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D C Gruenert

Publications and source records attributed to D C Gruenert.

At least 19 recordsLinked to original sources
PubMed2004

Established cell lines used in cystic fibrosis research.

The development of immortalized cell lines has been a significant benefit to the study of human disease, due to limitations in using primary cells and the availability of tissue. The immortalization of cells from cystic fibrosis (CF) patients as well as cells from non-CF individuals from tissues relevant to CF has been critical to enhancing our understanding of the physiological, biochemical and genetic mechanisms underlying CF and for the development of therapeutic strategies designed to manage CF pathology. A comprehensive list of immortalized cells from various tissue and species, with an emphasis on epithelial cells, is presented and discussed here.

Animals↗
PubMed2002

Genome medicine: gene therapy for the millennium, 30 September-3 October 2001, Rome, Italy.

The recent surge of DNA sequence information resulting from the efforts of agencies interested in deciphering the human genetic code has facilitated technological developments that have been critical in the identification of genes associated with numerous disease pathologies. In addition, these efforts have opened the door to the opportunity to develop novel genetic therapies to treat a broad range of inherited disorders. Through a joint effort by the University of Vermont, the University of Rome, Tor Vergata, University of Rome, La Sapienza, and the CSS Mendel Institute, Rome, an international meeting, 'Genome Medicine: Gene Therapy for the Millennium' was organized. This meeting provided a forum for the discussion of scientific and clinical advances stimulated by the explosion of sequence information generated by the Human Genome Project and the implications these advances have for gene therapy. The meeting had six sessions that focused on the functional evaluation of specific genes via biochemical analysis and through animal models, the development of novel therapeutic strategies involving gene targeting, artificial chromsomes, DNA delivery systems and non-embryonic stem cells, and on the ethical and social implications of these advances.

Forecasting↗
PubMed2002

Isolation of CF cell lines corrected at DeltaF508-CFTR locus by SFHR-mediated targeting.

Cystic fibrosis is the most common inherited disease in the Caucasian population. About 70% of all CF chromosomes carry the DeltaF508 mutation, a 3-bp deletion that results in the loss of a phenylalanine at amino acid 508 in the CF transmembrane conductance regulator (CFTR) protein. Direct modification of the DeltaF508 locus of endogenous CFTR was achieved by small fragment homologous replacement (SFHR). Transformed human airway epithelial cells (CFBE41o(-)), homozygous for DeltaF508 mutation, were transfected with small fragments (491-bp) of wild-type (WT) CFTR DNA comprising exon 10 and the flanking introns. The DNA fragments were in a liposome-DNA complex at a charge ratio of 6:1 (+:-), respectively). The population of transfected cells was subcloned by limiting dilution at approximately 1 cell/well in 96-well plates. Individual colonies were isolated and analyzed. The DNA from several colonies was characterized by radiolabeled, nonallele-specific and radiolabeled, allele-specific PCR amplification, as well as by genomic DNA fingerprinting. The CFTR-WT allele was detected in five of these colonies by allele-specific PCR amplification thus indicating that the cell lines carried both WT and DeltaF alleles. DNA fingerprint analysis confirmed that the colonies were isogenic and derived from the parental CFBE41o(-) cell line. Although, the WT allele was detected by allele-specific PCR, it was not detected initially when the same samples were analyzed by non allele-specific PCR. A sensitivity assay, mixing the genomic DNA of wild-type (16HBE14o(-)) and mutant (CFBE41o(-)) cell lines, indicated that the allele-specific PCR was at least 25-fold more sensitive than non allele-specific PCR. These results suggest that the colony is not yet clonal, but still contains a population of parental, CFBE41o(-) cells that have not been modified. Based on the mixing analysis, the proportion of corrected cells appears to be between 1 and 10% of the total population. Nonallele-specific reverse transcriptase PCR (RT-PCR) analysis of the CFTR mRNA indicated that two of the colonies expressed both WT and DeltaF508 CFTR mRNA, while one colony appeared to express only the WT mRNA. The mRNA results were confirmed by sequence analysis of 3' end primer extension products from the mRNA of CFTR exon 10 showing that the mRNA containing exon 10. Furthermore, a survey of primer extension products indicated no random insertion of the fragment in an expressed gene. This study demonstrates SFHR-mediated modification of the DeltaF508 allele in DeltaF508 homozygote human airway epithelial cells over multiple generations. The resultant cells express WT-CFTR mRNA and can be subcloned further to isolate isogenic clonal populations of cells.

