogate for the lung respiratory epithelium.

Topics: Adolescent; Adult; Bronchi; Case-Control Studies; Child; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Female; Gene Expression Regulation; Humans; Immunohistochemistry; Inflammation; Keratins; Male; Nasal Mucosa; Oligonucleotide Array Sequence Analysis; Young Adult

Unbiased approaches that study aberrant protein expression in primary airway epithelial cells at single cell level may profoundly improve diagnosis and understanding of airway diseases. We here present a flow cytometric procedure to study CFTR expression in human primary nasal epithelial cells from patients with Cystic Fibrosis (CF). Our novel approach may be important in monitoring of therapeutic responses, and better understanding of CF disease at the molecular level.. Validation of a panel of CFTR-directed monoclonal antibodies for flow cytometry and CFTR expression analysis in nasal epithelial cells from healthy controls and CF patients.. We analyzed CFTR expression in primary nasal epithelial cells at single cell level using flow cytometry. Nasal cells were stained for pan-Cytokeratin, E cadherin, and CD45 (to discriminate epithelial cells and leukocytes) in combination with intracellular staining of CFTR. Healthy individuals and CF patients were compared.. We observed various cellular populations present in nasal brushings that expressed CFTR protein at different levels. Our data indicated that CF patients homozygous for F508del express varying levels of CFTR protein in nasal epithelial cells, although at a lower level than healthy controls.. CFTR protein is expressed in CF patients harboring F508del mutations but at lower levels than in healthy controls. Multicolor flow cytometry of nasal cells is a relatively simple procedure to analyze the composition of cellular subpopulations and protein expression at single cell level.

Topics: Animals; Bronchi; Cadherins; Case-Control Studies; Cell Line; Cell Line, Tumor; Cricetinae; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelial Cells; Flow Cytometry; Gene Expression Regulation; Homozygote; Humans; Keratins; Leukocyte Common Antigens; Nasal Mucosa

Recent studies have suggested that both embryonic stem cells and adult bone marrow stem cells can participate in the regeneration and repair of diseased adult organs, including the lungs. However, the extent of airway epithelial remodeling with adult marrow stem cells is low, and there are no available in vivo data with embryonic stem cells. Human umbilical cord blood contains both hematopoietic and nonhematopoietic stem cells, which have been used clinically as an alternative to bone marrow transplantation for hematologic malignancies and other diseases.. We hypothesized that human umbilical cord blood stem cells might be an effective alternative to adult bone marrow and embryonic stem cells for regeneration and repair of injured airway epithelium.. Human cord blood was obtained from normal deliveries at the University of Vermont. Cultured plastic adherent cells were characterized as mesenchymal stem cells (MSCs) by flow cytometry and differentiation assays. Cord blood-derived MSCs (CB-MSCs) were cultured in specialized airway growth media or with specific growth factors, including keratinocyte growth factor and retinoic acid. mRNA and protein expression were analyzed with PCR and immunofluorescent staining. CB-MSCs were systematically administered to immunotolerant, nonobese diabetic/severe combined immunodeficiency (NOD-SCID) mice. Lungs were analyzed for presence of human cells.. When cultured in specialized airway growth media or with specific growth factors, CB-MSCs differentially expressed Clara cell secretory protein (CCSP), cystic fibrosis transmembrane conductance regulator (CFTR), surfactant protein C, and thyroid transcription factor-1 mRNA, and CCSP and CFTR protein. Furthermore, CB-MSCs were easily transduced with recombinant lentiviral vectors to express human CFTR. After systemic administration to immunotolerant, NOD-SCID, mice, rare cells were found in the airway epithelium that had acquired cytokeratin and human CFTR expression.. CB-MSCs appear to be comparable to MSCs obtained from adult bone marrow in ability to express phenotypic markers of airway epithelium and to participate in airway remodeling in vivo.

Topics: Animals; Cell Culture Techniques; Cord Blood Stem Cell Transplantation; Cystic Fibrosis; Humans; Keratins; Mesenchymal Stem Cells; Mice; Mice, Inbred NOD; Mice, SCID; Regeneration; Respiratory Mucosa; Transduction, Genetic

