26 results on '"Nancy L. Quinney"'
Search Results
2. A Novel Co-Culture Model Reveals Enhanced CFTR Rescue in Primary Cystic Fibrosis Airway Epithelial Cultures with Persistent Pseudomonas aeruginosa Infection
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Deborah M. Cholon, Matthew A. Greenwald, Matthew G. Higgs, Nancy L. Quinney, Susan E. Boyles, Suzanne L. Meinig, John T. Minges, Ashlesha Chaubal, Robert Tarran, Carla M. P. Ribeiro, Matthew C. Wolfgang, and Martina Gentzsch
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CFTR ,cystic fibrosis ,airway epithelia ,Pseudomonas aeruginosa ,cytokines ,CFTR modulators ,Cytology ,QH573-671 - Abstract
People with cystic fibrosis (pwCF) suffer from chronic and recurring bacterial lung infections that begin very early in life and contribute to progressive lung failure. CF is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, which encodes an ion channel important for maintaining the proper hydration of pulmonary surfaces. When CFTR function is ablated or impaired, airways develop thickened, adherent mucus that contributes to a vicious cycle of infection and inflammation. Therapeutics for pwCF, called CFTR modulators, target the CFTR defect directly, restoring airway surface hydration and mucociliary clearance. However, even with CFTR modulator therapy, bacterial infections persist. To develop a relevant model of diseased airway epithelium, we established a primary human airway epithelium culture system with persistent Pseudomonas aeruginosa infection. We used this model to examine the effects of CFTR modulators on CFTR maturation, CFTR function, and bacterial persistence. We found that the presence of P. aeruginosa increased CFTR mRNA, protein, and function. We also found that CFTR modulators caused a decrease in P. aeruginosa burden. These results demonstrate the importance of including live bacteria to accurately model the CF lung, and that understanding the effects of infection on CFTR rescue by CFTR modulators is critical to evaluating and optimizing drug therapies for all pwCF.
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- 2023
- Full Text
- View/download PDF
3. Airway Epithelial Inflammation In Vitro Augments the Rescue of Mutant CFTR by Current CFTR Modulator Therapies
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Martina Gentzsch, Deborah M. Cholon, Nancy L. Quinney, Mary E. B. Martino, John T. Minges, Susan E. Boyles, Tara N. Guhr Lee, Charles R. Esther, and Carla M. P. Ribeiro
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airway inflammation ,CFTR ,F508del ,CFTR corrector ,CFTR potentiator ,CFTR rescue ,Therapeutics. Pharmacology ,RM1-950 - Abstract
In cystic fibrosis (CF), defective biogenesis and activity of the cystic fibrosis transmembrane conductance regulator (CFTR) leads to airway dehydration and impaired mucociliary clearance, resulting in chronic airway infection and inflammation. The most common CFTR mutation, F508del, results in a processing defect in which the protein is retained in the endoplasmic reticulum and does not reach the apical surface. CFTR corrector compounds address this processing defect to promote mutant CFTR transfer to the apical membrane. When coupled with potentiators to increase CFTR channel activity, these drugs yield significant clinical benefits in CF patients carrying the F508del mutation. However, processing of CFTR and other proteins can be influenced by environmental factors such as inflammation, and the impact of airway inflammation on pharmacological activity of CFTR correctors is not established. The present study evaluated CFTR-rescuing therapies in inflamed CF airway epithelial cultures, utilizing models that mimic the inflammatory environment of CF airways. Primary bronchial epithelial cultures from F508del/F508del CF patients were inflamed by mucosal exposure to one of two inflammatory stimuli: 1) supernatant from mucopurulent material from CF airways with advanced lung disease, or 2) bronchoalveolar lavage fluid from pediatric CF patients. Cultures inflamed with either stimulus exhibited augmented F508del responses following therapy with correctors VX-809 or VX-661, and overcame the detrimental effects of chronic exposure to the CFTR potentiator VX-770. Remarkably, even the improved CFTR rescue responses resulting from a clinically effective triple therapy (VX-659/VX-661/VX-770) were enhanced by epithelial inflammation. Thus, the airway inflammatory milieu from late- and early-stage CF lung disease improves the efficacy of CFTR modulators, regardless of the combination therapy used. Our findings suggest that pre-clinical evaluation of CFTR corrector therapies should be performed under conditions mimicking the native inflammatory status of CF airways, and altering the inflammatory status of CF airways may change the efficacy of CFTR modulator therapies.
