Janet F. Forstner

Modulation of disease severity in cystic fibrosis transmembrane conductance regulator deficient mice by a secondary genetic factor

Mice that have been made deficient for the cystic fibrosis transmembrane conductance regulator (Cftr) usually die of intestinal obstruction. We have created Cftr-deficient mice and demonstrate prolonged survival among backcross and intercross progeny with different inbred strains, suggesting that modulation of disease severity is genetically determined. A genome scan showed that the major modifier locus maps near the centromere of mouse chromosome 7. Electrophysiological studies on mice with prolonged survival show that the partial rectification of Cl− and Na+ ion transport abnormalities can be explained in part by up-regulation of a calcium-activated Cl− conductance. Identification of modifier genes in our Cftr m1HSC/Cftr m1HSC mice should provide important insight into the heterogeneous disease presentation observed among CF patients.

P-113d, an Antimicrobial Peptide Active against Pseudomonas aeruginosa, Retains Activity in the Presence of Sputum from Cystic Fibrosis Patients

ABSTRACT Antimicrobial peptides are a source of novel agents that could be useful for treatment of the chronic lung infections that afflict cystic fibrosis (CF) patients. Efficacy depends on antimicrobial activity against the major pathogens of CF patients, Pseudomonas aeruginosa, Staphylococcus aureus, and Haemophilus influenzae, in the environment of the CF patients airway. We describe the in vitro efficacies of derivatives of histatins, which are histidine-rich peptides produced by the salivary glands of humans and higher primates. P-113, a peptide containing 12 of the 24 amino acid residues of the parent molecule, histatin 5, retained full antibacterial activity and had a good spectrum of activity in vitro against the prominent pathogens of CF patients. However, P-113 was not active in the presence of purulent sputum from CF patients. In contrast, P-113d, the mirror-image peptide with the amino acid residues in the d configuration, was stable in sputum, was as active as P-113 against pathogens of CF patients in the absence of sputum and retained significant activity in the presence of sputum from CF patients. Recombinant human DNase, which effectively liquefies sputum, enhanced the activity of P-113d in undiluted sputum against both exogenous (added) bacteria and endogenous bacteria. Because of its properties, P-113d shows potential as an inhalant in chronic suppressive therapy for CF patients.

Phenotypic abnormalities in long-term surviving cystic fibrosis mice.

Mouse models for cystic fibrosis (CF) with no CFTR function(Cftr-/-) have the disadvantage that most animals die of intestinal obstruction shortly after weaning. The objective of this research was to extend the lifespan of CF mice and characterize their phenotype. Weanlings were placed on a nutrient liquid diet, and histologic and functional aspects of organs implicated in the disease were subsequently examined. Approximately 90% of Cftr-/- mice survived to 60 d, the majority beyond 100 d. Cftr-/- mice were underweight and had markedly abnormal intestinal histology. The intestinal epithelia did not respond to challenges with agents that raised intracellular cAMP, consistent with the absence of functional CFTR. No lesions or functional abnormalities were evident in the lungs. Liquid-fed Cftr-/- mice were infertile, although some males weaned to a solid diet were fertile before they died. Thus, we have succeeded in using dietary means to prolong the lives ofCftr-/- mice.

Protection of Cftr knockout mice from acute lung infection by a helper-dependent adenoviral vector expressing Cftr in airway epithelia

We developed a helper-dependent adenoviral vector for cystic fibrosis lung gene therapy. The vector expresses cystic fibrosis transmembrane conductance regulator (Cftr) using control elements from cytokeratin 18. The vector expressed properly localized CFTR in cultured cells and in the airway epithelia of mice. Cftr RNA and protein were present in whole lung and bronchioles, respectively, for 28 days after a vector dose. Acute inflammation was minimal to moderate. To test the therapeutic potential of the vector, we challenged mice with a clinical strain of Burkholderia cepacia complex (Bcc). Cftr knockout mice (but not Cftr+/+ littermates) challenged with Bcc developed severe lung histopathology and had high lung bacteria counts. Cftr knockout mice receiving gene therapy 7 days before Bcc challenge had less severe histopathology, and the number of lung bacteria was reduced to the level seen in Cftr+/+ littermates. These data suggest that gene therapy could benefit cystic fibrosis patients by reducing susceptibility to opportunistic pathogens.

Human intestinal mucin in cystic fibrosis.