Cystic fibrosis airways are recurrently exposed to noxious stimuli, leading to epithelial injury. Previous reports suggest that cystic fibrosis airway epithelia may respond to injury by increasing proliferation.. We sought to determine the characteristics of the proliferating cell population in cystic fibrosis airways.. Six cystic fibrosis and six normal lung sections from lung transplant recipients or lung surgery were obtained from the Duke Hospital pathology archives. Sections containing bronchi were evaluated for epithelial cell proliferation using immunohistochemistry for a nuclear proliferation antigen, Ki-67, and image analysis; immunohistochemistry for basal cells using a cytokeratin 5/14 antibody; and immunohistochemistry for the epidermal growth factor receptor and ErbB2, two receptor tyrosine kinases implicated in epithelial proliferation and differentiation.. Overall, cystic fibrosis sections had a greater proliferation index than control sections with 25.1 +/- 2.1% positively staining nuclei/total nuclei compared with control sections, 4.6 +/- 0.9% (p = 0.002). In cystic fibrosis sections only, there were areas of hyperplastic cuboidal cells adjacent to normal pseudostratified columnar epithelial sections; in these areas of epithelial hyperplasia, there was uniform Ki-67 staining, indicating a zone of proliferating cells. The proliferating cell population also expressed the basal cell cytokeratins 5/14 and epidermal growth factor receptor. Expression of ErbB2 was diminished in the proliferating cells.. Our results suggest that basal-like cells, expressing the epidermal growth factor receptor, constitute the proliferating cell population in cystic fibrosis airways.

Topics: Adolescent; Adult; Basement Membrane; Biopsy; Bronchi; Case-Control Studies; Cell Count; Cell Proliferation; Child, Preschool; Cystic Fibrosis; ErbB Receptors; Female; Goblet Cells; Humans; Hyperplasia; Image Cytometry; Immunohistochemistry; Infant; Keratins; Ki-67 Antigen; Male; Middle Aged; Photomicrography; Receptor, ErbB-2; Respiratory Mucosa

A feature associated frequently with the pathologic pancreas is the presence of tubular complexes produced by a phenotypic modulation of acinar cells that take on the characteristics of ductular cells. Since the type I Reg gene, an acinar cell product, is increased in the pancreas following an acinar injury, we aimed to evaluate whether the Reg I protein might be involved in this dedifferentiation process in the human pancreas. We studied duct-like structures in fixed human pathologic pancreatic tissues and human cells with a ductal phenotype obtained by culturing human exocrine preparations. Immunocytochemistry, Western blotting, and RT-PCR were applied for detection of type I Reg. Reg I was observed not only in acinar cells but also in the duct-like cells and dilated duct cells, both positive for cytokeratin 19. However, none of the other acinar markers was observed in these cells. In vitro, human acinar cells dedifferentiated, losing their acinar phenotype, but expression of Reg I remained constant throughout the culture duration. Furthermore, Reg I was not associated with proliferation. We demonstrated that Reg I expression was linked to acinar cell dedifferentiation. We postulate that Reg I might be used as a marker to understand the events leading to phenotypic changes of acinar cells to address the physiological role of Reg I in the pancreas.

Topics: Adolescent; Adult; Biomarkers; Calcium-Binding Proteins; Cell Differentiation; Cell Division; Cell Polarity; Cystic Fibrosis; Female; Gene Expression Regulation; Glycoproteins; Humans; Keratins; Lithostathine; Male; Pancreas; Pancreatic Ducts; Phenotype; Reverse Transcriptase Polymerase Chain Reaction

Alveolar type II pneumocytes are thought to be progenitor cells capable of self-renewal and differentiation into type I pneumocytes. Nevertheless, the existence of an alveolar stem cell has been postulated. In lungs from patients with cystic fibrosis, the alveolar epithelium is damaged with ulceration and subsequent regeneration. We characterized alveolar modifications histologically and immunohistochemically in the pulmonary tissue of a patient homozygous for the DeltaF508 mutation. Alveoli were of variable size and surrounded by an inflammatory infiltrate. They were lined by a continuous layer of cuboidal cells with very weak proliferative activity. These cells resembled type II pneumocytes. They expressed thyroid transcription factor-1, cystic fibrosis transmembrane conductance regulator, cytokeratin 7 and contained lamellar bodies. Weak expression of cytokeratin 5 considered to be a marker of progenitor cells of the bronchial and bronchiolar epithelium was detected. Explantation of this alveolar epithelium produced primary cultures and subcultures of epithelial cells that had acquired proliferative properties showing signs of dedifferentiation with a loss of lamellar bodies and a lack of expression of thyroid transcription factor-1. Persistence of the expression of cytokeratin 7 and a strong expression of cytokeratin 5 were observed. The culture conditions were thought to have circumvented the inhibition of proliferation observed in vivo due to the inflammatory peri-alveolar environment. They thus favored the multiplication of a population of cells co-expressing cytokeratin 5 and certain characteristics of type II pneumocytes. The presence of these cells of intermediate phenotype is indicative of the existence of immature precursors for type II pneumocytes.