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- 2021
- Full Text
- View/download PDF
4. Generation of renewable mouse intestinal epithelial cell monolayers and organoids for functional analyses
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Emily C. Moorefield, R. Eric Blue, Nancy L. Quinney, Martina Gentzsch, and Shengli Ding
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Intestinal epithelial cells ,Monolayer ,Murine ,Cytology ,QH573-671 - Abstract
Abstract Background Conditional reprogramming has enabled the development of long-lived, normal epithelial cell lines from mice and humans by in vitro culture with ROCK inhibitor on a feeder layer. We applied this technology to mouse small intestine to create 2D mouse intestinal epithelial monolayers (IEC monolayers) from genetic mouse models for functional analysis. Results IEC monolayers form epithelial colonies that proliferate on a feeder cell layer and are able to maintain their genotype over long-term passage. IEC monolayers form 3D spheroids in matrigel culture and monolayers on transwell inserts making them useful for functional analyses. IEC monolayers derived from the Cystic Fibrosis (CF) mouse model CFTR ∆F508 fail to respond to CFTR activator forskolin in 3D matrigel culture as measured by spheroid swelling and transwell monolayer culture via Ussing chamber electrophysiology. Tumor IEC monolayers generated from the Apc Min/+ mouse intestinal cancer model grow more quickly than wild-type (WT) IEC monolayers both on feeders and as spheroids in matrigel culture. Conclusions These results indicate that generation of IEC monolayers is a useful model system for growing large numbers of genotype-specific mouse intestinal epithelial cells that may be used in functional studies to examine molecular mechanisms of disease and to identify and assess novel therapeutic compounds.
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- 2018
- Full Text
- View/download PDF
5. A small-molecule inhibitor and degrader of the RNF5 ubiquitin ligase
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Jingjing Ruan, Dongdong Liang, Wenjing Yan, Yongwang Zhong, Daniel C. Talley, Ganesha Rai, Dingyin Tao, Christopher A. LeClair, Anton Simeonov, Yinghua Zhang, Feihu Chen, Nancy L. Quinney, Susan E. Boyles, Deborah M. Cholon, Martina Gentzsch, Mark J. Henderson, Fengtian Xue, and Shengyun Fang
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DNA-Binding Proteins ,Ubiquitin ,Ubiquitin-Protein Ligases ,Cell Biology ,Thiophenes ,Molecular Biology ,Cyclic S-Oxides - Abstract
RNF5 has multiple biological roles and has been linked to the development of severe diseases such as cystic fibrosis, acute myeloid leukemia, and certain viral infections. This study developed a small molecule inhibitor and degrader of the RNF5 ubiquitin ligase, providing a chemical biology tool for RNF5 research and therapeutic development.
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- 2023
6. Secretory Cells Dominate Airway CFTR Expression and Function in Human Airway Superficial Epithelia
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Wanda K. O'Neal, Rhianna E. Lee, Teresa M. Mascenik, Takanori Asakura, Hong Dang, Lisa C. Morton, Barbara R. Grubb, Hirotoshi Matsui, Martina Gentzsch, Richard C. Boucher, Carlton W Anderson, Kenichi Okuda, Gang Chen, Purushothama Rao Tata, Yvonne K. O’Neal, Satoko Nakano, Takahide Nagase, Yoshihiko Kobayashi, Michael Chua, Selene Margarita Barbosa Cardenas, Nancy L. Quinney, John C. Olsen, Andrew J. Ghio, Gianni Carraro, Caroline E Minnick, Lawrence E. Ostrowski, Rodney C. Gilmore, Takafumi Kato, Scott H. Randell, Barry R. Stripp, and Weining Yin
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Pulmonary and Respiratory Medicine ,Cell specific ,congenital, hereditary, and neonatal diseases and abnormalities ,business.industry ,Regulator ,Human airway ,Critical Care and Intensive Care Medicine ,medicine.disease ,Cystic fibrosis ,Transmembrane protein ,Cell biology ,03 medical and health sciences ,0302 clinical medicine ,030228 respiratory system ,Medicine ,030212 general & internal medicine ,business ,Airway ,Function (biology) - Abstract
Rationale: Identification of the specific cell types expressing CFTR (cystic fibrosis [CF] transmembrane conductance regulator) is required for precision medicine therapies for CF. However, a full ...