Cell Line↗
PubMed2002

Application of SFHR to gene therapy of monogenic disorders.

Gene therapy treatment of disease will be greatly facilitated by the identification of genetic mutations through the Human Genome Project. The specific treatment will ultimately depend on the type of mutation as different genetic lesions will require different gene therapies. For example, large rearrangements and translocations may call for complementation with vectors containing the cDNA for the wild-type (wt) gene. On the other hand, smaller lesions, such as the reversion, addition or deletion of only a few base pairs, on single genes, or monogenic disorders, lend themselves to gene targeting. The potential for one gene targeting technique, small fragment homologous replacement (SFHR) to the gene therapy treatment of sickle cell disease (SCD) is presented. Successful conversion of the wt-beta-globin locus to a SCD genotype of human lymphocytes (K562) and progenitor/stem hematopoietic cells (CD34(+) and lin-CD38-) was achieved by electroporation or microinjection small DNA fragments (SDF).

Antigens, CD34↗
PubMed2001

Expression sequence tag-specific full-length cDNA cloning: actin cDNAs.

Current strategies for cDNA cloning are based on construction of cDNA libraries and colony screening. The process of obtaining a full-length cDNA clone can be highly time and labor intensive. Using the human actin gene as a model target cDNA, we have developed an RNA-capture method for rapid cloning of full-length cDNAs. The approach involves the capture of mRNA with expressed sequence tag (EST)-derived, biotin labeled antisense "capture" primers and streptavidin-coated magnetic beads. Full-length cDNA is then synthesized from purified EST-specific mRNA and cloned directly into plasmid vectors. The results of using beta-actin-based capture primers on cytoplasmic RNA were the isolation of both beta- and gamma-actin cDNA clones. Of the 16 actin-specific cDNA clones analyzed, 15 (93%) were full-length. This approach for cloning full-length cDNAs from available ESTs or partial cDNA sequences will facilitate a more rapid and efficient characterization of gene structure and function.

Actins↗
PubMed2001

Targeted correction of a defective selectable marker gene in human epithelial cells by small DNA fragments.

A novel gene targeting strategy, small fragment homologous replacement (SFHR), has been used to correct specific genomic lesions in human epithelial cells. The frequency of targeting was estimated to be 1-10%. However, given the genomic target, the cystic fibrosis transmembrane conductance regulator (CFTR) gene, it is difficult to accurately quantify targeting frequency. As an alternative to targeting CFTR, targeted correction of a mutant selectable marker or reporter gene would be more amenable to accurate and rapid quantification of gene targeting efficiency. The present study evaluates the conditions that modulate SFHR-mediated correction of a defective Zeocin antibiotic resistance (Zeo(r)) gene that has been inactivated by a 4-bp insertion. The conditions include delivery systems, plasmid-to-fragment ratio, fragment length, and fragment strandedness (single- or double-stranded DNA). Targeting fragments comprise the wild-type Zeo(r) gene sequence and were either 410 (Zeo1) or 458 bp (Zeo3). Expression vectors containing the corrected Zeo(r) gene were isolated as episomal plasmids or were allowed to stably integrate into cultured human airway epithelial cells. Correction of the Zeo(r) gene was phenotypically defined as restoration of resistance to Zeocin in either bacteria or epithelial cell clones. Extrachromosomal gene correction was assayed using polymerase chain reaction amplification, restriction enzyme digestion, DNA sequencing, and Southern blot hybridization analysis of DNA from isolated prokaryotic and eukaryotic clones. Neither random sequence alteration in the target episomal gene nor random integration of the small fragments was detected. Targeted correction efficiencies of up to 4% were attained. These studies provide insight into parameters that can be modulated for the optimization of SFHR-mediated targeting.

Base Sequence↗
PubMed2001

Expression of DeltaF508 CFTR in normal mouse lung after site-specific modification of CFTR sequences by SFHR.

The development of gene targeting strategies for specific modification of genomic DNA in human somatic cells has provided a potential gene therapy for the treatment of inherited diseases. One approach, small fragment homologous replacement (SFHR), directly targets and modifies specific genomic sequences with small fragments of exogenous DNA (400-800 bp) that are homologous to genomic sequences except for the desired modification. This approach has been effective for the in vitro modification of exon 10 in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in human airway epithelial cells. As another step in the development of SFHR for gene therapy, studies were carried out to target and modify specific genomic sequences in exon 10 of the mouse CFTR (mCFTR) in vivo. Small DNA fragments (783 bp), homologous to mCFTR except for a 3-bp deletion (DeltaF508) and a silent mutation which introduces a unique restriction site (KpnI), were instilled into the lungs of normal mice using four different DNA vehicles (AVE, LipofectAMINE, DDAB, SuperFect). Successful modification was determined by PCR amplification of DNA or mRNA-derived cDNA followed by KpnI digestion. The results of these studies showed that SFHR can be used as a gene therapy to introduce specific modifications into the cells of clinically affected organs and that the cells will express the new sequence.

