RESUMO
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.
Assuntos
Caveolina 1/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/microbiologia , Endocitose/fisiologia , Células Epiteliais/fisiologia , Pseudomonas aeruginosa/metabolismo , Aminoglicosídeos/metabolismo , Animais , Caveolina 1/genética , Linhagem Celular , Cloretos/metabolismo , Colforsina/metabolismo , Fibrose Cística/imunologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Células Epiteliais/citologia , Humanos , Imunidade Inata/fisiologia , Microdomínios da Membrana/metabolismo , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/patogenicidade , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Infecções Respiratórias/imunologiaRESUMO
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.
Assuntos
Úlcera da Córnea/prevenção & controle , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Epitélio Corneano/microbiologia , Infecções Oculares Bacterianas/prevenção & controle , Microdomínios da Membrana/metabolismo , Infecções por Pseudomonas/prevenção & controle , Pseudomonas aeruginosa/fisiologia , Animais , Aderência Bacteriana/efeitos dos fármacos , Western Blotting , Linhagem Celular Transformada , Contagem de Colônia Microbiana , Úlcera da Córnea/metabolismo , Úlcera da Córnea/microbiologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Modelos Animais de Doenças , Infecções Oculares Bacterianas/metabolismo , Infecções Oculares Bacterianas/microbiologia , Imunofluorescência , Humanos , Camundongos , Microscopia Confocal , Infecções por Pseudomonas/metabolismo , Infecções por Pseudomonas/microbiologia , RNA Interferente Pequeno/farmacologia , beta-Ciclodextrinas/farmacologiaRESUMO
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.
Assuntos
Insuficiência da Valva Aórtica/cirurgia , Estenose da Valva Aórtica/cirurgia , Bioprótese , Cardiopatias Congênitas/cirurgia , Doenças das Valvas Cardíacas/cirurgia , Implante de Prótese de Valva Cardíaca , Próteses Valvulares Cardíacas , Adulto , Fatores Etários , Animais , Valva Aórtica/cirurgia , Insuficiência da Valva Aórtica/etiologia , Estenose da Valva Aórtica/etiologia , Doença da Válvula Aórtica Bicúspide , Estudos de Coortes , Feminino , Cardiopatias Congênitas/complicações , Doenças das Valvas Cardíacas/complicações , Cavalos , Humanos , Masculino , Pericárdio , Resultado do Tratamento , Adulto JovemRESUMO
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.
Assuntos
Anormalidades Múltiplas , Aorta Torácica/cirurgia , Aneurisma Aórtico/cirurgia , Coartação Aórtica/cirurgia , Valva Aórtica/anormalidades , Implante de Prótese Vascular , Doenças das Valvas Cardíacas/complicações , Síndrome de Klippel-Feil/complicações , Aorta Torácica/anormalidades , Aorta Torácica/diagnóstico por imagem , Aneurisma Aórtico/diagnóstico , Aneurisma Aórtico/etiologia , Coartação Aórtica/complicações , Coartação Aórtica/diagnóstico , Aortografia/métodos , Doença da Válvula Aórtica Bicúspide , Prótese Vascular , Implante de Prótese Vascular/instrumentação , Feminino , Parada Cardíaca Induzida , Doenças das Valvas Cardíacas/diagnóstico , Humanos , Hipotermia Induzida , Síndrome de Klippel-Feil/diagnóstico , Pessoa de Meia-Idade , Polietilenotereftalatos , Desenho de Prótese , Tomografia Computadorizada por Raios X , Resultado do TratamentoRESUMO
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.
Assuntos
Aneurisma Aórtico/cirurgia , Dissecção Aórtica/cirurgia , Implante de Prótese Vascular/efeitos adversos , Aneurisma Coronário/cirurgia , Ponte de Artéria Coronária/efeitos adversos , Coração Auxiliar , Infarto do Miocárdio/cirurgia , Complicações Cardiovasculares na Gravidez/cirurgia , Choque Cardiogênico/terapia , Doença Aguda , Dissecção Aórtica/fisiopatologia , Aneurisma Aórtico/fisiopatologia , Cesárea , Aneurisma Coronário/fisiopatologia , Feminino , Hemodinâmica , Humanos , Nascido Vivo , Infarto do Miocárdio/fisiopatologia , Gravidez , Complicações Cardiovasculares na Gravidez/fisiopatologia , Terceiro Trimestre da Gravidez , Choque Cardiogênico/etiologia , Resultado do TratamentoRESUMO
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.
Assuntos
Pneumopatias/imunologia , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/imunologia , Mucosa Respiratória/metabolismo , Mucosa Respiratória/microbiologia , Partículas de Ribonucleoproteínas em Forma de Abóbada/fisiologia , Animais , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Humanos , Imunidade Inata , Pulmão/imunologia , Pulmão/microbiologia , Pneumopatias/metabolismo , Pneumopatias/microbiologia , Microdomínios da Membrana/metabolismo , Camundongos , Infecções por Pseudomonas/metabolismo , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/metabolismo , RNA Interferente Pequeno/genética , Mucosa Respiratória/imunologia , Transdução de SinaisRESUMO
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.