Topics: Biomarkers; Cells, Cultured; Child; Cystic Fibrosis; Epithelial Cells; Humans; Keratin-7; Keratins; Lung; Pulmonary Alveoli; Stem Cells

Efforts have been made to deliver transgenes to the airway epithelia of laboratory animals and humans to develop gene therapy for cystic fibrosis. These investigations have been disappointing due to combinations of transient and low-level gene expression, acute toxicity, and inflammation. We have developed new helper-dependent adenoviral vectors to deliver an epithelial cell-specific keratin 18 expression cassette driving the beta-galactosidase (beta-gal) or human alpha-fetoprotein (AFP) reporter genes. Following intranasal administration to mice, we found that the reporter genes were widely expressed in airway epithelial and submucosal cells, and secreted human AFP was also detectable in serum. In contrast to a first-generation adenoviral vector, inflammation was negligible at doses providing efficient transduction, and expression lasted longer than typically reported-up to 28 days with beta-gal and up to 15 weeks with human AFP. These results suggest that delivery to the airway of helper-dependent adenoviral vectors utilizing a tissue-specific promoter could be a significant advance in the development of gene therapy for cystic fibrosis.

Topics: Adenoviridae; alpha-Fetoproteins; Animals; beta-Galactosidase; Cystic Fibrosis; Epithelial Cells; Female; Gene Expression; Genetic Therapy; Genetic Vectors; Helper Viruses; Humans; Inflammation; Keratins; Mice; Mice, Inbred C57BL; Mucous Membrane; Respiratory Mucosa

Liver disease causes significant morbidity and mortality from multilobular cirrhosis in patients with cystic fibrosis. Abnormal bile transport and biliary fibrosis implicate abnormal biliary physiology in the pathogenesis of cystic fibrosis-associated liver disease (CFLD), yet the mediators linking biliary events to fibrosis remain unknown. Activated hepatic stellate cells (HSCs) are the pre-eminent mediators of fibrosis in a range of hepatic disorders. The dominant stimulus for matrix production by HSCs is the cytokine transforming growth factor (TGF)-beta(1). In CFLD, the role of HSCs and the source of TGF-beta(1) have not been evaluated. Liver biopsy tissue obtained from 38 children with CFLD was analyzed. Activated HSCs, identified by co-localization of procollagen alpha(1)(I) mRNA and alpha-smooth muscle actin, were demonstrated as the cellular source of excess collagen production in the fibrosis surrounding the bile ducts and the advancing edge of scar tissue. TGF-beta protein and TGF-beta(1) mRNA expression were shown to be predominantly expressed by bile duct epithelial cells. TGF-beta(1) expression was significantly correlated with both hepatic fibrosis and the percentage of portal tracts showing histological abnormalities associated with CFLD. This study demonstrates a definitive role for HSCs in fibrogenesis associated with CFLD and establishes a potential mechanism for the induction of HSC collagen gene expression through the production of TGF-beta(1) by bile duct epithelial cells.

Topics: Actins; Adolescent; Child; Child, Preschool; Collagen; Cystic Fibrosis; Female; Gene Expression; Humans; Immunohistochemistry; In Situ Hybridization; Infant; Keratins; Liver; Liver Diseases; Male; Muscle, Smooth; Procollagen; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1

The influence of cell differentiation and proliferation on cationic vector mediated gene transfer into the explant-outgrowth cell culture from nasal polyps was investigated. Respiratory cells were categorized into two groups based on the expression of cytokeratin filaments (CKs), which were used as differentiation markers. Outgrowths grown for 2 weeks expressed similar levels of CKs 14, 13 and 18 showing a de-differentiated phenotype, while outgrowths cultured for 4 weeks presented very high levels of CK 13, high CK 14 and low CK 18 expression and were squamous differentiated. De-differentiated cells presented higher proliferation indexes than squamous cells. Gene transfer levels, as evaluated using a quantitative reporter gene (firefly luciferase), were significantly higher in the 2- than in the 4-week-old outgrowths. Cationic vector transfected respiratory cells were located both proximally and distally to the explant, as shown by enzymatic staining of beta-galactosidase-positive cells. Respiratory cell outgrowths from nasal polyps can be considered a suitable model to study gene transfer protocols in vitro.