- Published
- 2021
7. DNAJB12 and Hsp70 triage arrested intermediates of N1303K-CFTR for endoplasmic reticulum-associated autophagy
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Andrei A. Aleksandrov, Scott A. Houck, Martina Gentzsch, Lihua He, Nancy L. Quinney, Hong Yu Ren, Andrew S. Kennedy, Douglas M. Cyr, Alexandra Cyr-Scully, Scott H. Randell, and Deborah M. Cholon
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congenital, hereditary, and neonatal diseases and abnormalities ,Protein Folding ,Cystic Fibrosis Transmembrane Conductance Regulator ,Biology ,Endoplasmic Reticulum ,Cell Line ,03 medical and health sciences ,0302 clinical medicine ,Cricetinae ,Chlorocebus aethiops ,Autophagy ,Animals ,Humans ,HSP70 Heat-Shock Proteins ,Molecular Biology ,Heat-Shock Proteins ,030304 developmental biology ,0303 health sciences ,Endoplasmic reticulum ,Autophagosomes ,Endoplasmic Reticulum-Associated Degradation ,Articles ,Cell Biology ,respiratory system ,HSP40 Heat-Shock Proteins ,Transmembrane protein ,Cell biology ,Hsp70 ,Cytosol ,HEK293 Cells ,Membrane protein ,Cytoplasm ,COS Cells ,030217 neurology & neurosurgery - Abstract
The transmembrane Hsp40 DNAJB12 and cytosolic Hsp70 cooperate on the endoplasmic reticulum’s (ER) cytoplasmic face to facilitate the triage of nascent polytopic membrane proteins for folding versus degradation. N1303K is a common mutation that causes misfolding of the ion channel CFTR, but unlike F508del-CFTR, biogenic and functional defects in N1303K-CFTR are resistant to correction by folding modulators. N1303K is reported to arrest CFTR folding at a late stage after partial assembly of its N-terminal domains. N1303K-CFTR intermediates are clients of JB12-Hsp70 complexes, maintained in a detergent-soluble state, and have a relatively long 3-h half-life. ER-associated degradation (ERAD)-resistant pools of N1303K-CFTR are concentrated in ER tubules that associate with autophagy initiation sites containing WIPI1, FlP200, and LC3. Destabilization of N1303K-CFTR or depletion of JB12 prevents entry of N1303K-CFTR into the membranes of ER-connected phagophores and traffic to autolysosomes. In contrast, the stabilization of intermediates with the modulator VX-809 promotes the association of N1303K-CFTR with autophagy initiation machinery. N1303K-CFTR is excluded from the ER-exit sites, and its passage from the ER to autolysosomes does not require ER-phagy receptors. DNAJB12 operates in biosynthetically active ER microdomains to triage membrane protein intermediates in a conformation-specific manner for secretion versus degradation via ERAD or selective-ER-associated autophagy.
