Cystic fibrosis transmembrane conductance regulator and caveolin-1 regulate epithelial cell internalization of Pseudomonas aeruginosa.

Patients with cystic fibrosis (CF) exhibit defective innate immunity and are susceptible to chronic lung infection with Pseudomonas aeruginosa. To investigate the molecular bases for the hypersusceptibility of CF patients to P. aeruginosa, we used the IB3-1 cell line with two defective CF transmembrane conductance regulator (CFTR) genes (DeltaF508/W1282X) to generate isogenic stable, clonal lung epithelial cells expressing wild-type (WT)-CFTR with an NH(2)-terminal green fluorescent protein (GFP) tag. GFP-CFTR exhibited posttranslational modification, subcellular localization, and anion transport function typical of WT-CFTR. P. aeruginosa internalization, a component of effective innate immunity, required functional CFTR and caveolin-1, as shown by: 1) direct correlation between GFP-CFTR expression levels and P. aeruginosa internalization; 2) enhanced P. aeruginosa internalization by aminoglycoside-induced read through of the CFTR W1282X allele in IB3-1 cells; 3) decreased P. aeruginosa internalization following siRNA knockdown of GFP-CFTR or caveolin-1; and 4) spatial association of P. aeruginosa with GFP-CFTR and caveolin-1 at the cell surface. P. aeruginosa internalization also required free lateral diffusion of GFP-CFTR, allowing for bacterial coclustering with GFP-CFTR and caveolin-1 at the plasma membrane. Thus efficient initiation of innate immunity to P. aeruginosa requires formation of an epithelial "internalization platform" involving both caveolin-1 and functional, laterally mobile CFTR.

Disruption of CFTR-dependent lipid rafts reduces bacterial levels and corneal disease in a murine model of Pseudomonas aeruginosa keratitis.

PURPOSE: Pseudomonas aeruginosa enters corneal epithelial cells in vitro via membrane microdomains or lipid rafts. Bacterial entry, mediated by the cystic fibrosis transmembrane conductance regulator (CFTR), promotes infection and disease. This study was conducted to determine whether P. aeruginosa and CFTR are colocalized to rafts in isogenic corneal cells expressing wild-type (WT) or mutant DeltaF508-CFTR and whether disruption of the rafts both in vitro and in vivo affects the bacterial levels and the course of the disease. METHODS: Transformed human corneal epithelial cells from a patient homozygous for DeltaF508-CFTR, and the same cells corrected with WT-CFTR, were exposed to six isolates of P. aeruginosa-three invasive and three cytotoxic strains-in the presence of beta-cyclodextrin (CD), which disrupts rafts. Association and cellular uptake of the invasive strains were measured, as was lactate dehydrogenase release induced by the cytotoxic strains. Scratch-injured mouse eyes were infected with the six P. aeruginosa strains, and the effect of prophylactic or therapeutic administration of CD on bacterial levels and disease was evaluated. RESULTS: P. aeruginosa and CFTR were colocalized with lipid rafts in cells with WT-CFTR, and CD treatment of these cells disrupted bacterial association, internalization, and cytotoxic effects. Cells expressing DeltaF508-CFTR were marginally affected by CD. Prophylactic and therapeutic topical application of CD ameliorated corneal disease and reduced the bacterial count in the eye. CONCLUSIONS: P. aeruginosa enters human corneal epithelial cells via lipid rafts containing CFTR, and disruption of raft-mediated uptake of this organism by CD protects against disease and reduces bacterial levels in the mouse model of keratitis.

Aortic Valve Repair of a Stenotic Unicuspid Aortic Valve in Young Patients.

BACKGROUND: The unicuspid aortic valve (UAV) is a well-described pediatric congenital abnormality, with incidence of 0.02% in the general population. Bicuspidization has been described as a potential surgical option to repair this defect. METHODS: Seventeen symptomatic young patients with a unicuspid valve combined with either valve insufficiency or valve stenosis underwent aortic valve (AV) bicuspidization procedure by using an equine pericardium. In addition to bicuspidization, 8 patients underwent aortic ring implantation and 5 patients underwent supracoronary replacement of the aorta. RESULTS: Our results show safety of the bicuspidization procedure. No deaths occurred during our average follow-up period of 26 months. Freedom from reoperation for any valve-related reason was 100% during this follow-up period. We observed a statistically significant increase in the AV area from 0.8 ± 0.1 cm2 to 2.8 ± 0.7 cm2 (p < 0.01), a statistically significant decrease in the mean systolic pressure gradient from 36 ± 13.3 mm Hg to 9 ± 4 mm Hg (p < 0.001), a statistically significant decrease in aortic insufficiency grade from 2.1 ± 1.0 to 0.6 ± 0.7 (p < 0.01) before and after bicuspidization, respectively, and a statistically significant decrease in the left ventricular end-diastolic diameter from 49.88 ± 5.11 mm to 40.46 ± 7.20 mm (p < 0.0005) and a statistically significant increase of the left ventricular ejection fraction from 56% ± 8.20% to 64% ± 7.83% at the time of follow-up. CONCLUSIONS: From our study, bicuspidization is an attractive surgical option to repair UAV, particularly in young patients who do not want to be subjected to long-term anticoagulation therapy or who refuse a more traditional surgical approach, such as Ross procedure, for reasons described previously.