Topics: Biomarkers; Cell Differentiation; Cells, Cultured; Cystic Fibrosis; Fatty Acids, Monounsaturated; Gene Transfer Techniques; Genes, Reporter; Genetic Vectors; Humans; Keratins; Nasal Polyps; Phenotype; Quaternary Ammonium Compounds; Transfection

We developed an epithelium-specific, inducible cystic fibrosis transmembrane conductance regulator (CFTR) expression system. In this system we used a human cytokeratin 18 expression cassette to drive epithelium-specific expression of the reverse tetracycline transactivator (rtTA), which turns on CFTR expression from a Tet-inducible promoter in the presence of doxycycline. CFTR expression was monitored by reverse-transcription polymerase chain reaction, immunostaining, and Western blotting. We confirmed that protein expression was dose-dependent in double stable transfected cell lines, with no detectable protein in the absence of doxycycline. However, low levels of CFTR mRNA could be detected in the uninduced state. When clones capable of inducing high levels of CFTR expression were analyzed, we observed a decrease in cell proliferation, consistent with reports in other cell lines (NIH3T3 and BTS). We generated transgenic mice expressing rtTA from the K18 expression cassette and demonstrated that the system retained its tissue specificity for lacZ reporter expression in vivo. When mice were induced with doxycycline, high levels of expression were found in the trachea, upper bronchi, and submucosal glands. Therefore, this inducible system can improve our understanding of the role of CFTR in the lung and should help in the design of safe and effective CF therapies.

Topics: 3T3 Cells; Animals; beta-Galactosidase; Blotting, Western; Bronchi; Cell Division; Cell Line; Cells, Cultured; COS Cells; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Dose-Response Relationship, Drug; Doxycycline; Epithelial Cells; Epithelium; Gene Transfer Techniques; Genes, Reporter; Humans; Keratins; Lung; Mice; Mice, Transgenic; Microscopy, Fluorescence; Plasmids; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tetracycline; Time Factors; Transcriptional Activation; Transfection

We have developed an expression cassette for cystic fibrosis (CF) gene therapy using control elements from the human cytokeratin 18 gene (KRT18, also known as K18). KRT18 is naturally expressed in a spatial pattern similar to that of CFTR, the gene mutated in CF. We delivered a KRT18-driven lacZ plasmid complexed with cationic liposomes intravenously to mice and examined expression in various tissues. We found expression in nasal and bronchial epithelium, airway submucosal glands, gall bladder, and kidneys. Expression was low in pancreas and gut, and absent from liver and alveolar lung. This is consistent with the expression pattern reported for a K18lacZ transgenic mouse. Following delivery of a cytomegalovirus (CMV) major immediate-early promoter/enhancer-driven lacZ plasmid, we found expression in bronchi, submucosal glands, alveolar cells, liver, and kidney. We did not detect expression in nose, pancreas, gall bladder, or gut. Using fluorescently labeled plasmid delivered by means of liposomes, we identified the liver, alveolar lung, and kidneys as the major plasmid deposition sites. Our data demonstrate that a KRT18-driven expression vector delivered systemically can target gene expression to CF-affected tissues, despite an uneven distribution of plasmid DNA. A KRT18-based vector may be a useful alternative to viral promoter-based vectors in clinical gene therapy trials to treat CF.

Topics: Animals; Cystic Fibrosis; Gene Expression; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Glycerophospholipids; Humans; Injections, Intravenous; Keratins; Lac Operon; Liposomes; Mice; Mice, Inbred Strains; Organ Specificity; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Surface-Active Agents; Tissue Distribution; Transgenes

Although the Burkholderia cepacia complex consists of several genomovars, one highly transmissible strain of B. cepacia has been isolated from the sputa of cystic fibrosis (CF) patients throughout the United Kingdom and Canada. This strain expresses surface cable (Cbl) pili and is thought to be the major strain associated with the fatal "cepacia syndrome." In the present report we characterize the specific 55-kDa buccal epithelial cell (BEC) protein that binds cable pilus-positive B. cepacia. N-terminal sequences of CNBr-generated internal peptides identified the protein as cytokeratin 13 (CK13). Western blots of BEC extracts probed with a specific monoclonal antibody to CK13 confirmed the identification. Mixed epidermal cytokeratins (which contain CK13), cytokeratin extract from BEC (which consists essentially of CK13 and CK4), and a polyclonal antibody to mixed cytokeratins inhibited B. cepacia binding to CK13 blots and to normal human bronchial epithelial (NHBE) cells. Preabsorption of the antikeratin antibody with the BEC cytokeratin fraction reversed the inhibitory effect of the antibody. A cytokeratin mixture lacking CK13 was ineffective as an inhibitor of binding. Colocalization of CK13 and B. cepacia by confocal microscopy demonstrated that intact nonpermeabilized NHBE cells express small amounts of surface CK13 and bind Cbl-positive B. cepacia in the same location. Binding to intact NHBE cells was dependent on bacterial concentration and was saturable, whereas a Cbl-negative isolate exhibited negligible binding. These findings raise the possibility that surface-accessible CK13 in respiratory epithelia may be a biologically relevant target for the binding of cable piliated B. cepacia.