Animals↗
PubMed2001

The transmembrane protein occludin of epithelial tight junctions is a functional target for serine peptidases from faecal pellets of Dermatophagoides pteronyssinus.

There have been only a few studies of how allergens cross the airway epithelium to cause allergic sensitization. House dust mite fecal pellets (HDMFP) contain several proteolytic enzymes. Group 1 allergens are cysteine peptidases, whilst those of groups 3, 6 and 9 have catalytic sites indicative of enzymes that mechanistically behave as serine peptidases. We have previously shown that the group 1 allergen Der p 1 leads to cleavage of tight junctions (TJs), allowing allergen delivery to antigen presenting cells. In this study we determined whether HDMFP serine peptidases similarly compromise the airway epithelium by attacking TJs, desmosomes and adherens junctions. Experiments were performed in monolayers of MDCK, Calu-3 or 16HBE14o-epithelial cells. Cell junction morphology was examined by 2-photon molecular excitation microscopy and digital image analysis. Barrier function was measured as mannitol permeability. Cleavage of cell adhesion proteins was studied by immunoblotting and mass spectrometry. HDMFP serine peptidases led to a progressive cleavage of TJs and increased epithelial permeability. Desmosomal puncta became more concentrated. Cleavage of TJs involved proteolysis of the TJ proteins, occludin and ZO-1. This was associated with activation of intracellular proteolysis of ZO-1. In contrast to occludin, E-cadherin of adherens junctions was cleaved less extensively. Although Calu-3 and 16HBE14o-cells expressed tethered ligand receptors for serine peptidases, these were not responsible for transducing the changes in TJs. HDMFP serine peptidases cause cleavage of TJs. This study identifies a second general class of HDM peptidase capable of increasing epithelial permeability and thereby creating conditions that would favour transepithelial delivery of allergens.

Amino Acid Sequence↗
PubMed2000

Neutrophils enhance expression of inducible nitric oxide synthase in human normal but not cystic fibrosis bronchial epithelial cells.

The bronchial epithelium in cystic fibrosis (CF) expresses very low levels of the inducible form of nitric oxide synthase (iNOS). The product of iNOS, nitric oxide (NO), mediates anti-microbial effects and can reduce neutrophil sequestration in the lung. Heavy neutrophilic infiltration of the pulmonary epithelium is a major feature of the end-stage CF lung. This study hypothesized that the system whereby the pulmonary epithelium protects itself against exaggerated neutrophilic infiltration by producing NO is compromised in CF. Human neutrophils were activated by incubation with cytokines, added to monolayers of normal (16HBE14o-) and CF (CFBE41o-) bronchial epithelial cells and co-cultured for up to 72 h. Marked up-regulation of iNOS protein expression was seen in normal bronchial epithelial cells following neutrophil co-culture but the CF cells showed a significantly smaller increase (p<0.001). To determine whether the relative lack of protein was due to a defect in translation, RT-PCR of iNOS mRNA was carried out and a pattern of mRNA expression was seen paralleling that of the protein. The reduced production of NO by CF compared with normal epithelium was shown by the presence of significantly (p<0.001) less accumulated nitrites in medium after co-culture with neutrophils. In summary, this study shows that the normal production of NO by bronchial epithelium in response to contact with neutrophils is lacking in CF. As NO has been shown to oppose neutrophil sequestration, its relative lack in CF may underlie the heavy neutrophilic infiltration that characterizes the disease.

Bronchi↗
PubMed2000

Tight junction properties of the immortalized human bronchial epithelial cell lines Calu-3 and 16HBE14o-.