Summary: Human intestinal mucins from six subjects with Cystic Fibrosis (CF) and eight subjects without CF were prepared from tissue obtained at surgery (one case) and postmortem. Subjects were not age-matched, but the nonCF mucin was obtained from subjects with ages which bracketed those of the CF subjects. Cesium chloride analytical gradient ultracentrifugation showed that CF mucins were generally denser than nonCF mucins. Sedimentation coefficients were also higher in the CF samples. CF mucins were enriched in fucose, galactose, N-acetylglucosamine and total carbohydrate per mg protein and per oligosaccharide chain (mole/ mole GalNAc). Fucose/sialic acid molar ratios were significantly higher in CF mucins, and the average oligosaccharide chain length was approximately three residues greater in CF as compared with nonCF mucins. There was no difference in amino acid profiles or the number of side chains per molecule. The mean sulfate content was higher in the CF mucins but not to a level of significance; however, in the eight mucins, sulfate content correlated positively with total carbohydrate, N-acetylglucosamine and galactose, and therefore increased with oligosaccharide chain length. CF intestinal mucin was therefore denser and more highly glycosylated than nonCF musin and probably contained more sulfate. The increase in glycosylation resulted from a rise in fucose, galactose, and N-acetylglucosamine without a concomitant rise in sialic acid.Speculation: Hyperglycosylation with an associated increase in average chain length of carbohydrate side chains ought to increase the equivalent hydrodynamic volume of the mucin molecule and so result in enhanced viscosity and gelling. These features may contribute to the hyperviscosity often noted in mucus-rich secretions in CF and could enhance mucus plugging of small ducts. Increased mucin carbohydrate could represent one of the fundamental abnormalities contributing to the pathology of this disease.

Enhanced susceptibility to pulmonary infection with Burkholderia cepacia in Cftr(-/-) mice.

ABSTRACT Progressive pulmonary infection is the dominant clinical feature of cystic fibrosis (CF), but the molecular basis for this susceptibility remains incompletely understood. To study this problem, we developed a model of chronic pneumonia by repeated instillation of a clinical isolate of Burkholderia cepacia (genomovar III, ET12 strain), an opportunistic gram-negative bacterium, from a case of CF into the lungs of Cftr m1unc−/−(Cftr−/−) and congenicCftr+/+ controls. Nine days after the last instillation, the CF transmembrane regulator knockout mice showed persistence of viable bacteria with chronic severe bronchopneumonia while wild-type mice remained healthy. The histopathological changes in the lungs of the susceptibleCftr−/− mice were characterized by infiltration of a mixed inflammatory-cell population into the peribronchiolar and perivascular spaces, Clara cell hyperplasia, mucus hypersecretion in airways, and exudation into alveolar airspaces by a mixed population of macrophages and neutrophils. An increased proportion of neutrophils was observed in bronchoalveolar lavage fluid from the Cftr−/− mice, which, despite an increased bacterial load, demonstrated minimal evidence of activation. Alveolar macrophages from Cftr−/− mice also demonstrated suboptimal activation. These observations suggest that the pulmonary host defenses are compromised in lungs from animals with CF, as manifested by increased susceptibility to bacterial infection and lung injury. This murine model of chronic pneumonia thus reflects, in part, the situation in human patients and may help elucidate the mechanisms leading to defective host defense in CF.

Cable-Piliated Burkholderia cepacia Binds to Cytokeratin 13 of Epithelial Cells

ABSTRACT 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 inhibitedB. 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 andB. 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.

Responses of Well-Differentiated Airway Epithelial Cell Cultures from Healthy Donors and Patients with Cystic Fibrosis to Burkholderia cenocepacia Infection

ABSTRACT Well-differentiated cultures established from airway epithelia of patients with cystic fibrosis (CF cultures) exhibited goblet cell hyperplasia, increased secretion of mucus, and higher basal levels of interleukin-8 than similarly cultured cells from healthy donors. Upon apical infection with low doses (104 to 105 CFU) of Burkholderia cenocepacia isolate BC7, the two cultures gave different responses. While normal cultures trapped the added bacteria in the mucus layer, killed and/or inhibited bacterial replication, and prevented bacterial invasion of the cells, CF cultures failed to kill and/or supported the growth of bacteria, leading to invasion of underlying epithelial cells, compromised transepithelial permeability, and cell damage. Depletion of the surface mucus layer prior to bacterial infection rendered the normal cultures susceptible to bacterial invasion, but the invading bacteria were mainly confined to vacuoles within the cells and appeared to be nonviable. In contrast, bacteria that invaded cells in CF cultures were found free in the cytoplasm surrounded by intermediate filaments and also between cells. Cultured CF airway epithelium was therefore more susceptible to infection than normal epithelium. This mimics CF tissue in vivo and illustrates differences in the way epithelia in CF patients and normal subjects handle bacterial infection. In addition, we found that the CF and normal cell cultures responded differently not only to isolate BC7 but also to isolates of other B. cepacia complex species. We therefore conclude that this cell culture model is suitable for investigation of B. cepacia complex pathogenesis in CF patients.