Topics: Amino Acid Sequence; Bacterial Adhesion; Bronchi; Burkholderia cepacia; Cells, Cultured; Cystic Fibrosis; Cytoskeleton; Cytosol; Epithelial Cells; Humans; Keratins; Microscopy, Fluorescence; Molecular Sequence Data; Receptors, Immunologic; Tumor Cells, Cultured

The Burkholderia cepacia complex consists of at least five well-documented bacterial genomovars, each of which has been isolated from the sputum of different patients with cystic fibrosis (CF). Although the world-wide prevalence of this opportunist pathogen in CF patients is low (1-3%), 'epidemic' clusters occur in geographically isolated regions. Prevalence in some of these clusters is as high as 30-40%. The majority of CF B. cepacia isolates belong to genomovar III, but the relationship between genomovar and virulence has not yet been defined. Because the initial stage of infection involves bacterial binding to host tissues, the present study investigated differences in the binding of representative isolates of all five genomovars to fixed nasal sections of UNC cftr (-/-) and (+/+) mice and to human lung explants, biopsy and autopsy tissue of CF and non-CF patients. Binding was highest for isolates of genomovar III, subgroup RAPD type 2, but only if the isolates expressed the cable pili phenotype. Antibodies to the 22-kDa adhesin of cable pili virtually abolished binding. Binding occurred only to cftr (-/-) nasal sections or to CF lung sections and was negligible in cftr (+/+) or human non-CF, histologically normal lung sections. Unlike normal epithelia, the hyperplastic epithelia of CF bronchioles were enriched in cytokeratin 13, a 55-kDa protein that has previously been shown to act as a receptor in vitro for cable-piliated B. cepacia. These findings may help to explain the high transmissibility ofCbl-positive, genomovar III strains of B. cepacia among CF patients.

Topics: Adhesins, Bacterial; Adolescent; Adult; Animals; Antibodies, Bacterial; Bacterial Adhesion; Burkholderia cepacia; Burkholderia Infections; Canada; Cystic Fibrosis; DNA Primers; DNA, Bacterial; Fimbriae, Bacterial; Humans; Keratins; Lung; Mice; Mice, Inbred C57BL; Mice, Knockout; Nasal Mucosa; Specific Pathogen-Free Organisms; Virulence

Human tracheal gland serous (HTGS) cells are now considered one principal pulmonary target for the gene therapy of cystic fibrosis (CF). We developed a CF tracheal gland serous cell line, CF-KM4, obtained by the transformation of primary cultures of CF tracheal gland serous cells homozygous for the DeltaF508 mutation by using the wild-type SV40 virus. This cell line retained epithelial and secretory features of the native CF-HTGS cells in primary culture, namely, presence of cytokeratin, constitutive secretion of secretory leukocyte proteinase inhibitor, absence of responsiveness to carbachol and isoproterenol, and defective cyclic adenosine monophosphate-dependent chloride channel activity. Adenovirus-mediated CF transmembrane conductance regulator (CFTR) gene transfer into CF-KM4 cells corrected the defective chloride channel activity as well as the responsiveness to adrenergic and cholinergic agonists. In contrast, control transfection using adenovirus-mediated beta-galactosidase gene transfer was totally ineffective. In conclusion, these results present a stable CF tracheal gland cell line that has retained its epithelial and CF-specific defective secretory characteristics which are corrected after CFTR gene transfer. This cell line therefore appears to be a useful tool for large-scale molecular and cellular pharmacologic investigations designed to test potential therapies of the disease CF.

Topics: Animals; beta-Galactosidase; Cell Division; Cell Line; CHO Cells; Clone Cells; Cricetinae; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Gene Transfer Techniques; Genetic Vectors; Homozygote; Humans; Keratins; Membrane Potentials; Recombinant Proteins; Sequence Deletion; Simian virus 40; Trachea; Transfection

The absence or mislocalization of cystic fibrosis transmembrane conductance regulator (CFTR) is regarded as being specific for cystic fibrosis (CF). In principle, the supply of a non-CF lung transplant to a CF patient should bring up normal CFTR expression in the lower airways. Immunolocalization of CFTR and of epithelial differentiation markers (ie, cytokeratins 13, 14, and 18, and desmoplakins 1 and 2) was carried out on 21 mucosal biopsies from the upper lobe of grafts in non-CF (n = 12) and CF patients (n = 9) retrieved between days 23 and 1,608 after lung transplantation. Biopsy specimens from seven non-CF and four CF patients presented either a pseudostratified respiratory epithelium or slight basal cell hyperplasia. CFTR was distributed at the apical membrane of the ciliated cells. In remodeled epithelia with basal cell hyperplasia or squamous metaplasia, CFTR was either weakly expressed in the cytoplasm of the superficial epithelial cells or was undetectable. The extent of epithelium remodeling was significantly correlated with an impairment of lung function. The results suggest that posttransplant airway epithelium dedifferentiation of the graft leads to the loss of properly targeted CFTR irrespective of the underlying disease of the recipient.

