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1.  Bioelectric Characterization of Epithelia from Neonatal CFTR Knockout Ferrets 
Cystic fibrosis (CF) is a life-shortening, recessive, multiorgan genetic disorder caused by the loss of CF transmembrane conductance regulator (CFTR) chloride channel function found in many types of epithelia. Animal models that recapitulate the human disease phenotype are critical to understanding pathophysiology in CF and developing therapies. CFTR knockout ferrets manifest many of the phenotypes observed in the human disease, including lung infections, pancreatic disease and diabetes, liver disease, malnutrition, and meconium ileus. In the present study, we have characterized abnormalities in the bioelectric properties of the trachea, stomach, intestine, and gallbladder of newborn CF ferrets. Short-circuit current (ISC) analysis of CF and wild-type (WT) tracheas revealed the following similarities and differences: (1) amiloride-sensitive sodium currents were similar between genotypes; (2) responses to 4,4′-diisothiocyano-2,2′-stilbene disulphonic acid were 3.3-fold greater in CF animals, suggesting elevated baseline chloride transport through non-CFTR channels in a subset of CF animals; and (3) a lack of 3-isobutyl-1-methylxanthine (IBMX)/forskolin–stimulated and N-(2-Naphthalenyl)-((3,5-dibromo-2,4-dihydroxyphenyl)methylene)glycine hydrazide (GlyH-101)–inhibited currents in CF animals due to the lack of CFTR. CFTR mRNA was present throughout all levels of the WT ferret and IBMX/forskolin–inducible ISC was only observed in WT animals. However, despite the lack of CFTR function in the knockout ferret, the luminal pH of the CF ferret gallbladder, stomach, and intestines was not significantly changed relative to WT. The WT stomach and gallbladder exhibited significantly enhanced IBMX/forskolin ISC responses and inhibition by GlyH-101 relative to CF samples. These findings demonstrate that multiple organs affected by disease in the CF ferret have bioelectric abnormalities consistent with the lack of cAMP-mediated chloride transport.
doi:10.1165/rcmb.2012-0433OC
PMCID: PMC3931095  PMID: 23782101
cystic fibrosis; animal model; ferret; intestine; trachea
2.  Bioelectric Properties of Chloride Channels in Human, Pig, Ferret, and Mouse Airway Epithelia 
The development of effective therapies for cystic fibrosis (CF) requires animal models that can appropriately reproduce the human disease phenotype. CF mouse models have demonstrated cAMP-inducible, non–CF transmembrane conductance regulator (non-CFTR) chloride transport in conducting airway epithelia, and this property is thought to be responsible for the lack of a spontaneous CF-like phenotype in the lung. Thus, an understanding of species diversity in airway epithelial electrolyte transport and CFTR function is critical to developing better models for CF. Two species currently being used in attempts to develop better animal models of CF include the pig and ferret. In the study reported here, we sought to comparatively characterize the bioelectric properties of in vitro polarized airway epithelia—from human, mouse, pig and ferret—grown at the air–liquid interface (ALI). Bioelectric properties analyzed include amiloride-sensitive Na+ transport, 4,4′-diisothiocyanato-stilbene-2,2′-disulfonic acid (DIDS)-sensitive Cl− transport, and cAMP-sensitive Cl− transport. In addition, as an index for CFTR functional conservation, we evaluated the ability of four CFTR inhibitors, including glibenclamide, 5-nitro-2-(3-phenylpropyl-amino)-benzoic acid, CFTR inh-172, and CFTRinh-GlyH101, to block cAMP-mediated Cl− transport. Compared with human epithelia, pig epithelia demonstrated enhanced amiloride-sensitive Na+ transport. In contrast, ferret epithelia exhibited significantly reduced DIDS-sensitive Cl− transport. Interestingly, although the four CFTR inhibitors effectively blocked cAMP-mediated Cl− secretion in human airway epithelia, each species tested demonstrated unique differences in its responsiveness to these inhibitors. These findings suggest the existence of substantial species-specific differences at the level of the biology of airway epithelial electrolyte transport, and potentially also in terms of CFTR structure/function.
doi:10.1165/rcmb.2006-0286OC
PMCID: PMC1894945  PMID: 17008635
cystic fibrosis; airway biology; species; ion transport; airway epithelium
3.  Species-Specific Differences in Mouse and Human Airway Epithelial Biology of Recombinant Adeno-Associated Virus Transduction 
Differences in airway epithelial biology between mice and humans have presented challenges to evaluating gene therapies for cystic fibrosis (CF) using murine models. In this context, recombinant adeno-associated virus (rAAV) type 2 and rAAV5 vectors have very different transduction efficiencies in human air-liquid interface (ALI) airway epithelia (rAAV2 ≅ rAAV5) as compared with mouse lung (rAAV5≫rAAV2). It is unclear if these differences are due to species-specific airway biology or limitations of ALI cultures to reproduce in vivo airway biology. To this end, we compared rAAV2 and rAAV5 transduction biology in mouse and human ALI cultures, and investigated the utility of murine ΔF508 cystic fibrosis transmembrane conductance regulator (CFTR) ALI epithelia to study CFTR complementation. Our results demonstrate that mouse ALI epithelia retain in vivo preferences for rAAV serotype transduction from the apical membrane (rAAV5≫rAAV2) not seen in human epithelia (rAAV2 ≅ rAAV5). Viral binding of rAAV2 and rAAV5 to the apical surface of mouse ALI airway epithelia was not significantly different, and proteasome-modulating agents significantly enhanced rAAV2 transduction to a level equivalent to that of rAAV5 in the presence of these agents, suggesting that the ubiquitin/proteasome pathway represents a more significant intracellular block for rAAV2 transduction of mouse airway epithelia. Interestingly, cAMP-inducible chloride currents were enhanced in ΔF 508C FTR mouse ALI cultures, making this model incompatible with CFTR complementation studies. These studies emphasize species-specific differences in airway biology between mice and humans that significantly influence the use of mice as surrogate models for rAAV transduction and gene therapy for CF.
