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THAM Improves Bacterial Killing in Human Cystic Fibrosis Airway Epithelial Cell Cultures
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A3680 - THAM Improves Bacterial Killing in Human Cystic Fibrosis Airway Epithelial Cell Cultures
Author Block: Z. Holliday, M. Abou Alaiwa, A. Pezzulo, I. Thornell, N. Gansemer, J. Zabner, D. A. Stoltz; Internal Medicine, University of Iowa, Iowa City, IA, United States.
Rationale: Despite the development of new therapies, cystic fibrosis (CF) remains a life-shortening disease. Airway infection and inflammation begin during infancy and lead to respiratory failure. Thus, early interventions aimed at correcting the initial host defense defects and preventing/reducing infection could improve the course of CF lung disease. Mutations in the gene encoding cystic fibrosis transmembrane conductance regulator (CFTR), a HCO3- and Cl- conducting channel, cause CF. We discovered that the loss of CFTR-mediated HCO3- secretion and an abnormally acidic pH impair the activity of airway surface liquid (ASL) antimicrobials. In this study, we tested the hypothesis that airway alkalinization with Tromethamine (THAM, an FDA-approved alkalinizing agent in human clinical use for metabolic acidosis) will restore airway host defense leading to improved bacterial killing and have longer lasting effects than sodium bicarbonate.
Methods: Using human CF airway epithelial cell cultures, isosmolar solutions of either saline, sodium bicarbonate, or THAM were applied to the apical surface of cell cultures. The pH of the ASL was measured using a fluorescent ratiometric pH indicator SNARF at baseline and up to 15 minutes after the apical surface treatment application. Bacterial killing was tested using the grid method. Briefly, Staphylococcus aureus were attached to functionalized gold grids using avidin-Biotin interaction. At 10 minutes and 1 hour later, the grids were allowed to touch the cell culture epithelial surface for 15 minutes before percent bacterial killing was assessed with a fluorescent live/dead stain and confocal microscopic imaging.
Results: Both sodium bicarbonate and THAM initially increased ASL pH, but THAM’s effects on pH were longer lasting. Early bacteria killing at 10 minutes was enhanced with both sodium bicarbonate and THAM, but at 1 hour only THAM-treated airway cultures had enhanced bacterial killing.
Conclusion: In conclusion, ASL alkalinization in human CF airway epithelial cell cultures with THAM improves bacterial killing with a more sustained effect than sodium bicarbonate. The findings suggest that airway alkalinization with THAM could impact early CF airway disease.
Author Block: Z. Holliday, M. Abou Alaiwa, A. Pezzulo, I. Thornell, N. Gansemer, J. Zabner, D. A. Stoltz; Internal Medicine, University of Iowa, Iowa City, IA, United States.
Rationale: Despite the development of new therapies, cystic fibrosis (CF) remains a life-shortening disease. Airway infection and inflammation begin during infancy and lead to respiratory failure. Thus, early interventions aimed at correcting the initial host defense defects and preventing/reducing infection could improve the course of CF lung disease. Mutations in the gene encoding cystic fibrosis transmembrane conductance regulator (CFTR), a HCO3- and Cl- conducting channel, cause CF. We discovered that the loss of CFTR-mediated HCO3- secretion and an abnormally acidic pH impair the activity of airway surface liquid (ASL) antimicrobials. In this study, we tested the hypothesis that airway alkalinization with Tromethamine (THAM, an FDA-approved alkalinizing agent in human clinical use for metabolic acidosis) will restore airway host defense leading to improved bacterial killing and have longer lasting effects than sodium bicarbonate.
Methods: Using human CF airway epithelial cell cultures, isosmolar solutions of either saline, sodium bicarbonate, or THAM were applied to the apical surface of cell cultures. The pH of the ASL was measured using a fluorescent ratiometric pH indicator SNARF at baseline and up to 15 minutes after the apical surface treatment application. Bacterial killing was tested using the grid method. Briefly, Staphylococcus aureus were attached to functionalized gold grids using avidin-Biotin interaction. At 10 minutes and 1 hour later, the grids were allowed to touch the cell culture epithelial surface for 15 minutes before percent bacterial killing was assessed with a fluorescent live/dead stain and confocal microscopic imaging.
Results: Both sodium bicarbonate and THAM initially increased ASL pH, but THAM’s effects on pH were longer lasting. Early bacteria killing at 10 minutes was enhanced with both sodium bicarbonate and THAM, but at 1 hour only THAM-treated airway cultures had enhanced bacterial killing.
Conclusion: In conclusion, ASL alkalinization in human CF airway epithelial cell cultures with THAM improves bacterial killing with a more sustained effect than sodium bicarbonate. The findings suggest that airway alkalinization with THAM could impact early CF airway disease.
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