Topics: Adult; Age Factors; Biopsy; Cell Differentiation; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Cytoskeletal Proteins; Desmoplakins; Epithelial Cells; Epithelium; Female; Fluorescent Antibody Technique, Indirect; Forced Expiratory Volume; Humans; Keratins; Lung; Lung Transplantation; Male; Middle Aged

Cystic fibrosis (CF) involves abnormalities in mucus production and secretion of the airway. Studies of the regulation of airway mucin production and secretion has been difficult due to the lack of in vitro models of the airway epithelial cells which express functional differentiation. Because the majority of the mucin in the airway is apparently produced by the submucosal glands, we have focused our attention on the development of cell culture models of human airway submucosal glands. This report describes the propagation of CF airway submucosal gland epithelial cells which continue to express mucin production. The CF bronchus was obtained from a 31-yr-old patient who received a double lung transplant. The glands were dissected out and primary cultures prepared by the explant/outgrowth procedure. The cells were immortalized by infection with Ad12-SV40 hybrid virus. The cultures are maintained in serum-free keratinocyte basal medium supplemented with insulin (5 micrograms/ml), hydrocortisone (0.5 microgram/ml), epidermal growth factor (10 ng/ml), bovine pituitary extract (25 micrograms/ml), and antibiotics. Cultures were passaged using 0.125% trypsin in Ca+2 and Mg(+2)-free Hanks', balanced salt solution. Polymerase chain reaction (PCR) analysis demonstrated that the cells were homozygous for the delta F508 mutation. Morphologic observations showed that the cells were epithelial and were interconnected by sparsely distributed desmosomes. Their cytoplasm contained secretory-type structures including abundant Golgi, rough endoplasmic reticulum, and secretory vesicles. Immunofluorescent studies determined that all cells were positive for cytokeratins, mucin glycoconjugates, and cystic fibrosis transmembrane conductance regulator. The cultures secreted substantial amounts of mucin glycoproteins and expressed the MUC-2 mucin gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Base Sequence; Bronchi; Cell Differentiation; Cells, Cultured; Culture Media; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; DNA Primers; Epithelium; Gene Expression; Glycoconjugates; Homozygote; Humans; Immunohistochemistry; Keratins; Membrane Proteins; Molecular Sequence Data; Mucin-2; Mucins; Mutation; Polymerase Chain Reaction

Cystic fibrosis (CF) is the most common lethal inherited disease in the Caucasian population with an incidence of approximately 1 in 2,500 live births. Pulmonary complications of CF, which are the most morbid aspects of the disease, are caused by primary abnormalities in epithelial cells that lead to impaired mucociliary clearance. One potential therapeutic strategy is to reconstitute expression of the CF gene in airway epithelia by somatic gene transfer. To this end, we have developed an animal model of the human airway using bronchial xenografts and have tested the efficiency of in vivo retroviral gene transfer. Using the LacZ reporter gene, we find the efficiency of in vivo retroviral gene transfer to be dramatically dependent on the regenerative and mitotic state of the epithelium. Within an undifferentiated regenerating epithelium in which 40% of nuclei labeled with BrdU, 5-10% retroviral gene transfer was obtained. In contrast, no gene transfer was noted in a fully differentiated epithelium in which 1% of nuclei labeled with BrdU. These findings suggest that retroviral mediated gene transfer to the airway in vivo may be feasible if the proper regenerative state can be induced.

Topics: Animals; Bronchi; Cell Differentiation; Cell Division; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelial Cells; Epithelium; Keratins; Male; Membrane Proteins; Mice; Mice, Nude; Rats; Rats, Inbred F344; Regeneration; Retroviridae; Transfection; Transplantation, Heterologous

The epithelia lining the vas deferens and epididymis are directly involved in the pathology of the autosomal recessive disease cystic fibrosis (CF). We have established culture systems for these epithelial cells. Long-term cell lines have now been generated from these primary epithelial cells by transformation with a plasmid containing an origin-defective simian virus 40 (SV40). Lines have been established from vas deferens and epididymis and both maintain expression of the CF gene.

Topics: Base Sequence; Cell Line, Transformed; Cell Transformation, Viral; Cystic Fibrosis; DNA; Epididymis; Epithelial Cells; Epithelium; Gene Expression Regulation; Genes, Recessive; Humans; Keratins; Male; Molecular Sequence Data; Mucins; Plasmids; Vas Deferens

The understanding of pathways associated with differentiated function in human epithelial cells has been enhanced by the development of methods for the short-term culture of human epithelial cells. In general these methods involve the use of serum. The subculture and maintenance of epithelial cells in long-term culture has been more problematic. A serum-free medium developed for human bronchial epithelial cells was slightly modified and found to be useful for the subculture and long-term maintenance of not only bronchial epithelial cells, but also tracheal, nasal polyp, and sweat gland epithelial cells from either normal or cystic fibrosis individuals. The cells maintained epithelial-specific characteristics after multiple subcultures. Monolayers of epithelial cells showed junctional complex formation, the presence of keratin, and micro villi. Functional studies with Ussing chambers showed short circuit current (Isc) responses to isoproterenol, bradykinin, or calcium ionophore (A23187) in subcultured tracheal and bronchial cells.