doi:10.1165/rcmb.2005-0189OC
PMCID: PMC1752084  PMID: 16195538
recombinant adeno-associated virus; airway model; serotype; tropism
4.  Bioelectric Properties of Chloride Channels in Human, Pig, Ferret, and Mouse Airway Epithelia 
The development of effective therapies for cystic fibrosis (CF) requires animal models that can appropriately reproduce the human disease phenotype. CF mouse models have demonstrated cAMP-inducible, non–CF transmembrane conductance regulator (non-CFTR) chloride transport in conducting airway epithelia, and this property is thought to be responsible for the lack of a spontaneous CF-like phenotype in the lung. Thus, an understanding of species diversity in airway epithelial electrolyte transport and CFTR function is critical to developing better models for CF. Two species currently being used in attempts to develop better animal models of CF include the pig and ferret. In the study reported here, we sought to comparatively characterize the bioelectric properties of in vitro polarized airway epithelia—from human, mouse, pig and ferret—grown at the air–liquid interface (ALI). Bioelectric properties analyzed include amiloride-sensitive Na+ transport, 4,4′-diisothiocyanato-stilbene-2,2′-disulfonic acid (DIDS)-sensitive Cl− transport, and cAMP-sensitive Cl− transport. In addition, as an index for CFTR functional conservation, we evaluated the ability of four CFTR inhibitors, including glibenclamide, 5-nitro-2-(3-phenylpropyl-amino)-benzoic acid, CFTR inh-172, and CFTRinh-GlyH101, to block cAMP-mediated Cl− transport. Compared with human epithelia, pig epithelia demonstrated enhanced amiloride-sensitive Na+ transport. In contrast, ferret epithelia exhibited significantly reduced DIDS-sensitive Cl− transport. Interestingly, although the four CFTR inhibitors effectively blocked cAMP-mediated Cl− secretion in human airway epithelia, each species tested demonstrated unique differences in its responsiveness to these inhibitors. These findings suggest the existence of substantial species-specific differences at the level of the biology of airway epithelial electrolyte transport, and potentially also in terms of CFTR structure/function.
doi:10.1165/rcmb.2006-0286OC
PMCID: PMC1894945  PMID: 17008635
cystic fibrosis; airway biology; species; ion transport; airway epithelium
5.  Species-Specific Differences in Mouse and Human Airway Epithelial Biology of Recombinant Adeno-Associated Virus Transduction 
Differences in airway epithelial biology between mice and humans have presented challenges to evaluating gene therapies for cystic fibrosis (CF) using murine models. In this context, recombinant adeno-associated virus (rAAV) type 2 and rAAV5 vectors have very different transduction efficiencies in human air–liquid interface (ALI) airway epithelia (rAAV2 ≅ rAAV5) as compared with mouse lung (rAAV5 >> rAAV2). It is unclear if these differences are due to species-specific airway biology or limitations of ALI cultures to reproduce in vivo airway biology. To this end, we compared rAAV2 and rAAV5 transduction biology in mouse and human ALI cultures, and investigated the utility of murine ΔF508 cystic fibrosis transmembrane conductance regulator (CFTR) ALI epithelia to study CFTR complementation. Our results demonstrate that mouse ALI epithelia retain in vivo preferences for rAAV serotype transduction from the apical membrane (rAAV5 >> rAAV2) not seen in human epithelia (rAAV2 ≅ rAAV5). Viral binding of rAAV2 and rAAV5 to the apical surface of mouse ALI airway epithelia was not significantly different, and proteasome-modulating agents significantly enhanced rAAV2 transduction to a level equivalent to that of rAAV5 in the presence of these agents, suggesting that the ubiquitin/proteasome pathway represents a more significant intracellular block for rAAV2 transduction of mouse airway epithelia. Interestingly, cAMP-inducible chloride currents were enhanced in ΔF508CFTR mouse ALI cultures, making this model incompatible with CFTR complementation studies. These studies emphasize species-specific differences in airway biology between mice and humans that significantly influence the use of mice as surrogate models for rAAV transduction and gene therapy for CF.
doi:10.1165/rcmb.2005-0189OC
PMCID: PMC1752084  PMID: 16195538
recombinant adeno-associated virus; airway model; serotype; tropism
6.  Lysozyme Secretion by Submucosal Glands Protects the Airway from Bacterial Infection 
Submucosal glands are abundant (∼ 1 gland/mm2) secretory structures in the tracheobronchial airways of the human lung. Because submucosal glands express antibacterial proteins, it has been proposed that they contribute to lung defense. However, this concept is challenged by the fact that mice do not have submucosal glands in their bronchial airways, yet are quite resistant to bacterial lung infection. The contribution of airway submucosal glands to host defense is also debated as a pathophysiologic component of cystic fibrosis lung disease. Here, we asked whether submucosal glands protect airways against bacterial infection. By comparing tracheal xenograft airways with and without glands, we found that the presence of glands enhanced bacterial killing in vivo and by airway secretions in vitro. Moreover, immunodepletion studies suggested that lysozyme is a major antibacterial component secreted by submucosal glands. These studies provide evidence that submucosal glands are a major source of antibacterials critical for maintaining sterile airways.
doi:10.1165/rcmb.2005-0059OC
PMCID: PMC2715323  PMID: 15746432
innate immunity; lung; lysozyme; glands; antibacterial proteins

Results 1-6 (6)