Tight junctions (TJs) make a vital contribution to the barrier properties of the airway lining. Opening of TJs, or their frank cleavage, is suspected as a pathophysiological event in the lung, but research into the cellular and molecular mechanisms involved has been impeded by technical limitations of available experimental models. The authors have compared the properties of two epithelial cell lines derived from bronchial epithelium to explore whether these cell lines could constitute appropriate tools for the study of TJ regulation in bronchial epithelium. Investigations of TJs in 16HBE14o- cells and Calu-3 cells were made by fluorescent antibody labelling in conjunction with wide-field, confocal or 2-photon molecular excitation microscopy (2PMEM). The presence of TJ proteins was confirmed by immunoblotting and functional properties of the monolayers were studied by measurements of transepithelial electrical resistance and mannitol permeability. Cells of both lines formed confluent monolayers in which the cells expressed the TJ proteins occludin and ZO-1 in continuous circumferential patterns suggestive of functional TJs. This interpretation was supported by the development of transepithelial electrical resistances and of low paracellular permeability to solutes. Within the limits of resolution offered by 2PMEM, occludin and ZO-1 appeared to colocalize at TJs. These studies suggest that the 16HBE14o- cells and Calu-3 cell lines are potentially useful in vitro models to study how tight junction opening or cleavage changes the functional barrier properties of bronchial epithelium.

Animals↗
PubMed2000

Quantitative structural and biochemical analyses of tight junction dynamics following exposure of epithelial cells to house dust mite allergen Der p 1.

BACKGROUND: House dust mite allergen Der p 1 is a cysteine peptidase. Previously, we have suggested that the proteolytic activity of this allergen may contribute to asthma by damaging the barrier formed by the airways epithelium. OBJECTIVE: The present study applied novel techniques to compare changes in permeability with quantitative events in tight junctions (TJs) and desmosomes (DMs) of epithelial cells exposed to Der p 1. METHODS: Confluent monolayers of Madin-Darby canine kidney (MDCK) and 16HBE14o-human bronchial epithelial cells were used as experimental models. Permeability was estimated from mannitol clearance. Digital imaging with quantification of TJs and DMs was achieved by fluorescent antibody staining and 2-photon molecular excitation microscopy (2PMEM). Biochemical changes in TJs were studied by immunoblotting, radiolabelling and immunoprecipitation. RESULTS: Der p 1 caused a time-dependent breakage of TJs and reduction in their content of the protein ZO-1. Reduction in ZO-1 immunofluorescence at TJs occurred with a small increase in the amount of diffuse, cytoplasmic immunoreactive ZO-1 staining. Morpho-logical changes in TJs occurred in synchrony with increases in epithelial permeability. DM puncta increased both in size and intensity of staining. Immunoblotting demonstrated that the disruption of TJ morphology was associated with cleavage of ZO-1 and occludin. Cells recovered from allergen exposure by de novo synthesis of occludin. CONCLUSION: Der p 1 could contribute to sensitization and allergic responses by degrading the function of the airway epithelial barrier.

Animals↗
PubMed2000

Transfer and expression of foreign genes in mammalian cells.

The transfer of foreign genes into eukaryotic cells, in particular mammalian cells, has been essential to our understanding of the functional significance of genes and regulatory sequences as well as the development of gene therapy strategies. To this end, different mammalian expression vector systems have been designed. The choice of a particular expression system depends on the nature and purpose of the study and will involve selecting particular parameters of expression systems such as the type of promoter/enhancer sequences, the type of expression (transient versus stable) and the level of desired expression. In addition, the success of the study depends on efficient gene transfer. The purification of the expression vectors, as well as the transfer method, affects transfection efficiency. Numerous approaches have been developed to facilitate the transfer of genes into cells via physical, chemical or viral strategies. While these systems have all been effective in vitro they need to be optimized for individual cell types and, in particular, for in vivo transfection.

Animals↗
PubMed1999

Neutrophil recruitment by human IL-17 via C-X-C chemokine release in the airways.

IL-17 is a recently discovered cytokine that can be released from activated human CD4+ T lymphocytes. This study assessed the proinflammatory effects of human (h) IL-17 in the airways. In vitro, hIL-17 increased the release of IL-8 in human bronchial epithelial and venous endothelial cells, in a time- and concentration-dependent fashion. This effect of hIL-17 was inhibited by cotreatment with an anti-hIL-17 Ab and was potentiated by hTNF-alpha. In addition, hIL-17 increased the expression of hIL-8 mRNA in bronchial epithelial cells. Conditioned medium from hIL-17-treated bronchial epithelial cells increased human neutrophil migration in vitro. This effect was blocked by an anti-hIL-8 Ab. In vivo, intratracheal instillation of hIL-17 selectively recruited neutrophils into rat airways. This recruitment of neutrophils into the airways was inhibited by an anti-hIL-17 Ab and accompanied by increased levels of rat macrophage inflammatory protein-2 (rMIP-2) in bronchoalveolar lavage (BAL) fluid. The BAL neutrophilia was also blocked by an anti-rMIP-2 Ab. The effect of hIL-17 on the release of hIL-8 and rMIP-2 was also inhibited by glucocorticoids, in vitro and in vivo, respectively. These data demonstrate that hIL-17 can specifically and selectively recruit neutrophils into the airways via the release of C-X-C chemokines from bronchial epithelial cells and suggest a novel mechanism linking the activation of T-lymphocytes to recruitment of neutrophils into the airways.