Topics: Bronchi; Cells, Cultured; Culture Media; Cystic Fibrosis; Electric Conductivity; Epithelial Cells; Epithelium; Female; Humans; Immunohistochemistry; Intercellular Junctions; Keratins; Male; Microscopy, Electron; Microvilli; Nasal Polyps; Respiratory Physiological Phenomena; Respiratory System; Sweat Glands; Trachea

We undertook to extend the in vitro lifespan of epithelial cell cultures useful for the study of the cellular defect underlying cystic fibrosis (CF). Primary cultures from sweat glands of four CF and four non-CF and from nasal polyps of one non-CF and two CF individuals were transformed using a chimaeric virus, Ad5/SV40 1613 ori-. The extended lifespans ranged from 20 to more than 250 population doublings beyond that of the primary cultures. Despite some degree of aneuploidy (as assayed by total cellular DNA content) all samples tested retained at least one copy of the region of chromosome 7 containing the CF gene (as assayed by probing with flanking DNA markers). Epithelial characteristics, including an epithelioid morphology, tight junctions and desmosomes, apical microvilli, keratin networks, and dome formation were positive in the majority of cells examined, although variably expressed. All cells tested demonstrated outwardly rectifying chloride channels by patch clamp, with some from non-CF cells responsive to the catalytic subunit of cyclic AMP-dependent protein kinase. The cells were used for DNA transfection assays with selectable marker genes in appropriate vectors, in order to develop methodology for assaying the function of the CF gene product and the effects of mutations.

Topics: Cell Division; Cell Line, Transformed; Cell Survival; Cell Transformation, Viral; Cystic Fibrosis; DNA; Epithelial Cells; Humans; Keratins; Microscopy, Electron; Nasal Polyps; Sweat Glands

Continuous epithelial cell lines from individuals with cystic fibrosis (CF) and normal controls are required to understand the genetic and cellular defects in CF. We used retroviruses to transduce SV40 large T antigen into nasal epithelial cells. Transformed continuous cell lines were isolated that expressed epithelial markers, cytokeratin, and tight junctions. Northern blot analysis shows that all of the cell lines express the putative CF gene mRNA. Studies of transepithelial electrolyte transport show that CF and normal cell lines develop a transepithelial electrical resistance. Normal but not CF cell lines secreted Cl- in response to agonists that increase cellular levels of adenosine 3',5'-cyclic monophosphate (cAMP) (isoproterenol, forskolin, and a membrane-permeant analogue of cAMP) or in response to a tumor-promoting phorbol ester that activates protein kinase C. In contrast, the Ca2(+)-elevating agonist bradykinin and the Ca2+ ionophore A23187 stimulated secretion in both normal and CF cell lines. The continuous cell lines we have produced maintain their proper phenotypes and will serve as useful tools in understanding the pathophysiology of CF.

Topics: Antigens, Polyomavirus Transforming; Base Sequence; Biomarkers; Blood Proteins; Bradykinin; Calcimycin; Calgranulin A; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; Chloride Channels; Chlorides; Colforsin; Cystic Fibrosis; Epithelial Cells; Epithelium; Genotype; Humans; Ion Channels; Keratins; Membrane Potentials; Membrane Proteins; Molecular Sequence Data; Nasal Mucosa; Oligonucleotide Probes; Phenotype; Polymerase Chain Reaction; Reference Values; Retroviridae; Simian virus 40; Tetradecanoylphorbol Acetate; Theophylline

We have developed immortalized epithelial cystic fibrosis (CF) cell lines by infecting cultured nasal polyp cells with a SV40/Adenol2 hybrid virus. The cell lines obtained are epithelial in nature as shown by cytokeratin production and morphology, although cytokeratins 4 and 13 typical of primary nasal polyp cells are produced at a much reduced rate. Ussing chamber experiments showed that the precrisis CF cell line NCF3 was able to perform trans-cellular chloride transport when activated by agents which elevate intracellular calcium. cAMP agonists had no effect on chloride flux in NCF3 as expected for CF cells. The apical chloride channels found with the patch clamp technique in NCF3 and in the postcrisis cell line NCF3A have a conductance similar to that of chloride channels found earlier in normal and CF epithelial cells. The channels show a delay in the onset of activity in off-cell patches and are not activated by increased cAMP levels in the cell. This indicates that immortalized CF epithelial cells will provide a useful model for the study of cystic fibrosis.