- Published
- 2021
8. A PI3Kγ mimetic peptide triggers CFTR gating, bronchodilation, and reduced inflammation in obstructive airway diseases
- Author
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Alessandra Ghigo, Alessandra Murabito, Valentina Sala, Anna Rita Pisano, Serena Bertolini, Ambra Gianotti, Emanuela Caci, Alessio Montresor, Aiswarya Premchandar, Flora Pirozzi, Kai Ren, Angela Della Sala, Marco Mergiotti, Wito Richter, Eyleen de Poel, Michaela Matthey, Sara Caldrer, Rosa A. Cardone, Federica Civiletti, Andrea Costamagna, Nancy L. Quinney, Cosmin Butnarasu, Sonja Visentin, Maria Rosaria Ruggiero, Simona Baroni, Simonetta Geninatti Crich, Damien Ramel, Muriel Laffargue, Carlo G. Tocchetti, Renzo Levi, Marco Conti, Xiao-Yun Lu, Paola Melotti, Claudio Sorio, Virginia De Rose, Fabrizio Facchinetti, Vito Fanelli, Daniela Wenzel, Bernd K. Fleischmann, Marcus A. Mall, Jeffrey Beekman, Carlo Laudanna, Martina Gentzsch, Gergely L. Lukacs, Nicoletta Pedemonte, Emilio Hirsch, Ghigo, A., Murabito, A., Sala, V., Pisano, A. R., Bertolini, S., Gianotti, A., Caci, E., Montresor, A., Premchandar, A., Pirozzi, F., Ren, K., Sala, A. D., Mergiotti, M., Richter, W., de Poel, E., Matthey, M., Caldrer, S., Cardone, R. A., Civiletti, F., Costamagna, A., Quinney, N. L., Butnarasu, C., Visentin, S., Ruggiero, M. R., Baroni, S., Crich, S. G., Ramel, D., Laffargue, M., Tocchetti, C. G., Levi, R., Conti, M., Lu, X. -Y., Melotti, P., Sorio, C., De Rose, V., Facchinetti, F., Fanelli, V., Wenzel, D., Fleischmann, B. K., Mall, M. A., Beekman, J., Laudanna, C., Gentzsch, M., Lukacs, G. L., Pedemonte, N., and Hirsch, E.
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Inflammation ,Animal ,Cystic Fibrosis Transmembrane Conductance Regulator ,bronchus ,General Medicine ,PI3K ,Article ,lung ,Mice ,Phosphatidylinositol 3-Kinases ,CFTR, PI3K, cAMP, PKA, inflammation, lung, bronchus, airways ,cAMP ,Peptide ,Animals ,Class Ib Phosphatidylinositol 3-Kinase ,Humans ,PKA ,CFTR ,Phosphatidylinositol 3-Kinase ,airways ,Peptides ,Human - Abstract
Cyclic adenosine 3′,5′-monophosphate (cAMP)–elevating agents, such as β 2 -adrenergic receptor (β 2 -AR) agonists and phosphodiesterase (PDE) inhibitors, remain a mainstay in the treatment of obstructive respiratory diseases, conditions characterized by airway constriction, inflammation, and mucus hypersecretion. However, their clinical use is limited by unwanted side effects because of unrestricted cAMP elevation in the airways and in distant organs. Here, we identified the A-kinase anchoring protein phosphoinositide 3-kinase γ (PI3Kγ) as a critical regulator of a discrete cAMP signaling microdomain activated by β 2 -ARs in airway structural and inflammatory cells. Displacement of the PI3Kγ-anchored pool of protein kinase A (PKA) by an inhaled, cell-permeable, PI3Kγ mimetic peptide (PI3Kγ MP) inhibited a pool of subcortical PDE4B and PDE4D and safely increased cAMP in the lungs, leading to airway smooth muscle relaxation and reduced neutrophil infiltration in a murine model of asthma. In human bronchial epithelial cells, PI3Kγ MP induced unexpected cAMP and PKA elevations restricted to the vicinity of the cystic fibrosis transmembrane conductance regulator (CFTR), the ion channel controlling mucus hydration that is mutated in cystic fibrosis (CF). PI3Kγ MP promoted the phosphorylation of wild-type CFTR on serine-737, triggering channel gating, and rescued the function of F508del-CFTR, the most prevalent CF mutant, by enhancing the effects of existing CFTR modulators. These results unveil PI3Kγ as the regulator of a β 2 -AR/cAMP microdomain central to smooth muscle contraction, immune cell activation, and epithelial fluid secretion in the airways, suggesting the use of a PI3Kγ MP for compartment-restricted, therapeutic cAMP elevation in chronic obstructive respiratory diseases.