Adult↗
PubMed1999

Intracellular compartmentalization of DNA fragments in cultured airway epithelial cells mediated by cationic lipids.

PURPOSE: The amount and intracellular distribution of DNA fragments (491-bp) was characterized after transfection in vitro with a commercially available cationic lipid. Localization of fragment to the nucleus, its subcellular distribution, and integrity within the cells was determined for various times after transfection. METHODS: Cystic fibrosis (CF) airway epithelial cells were transfected with 32P and FITC labeled single-stranded (ss) or double-stranded (ds) DNA fragments complexed with Lipofectamine at various charge ratios. RESULTS: A 511 (+/-) charge ratio was found to be the optimal ratio for transfection of both ss-and dsDNA. After a 5 h exposure, 7.51 +/- 0.89% of the radioactivity was associated with the nuclear fraction whereas only 1.07 +/- 0.23%, was found in the nuclear fraction when dsDNA was used. The nuclear radioactivity detected after a 24 h exposure was only 1/3 of that after 5 h. Analysis of fragment stability in the cytosolic and nuclear fractions showed the presence of intact fragment in each subcellular compartment. No intranuclear/intracellular fragment could be detected in control experiments with naked DNA. Conclusions. The results from these experiments indicate that small fragments of DNA can be efficiently and rapidly transferred intact to the cell nucleus using cationic lipids and that ssDNA fragments are more effective than dsDNA fragments for nuclear delivery.

Bronchi↗
PubMed1999

Differential sensitivity of normal and cystic fibrosis airway epithelial cells to epinephrine.

1. Exposure to epinephrine has been shown to have a range of effects on cells and tissues. A recent study suggested that the proliferative ability of CF epithelial cells, exposed to high concentrations of epinephrine (200 - 300 microM), was reduced when compared to that of normal cells. This approach could potentially provide a means to effectively separate cells with functional cyclic AMP-dependent Cl-ion transport from those defective in this pathway. 2. The sensitivity to killing by epinephrine is reported here for four different CF cell lines, three normal cell lines, and two CF epithelial cell lines complemented with wild-type (wt) CF transmembrane conductance regulator (CFTR) cDNA. 3. While each cell line exhibited varying sensitivity to 200 microM epinephrine, no predictable pattern was observed between the expression of wt-CFTR and cell survival following epinephrine exposure. Overall, normal cell lines did exhibit a greater resistance to epinephrine-induced cell death although, the most resistant cell line was derived from CF tracheal epithelium (SigmaCFTE29o-). 4. The expression of exogenous wt-CFTR increased the survival of one cell line (CFDEo-) when compared to the parent line, but in another complemented line, survival was reduced. 5. These findings suggest that while epinephrine induces cell killing, it is not consistently effective for preferential selection of normal over CF cells. Although CFTR may play a role in the mechanism(s) of epinephrine killing, other factors such as cell density, proliferative ability, cell type origin and phenotype are involved.

Cell Count↗
PubMed1999

Development and characterization of an SV40 immortalized porcine ameloblast-like cell line.

Enamel is secreted as a protein matrix by the ameloblasts. These same cells then control the maturation of the enamel matrix, secreting proteinases that hydrolyze proteins as mineralization progresses, until mature enamel containing less than 1% protein by weight remains. Further understanding of the factors that control ameloblast function and differentiation requires an in vitro cell culture system. In this study, we report immortalization of enamel organ epithelial cells and the selection of a cell line with characteristics of ameloblasts. Porcine enamel organ cells were dissected from unerupted porcine molars, cultured in serum-free medium, and passaged twice. These cells were transfected with an origin-of-replication defective SV40 plasmid by calcium phosphate precipitation, and a cell line with mRNA expression characteristic of ameloblasts was cloned. This cell line (PABSo-E) expressed mRNA for amelogenin, matrix metalloproteinase-20 (enamelysin), and enamel matrix serine proteinase 1 (EMSP1), but not ameloblastin. PABSo-E cells have been passaged more than 55 times, while continuing to maintain characteristics of ameloblasts. These cells will be useful for future studies of ameloblast function.

Ameloblasts↗