Induction of mucin gene expression in human colonic cell lines by PMA is dependent on PKC-ε

Treatment of HT-29 cells with phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), induces MUC2 expression. To investigate the role of PKC in regulating mucin genes in intestinal cells, we examined the regulation of MUC1, MUC2, MUC5AC, MUC5B, and MUC6 expression in two human mucin-producing colonic cell lines, T84 and HT29/A1. T84 and HT29/A1 cells (at 80-90% confluency) were exposed to 100 nM PMA for 0, 3, and 6 h. Twofold or greater increases in mRNA levels for MUC2 and MUC5AC were observed in both cell lines during this time period, whereas the levels of MUC1, MUC5B, and MUC6 mRNAs were only marginally affected. These results indicated that PKC differentially regulates mucin gene expression and that it may be responsible for altered mucin expression. Our previous results suggested that the Ca2+-independent PKC-ε isoform appeared to mediate PMA-regulated mucin exocytosis in these cell lines. To determine if PKC-ε was also involved in MUC2/MUC5AC gene induction, HT29/A1 cells were stably transfected with either a wild-type PKC-ε or a dominant-negative ATP-binding mutant of PKC-ε (PKC-ε K437R). Overexpression of the dominant-negative PKC-ε K437R blocked induction of both mucin genes, whereas PMA-induced mucin gene expression was not prevented by overexpression of wild-type PKC-ε. PMA-dependent MUC2 mucin secretion was also blocked in cells overexpressing the dominant-negative PKC-ε K437R. On the basis of these observations, PKC-ε appears to mediate the expression of two major gastrointestinal mucins in response to PMA as well as PMA-regulated mucin exocytosis.Treatment of HT-29 cells with phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), induces MUC2 expression. To investigate the role of PKC in regulating mucin genes in intestinal cells, we examined the regulation of MUC1, MUC2, MUC5AC, MUC5B, and MUC6 expression in two human mucin-producing colonic cell lines, T84 and HT29/A1. T84 and HT29/A1 cells (at 80-90% confluency) were exposed to 100 nM PMA for 0, 3, and 6 h. Twofold or greater increases in mRNA levels for MUC2 and MUC5AC were observed in both cell lines during this time period, whereas the levels of MUC1, MUC5B, and MUC6 mRNAs were only marginally affected. These results indicated that PKC differentially regulates mucin gene expression and that it may be responsible for altered mucin expression. Our previous results suggested that the Ca(2+)-independent PKC-epsilon isoform appeared to mediate PMA-regulated mucin exocytosis in these cell lines. To determine if PKC-epsilon was also involved in MUC2/MUC5AC gene induction, HT29/A1 cells were stably transfected with either a wild-type PKC-epsilon or a dominant-negative ATP-binding mutant of PKC-epsilon (PKC-epsilon K437R). Overexpression of the dominant-negative PKC-epsilon K437R blocked induction of both mucin genes, whereas PMA-induced mucin gene expression was not prevented by overexpression of wild-type PKC-epsilon. PMA-dependent MUC2 mucin secretion was also blocked in cells overexpressing the dominant-negative PKC-epsilon K437R. On the basis of these observations, PKC-epsilon appears to mediate the expression of two major gastrointestinal mucins in response to PMA as well as PMA-regulated mucin exocytosis.

Inhibition of the binding of enterotoxigenic Escherichia coli Pb176 to human intestinal epithelial cell line HCT-8 by an extracellular protein fraction containing BIF of Bifidobacterium longum SBT2928: suggestive evidence of blocking of the binding receptor gangliotetraosylceramide on the cell surface.

The extracellular protein fraction (P100V) containing the protein BIF produced by Bifidobacterium longum SBT2928 (BL2928), which inhibits the binding of enterotoxigenic Escherichia coli Pb176 (ETEC) to the glycolipid binding receptor gangliotetraosylceramide (GA1) also inhibited the binding of ETEC to the human intestinal epithelial cell line HCT-8 (ATCC CCL 244) in a dose-dependent manner. ETEC-binding inhibitory experiments using crude colonization factor antigen (CFA)-II prepared from ETEC, rabbit anti-GA1 antiserum, medium containing GA1 and media containing lectins, as the binding-inhibitors, suggest that the interaction between the CFA-II antigen present on the cell surface of ETEC and GA1 expressed on HCT-8 cells plays a significant role in the adherence between them. It is strongly suggested that the P100V fraction works as a blocker for the ETEC receptor GA1 on HCT-8 cells.