- Published
- 2022
9. 179: Modulator therapy reverses aberrant mucus properties in vitro via hydration
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Nancy L. Quinney, C. Morrison, K. Araba, Wanda K. O'Neal, Lisa C. Morton, K. Shaffer, David B. Hill, Mehmet Kesimer, Martina Gentzsch, C. Ehre, and M. Markovitz
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Pulmonary and Respiratory Medicine ,business.industry ,Pediatrics, Perinatology and Child Health ,Medicine ,business ,medicine.disease ,Mucus ,Cystic fibrosis ,In vitro ,Cell biology - Published
- 2021
10. Airway Epithelial Inflammation
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Martina, Gentzsch, Deborah M, Cholon, Nancy L, Quinney, Mary E B, Martino, John T, Minges, Susan E, Boyles, Tara N, Guhr Lee, Charles R, Esther, and Carla M P, Ribeiro
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Pharmacology ,congenital, hereditary, and neonatal diseases and abnormalities ,CFTR corrector ,CFTR rescue ,CFTR potentiator ,respiratory system ,airway inflammation ,CFTR ,F508del ,respiratory tract diseases ,Original Research - Abstract
In cystic fibrosis (CF), defective biogenesis and activity of the cystic fibrosis transmembrane conductance regulator (CFTR) leads to airway dehydration and impaired mucociliary clearance, resulting in chronic airway infection and inflammation. The most common CFTR mutation, F508del, results in a processing defect in which the protein is retained in the endoplasmic reticulum and does not reach the apical surface. CFTR corrector compounds address this processing defect to promote mutant CFTR transfer to the apical membrane. When coupled with potentiators to increase CFTR channel activity, these drugs yield significant clinical benefits in CF patients carrying the F508del mutation. However, processing of CFTR and other proteins can be influenced by environmental factors such as inflammation, and the impact of airway inflammation on pharmacological activity of CFTR correctors is not established. The present study evaluated CFTR-rescuing therapies in inflamed CF airway epithelial cultures, utilizing models that mimic the inflammatory environment of CF airways. Primary bronchial epithelial cultures from F508del/F508del CF patients were inflamed by mucosal exposure to one of two inflammatory stimuli: 1) supernatant from mucopurulent material from CF airways with advanced lung disease, or 2) bronchoalveolar lavage fluid from pediatric CF patients. Cultures inflamed with either stimulus exhibited augmented F508del responses following therapy with correctors VX-809 or VX-661, and overcame the detrimental effects of chronic exposure to the CFTR potentiator VX-770. Remarkably, even the improved CFTR rescue responses resulting from a clinically effective triple therapy (VX-659/VX-661/VX-770) were enhanced by epithelial inflammation. Thus, the airway inflammatory milieu from late- and early-stage CF lung disease improves the efficacy of CFTR modulators, regardless of the combination therapy used. Our findings suggest that pre-clinical evaluation of CFTR corrector therapies should be performed under conditions mimicking the native inflammatory status of CF airways, and altering the inflammatory status of CF airways may change the efficacy of CFTR modulator therapies.
- Published
- 2020
11. DNAJB12 and Hsp70 Facilitate the Conformation Specific Degradation of Arrested N1303K-CFTR Intermediates by ER Associated-Autophagy
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Scott A. Houck, Martina Gentzsch, Lihua He, Andrew S. Kennedy, Scott H. Randell, Douglas M. Cyr, Nancy L. Quinney, Hong Yu Ren, Deborah M. Cholon, and Andrei A. Aleksandrov
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congenital, hereditary, and neonatal diseases and abnormalities ,Cytosol ,Membrane protein ,Chemistry ,Cytoplasm ,Autophagy ,Secretion ,Endoplasmic-reticulum-associated protein degradation ,Transmembrane protein ,Ion channel ,Cell biology - Abstract
SUMMARYThe transmembrane Hsp40 DNAJB12 and cytosolic Hsp70 cooperate on the ER’s cytoplasmic face to facilitate the triage of nascent polytopic membrane proteins for folding versus degradation. N1303K is the second most common mutation in the ion channel CFTR, but unlike F508del-CFTR, biogenic and functional defects in N1303K-CFTR are resistant to correction bolding modulators. N1303K is reported to arrest CFTR folding at a late stage after partial assembly of its N-terminal domains. N1303K-CFTR intermediates are clients of JB12-Hsp70 complexes, maintained in a detergent soluble-state, and have a relatively long 3-hour half-life. ERAD-resistant pools of N1303K-CFTR are concentrated in ER-tubules that associate with autophagy initiation sites containing WIPI1, FlP200, and LC3. Destabilization of N1303K-CFTR or depletion of JB12 prevents entry of N1303K-CFTR into the membranes of ER-connected phagophores and autolysosomes. Whereas, the stabilization of intermediates with the modulator VX-809 promotes the association of N1303K-CFTR with autophagy initiation machinery. N1303K-CFTR is excluded from the ER-exits site, and its passage from the ER to autolysosomes does not require ER-phagy receptors. DNAJB12 operates in biosynthetically active ER-microdomains to triage in a conformation-specific manner membrane protein intermediates for secretion versus degradation via ERAD or selective-ER associated autophagy.