Surgical management of a hypoplastic distal aortic arch and coarctation of aorta in a patient with Klippel-Feil syndrome, ascending aortic aneurysm and bicuspid aortic valve.

Klippel-Feil syndrome has been associated with cardiovascular malformations, but only 3 cases have been reported to be associated with aortic coarctation and surgical management is not defined. A 51-year old woman with Klippel-Feil syndrome associated with an aneurysm of the ascending aorta, hypoplastic aortic arch and aortic coarctation at the level of the left subclavian artery presented with shortness of breath 2 years after diagnosis. Imaging identified interim development of a 7.2-cm aneurysm at the level of the aortic coarctation. She underwent surgical repair with a Dacron interposition graft under hypothermic circulatory arrest. She continues to do well 18 months following repair.

Use of left ventricle assist device in a pregnant woman with acute aortic and coronary dissections.

We report the case of a 31-year-old female in her 32nd week of pregnancy, who experienced an acute type A aortic dissection resulting in severe aortic insufficiency, dissection of the left anterior descending coronary artery, and left ventricular infarct with ejection fraction of 20%. After a successful cesarean section, she underwent a Bentall operation and three-vessel coronary artery bypass grafting. Despite maximal inotropic support, her cardiovascular indices deteriorated postoperatively. On the first postoperative day she was started on mechanical circulatory support with the Levitronix CentriMag left ventricular assist device. Her myocardial function improved and she was successfully weaned from this mechanical support on post-implant day 14. She remains alive and well to date.

Host resistance to lung infection mediated by major vault protein in epithelial cells.

The airway epithelium plays an essential role in innate immunity to lung pathogens. Ribonucleoprotein particles primarily composed of major vault protein (MVP) are highly expressed in cells that encounter xenobiotics. However, a clear biologic function for MVP is not established. We report here that MVP is rapidly recruited to lipid rafts when human lung epithelial cells are infected with Pseudomonas aeruginosa, and maximal recruitment is dependent on bacterial binding to the cystic fibrosis transmembrane conductance regulator. MVP was also essential for optimal epithelial cell internalization and clearance of P. aeruginosa. These results suggest that MVP makes a substantial contribution to epithelial cell-mediated resistance to infection.

Endothelial cell cortactin coordinates intercellular adhesion molecule-1 clustering and actin cytoskeleton remodeling during polymorphonuclear leukocyte adhesion and transmigration.

Endothelial cell ICAM-1 interacts with leukocyte beta(2) integrins to mediate adhesion and transmit outside-in signals that facilitate leukocyte transmigration. ICAM-1 redistribution and clustering appear necessary for leukocyte transmigration, but the mechanisms controlling ICAM-1 redistribution and clustering have not been identified. We recently reported that Src kinase phosphorylation of endothelial cortactin regulates polymorphonuclear cell (PMN) transmigration. In this study, we tested the hypotheses that the Src family kinase-cortactin pathway mediates association of ICAM-1 with the actin cytoskeleton and that this association is required for ICAM-1 clustering and leukocyte transmigration. Cross-linking ICAM-1 induced cytoskeletal remodeling and a decrease in ICAM-1 lateral mobility, as assessed by fluorescence recovery after photobleaching. Cytoskeletal remodeling after ICAM-1 cross-linking was reduced by knockdown of cortactin by small interfering RNA, by expression of a cortactin mutant deficient in Src phosphorylation sites (cortactin3F), and by the Src kinase inhibitor PP2. Pretreatment of cytokine-activated human endothelial monolayers with cortactin small interfering RNA significantly decreased both actin and ICAM-1 clustering around adherent PMN and the formation of actin-ICAM-1 clusters required for PMN transmigration. Our data suggest a model in which tyrosine phosphorylation of cortactin dynamically links ICAM-1 to the actin cytoskeleton, enabling ICAM-1 to form clusters and facilitate leukocyte transmigration.