Topics: Cell Line; Cells, Cultured; Chloride Channels; Chlorides; Culture Techniques; Cystic Fibrosis; Epithelium; Humans; Ion Channels; Keratins; Membrane Proteins; Microscopy, Electron; Nasal Polyps

The recognized need for epithelial cell culture models for cystic fibrosis (CF) research has resulted in ongoing efforts to improve normal and CF submandibular duct cell culture capabilities. The duct is most likely the site of the CF defect in this and other exocrine glands. In a previous report conditions required for the successful primary explant culture of normal and CF submandibular glands were outlined; however, terminal keratinization and involution of these cultures were recognized as severe limiting factors to their utilization in CF research. This report explores the effects of calcium concentrations in the medium, growth factor supplements, and matrix components on growth and differentiation of these cultures. Results of the study further confirm the ductal origin of cells in the outgrowth and demonstrate that progressive keratinization is initiated only after cells proliferate beyond the environment of the explant fragment. Keratinization with subsequent multilayering, desmosome formation, and involution in the cell outgrowth are governed in degree by the calcium concentration of the growth medium. Upon reduction of medium calcium to 0.1 mM concentration, the cells proliferate as a monolayer and subculture through 8 to 9 passages and retain the capacity to undergo ductlike differentiation.

Topics: Blood; Calcium; Cell Differentiation; Cell Division; Cell Nucleus; Cells, Cultured; Collagen; Culture Media; Cystic Fibrosis; Desmosomes; Humans; Immunohistochemistry; Keratins; Laminin; Microscopy, Electron; Microvilli; Organoids; Submandibular Gland

The elucidation of the underlying defect in fluid secretion by cystic fibrosis (CF) sweat glands is hindered by the unavailability of an experimental model for investigating this disease. As a potential model system, a serum-free growth medium was developed that supports the explant growth of epithelial cells from fragments of human skin. Immunohistochemical analysis demonstrated that these epithelial cell outgrowths originated from the duct of the sweat gland. By electron microscopy, the cells were demonstrated to possess keratinocyte-like morphology as noted by the presence of a multilayered outgrowth of cells containing well-defined keratin bundles. Identical outgrowths from skin biopsies of CF patients were compared to normal outgrowths and alterations were noted to occur in dome formation and in the number of intercellular spaces between cells. Doming alterations were also noted to occur in the CF heterozygous state. No differences in cell fine structure or in growth factor requirements for cell proliferation were noted between normal and CF cells. The potential use of this system as a model for CF research is discussed.

Topics: Culture Techniques; Cystic Fibrosis; Epithelial Cells; Humans; Keratins; Microscopy, Electron; Sweat Glands

As an approach to investigating the disease cystic fibrosis, attempts were undertaken to culture from human submandibular glands epithelial cells with a potential for manifesting the cystic fibrosis genetic defect. To initiate the culture of submandibular gland epithelial cells, tissue fragments from glands were explanted as a function of both the composition of the serum-free growth medium and of the matrix utilized to coat the culture vessel growth surface. A morphologically homogeneous growth of submandibular gland epithelial cells, uncontaminated by fibroblasts, was obtained, once optimum culture conditions were defined. Light microscopic examination of these explant cultures in a transverse plane of section demonstrated variation in the outgrowth according to distance from the explant. At its outer margin, the outgrowth consisted of one or two layers of viable low cuboidal cells, and more centrally, it was multilayered. Mitotic figures were observed in the periphery of the outgrowth. In the region, a few cells removed from the periphery where the outgrowth consisted of about three to six cell layers, dilated intercellular spaces, indicative of secretion of fluid and ions into the spaces, separated the basal cuboidal cells. Overlying cells were increasingly flattened toward the culture surface and devoid of nuclei. Centrally, near the explant, the multilayer appeared completely involuted throughout. Ultrastructural examination in a transverse plane of the multilayered region with viable basal cells confirmed these observations showing wide spaces separating the cuboidal basal cells, keratinization of midstratum cells, and complete involution of the upper layer of ghost-like cells. These cells cultured from the submandibular gland reacted positively to immunochemical staining for keratin.

Topics: Cells, Cultured; Cystic Fibrosis; Epithelial Cells; Epithelium; Humans; Keratins; Microscopy, Electron; Organoids; Submandibular Gland

Topics: Cystic Fibrosis; Hand; Humans; Hydrogen-Ion Concentration; Immersion; Keratins; Protein Binding; Skin; Sodium Chloride; Sweat; Sweat Glands