- Published
- 2020
12. Mucin-Competent Secretory Cells Are a Major Cell Type for CFTR Expression in Normal Human Airway Epithelia
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Takafumi Kato, Michael Chua, Satoko Nakano, Kenichi Okuda, Wanda K. O'Neal, A.J. Ghio, Martina Gentzsch, Scott H. Randell, Richard C. Boucher, Purushothama Rao Tata, Nancy L. Quinney, Yoshihiko Kobayashi, V.K. O'Neal, Mehmet Kesimer, Carlton W Anderson, Gang Chen, S. Barbosa, Giorgia Radicioni, Rodney C. Gilmore, and Hong Dang
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Cell type ,Mucin ,Human airway ,Biology ,Cell biology - Published
- 2020
13. Defective Epithelial Mucociliary Differentiation Leads to Mucin Hyperconcentration in the Autosomal Dominant Hyper Ige Syndrome (AD-HIES) Airways
- Author
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Ling Sun, Gang Chen, Charles R. Esther, Kristijan Bogdanovski, Brian Button, Alexandra F. Freeman, Chevalia Robinson, Matthew C. Wolfgang, Wanda K. O'Neal, Kathryn A. Ramsey, Nancy L. Quinney, Richard C. Boucher, Kenneth N. Olivier, Martina Gentzsch, Mehmet Kesimer, and David Hill
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Mucin ,Immunology ,Autosomal dominant hyper-IgE syndrome ,Biology - Published
- 2020
14. Personalised medicine for non-classic cystic fibrosis resulting from rare CFTR mutations
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Nancy L. Quinney, Martina Gentzsch, Timothy J. Jensen, Matthew S. McCravy, Scott H. Donaldson, Peadar G. Noone, Susan E. Boyles, Andrei A. Aleksandrov, and Deborah M. Cholon
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Pulmonary and Respiratory Medicine ,Cystic Fibrosis ,Extramural ,business.industry ,MEDLINE ,Cystic Fibrosis Transmembrane Conductance Regulator ,Bioinformatics ,medicine.disease ,Precision medicine ,Cystic fibrosis ,Article ,03 medical and health sciences ,0302 clinical medicine ,Phenotype ,030228 respiratory system ,Mutation (genetic algorithm) ,Mutation ,medicine ,Humans ,030212 general & internal medicine ,Precision Medicine ,business ,Production team - Abstract
Footnotes This manuscript has recently been accepted for publication in the European Respiratory Journal . It is published here in its accepted form prior to copyediting and typesetting by our production team. After these production processes are complete and the …
- Published
- 2020
15. Accumulation and persistence of ivacaftor in airway epithelia with prolonged treatment
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Martina Gentzsch, Nancy L. Quinney, Tara N. Guhr Lee, Charles R. Esther, and Deborah M. Cholon
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0301 basic medicine ,Pulmonary and Respiratory Medicine ,Indoles ,Cystic Fibrosis ,Cell Culture Techniques ,Aminopyridines ,Bronchi ,Respiratory Mucosa ,Pharmacology ,Quinolones ,Aminophenols ,Cystic fibrosis ,Article ,Ivacaftor ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Pharmacokinetics ,medicine ,Humans ,Dosing ,Benzodioxoles ,Chloride Channel Agonists ,business.industry ,Lumacaftor ,Washout ,Epithelial Cells ,respiratory system ,medicine.disease ,Drug Combinations ,030104 developmental biology ,030228 respiratory system ,chemistry ,Pharmacodynamics ,Pediatrics, Perinatology and Child Health ,Airway ,business ,medicine.drug - Abstract
Background Current dosing strategies of CFTR modulators are based on serum pharmacokinetics, but drug concentrations in target tissues such as airway epithelia are not known. Previous data suggest that CFTR modulators may accumulate in airway epithelia, and serum pharmacokinetics may not accurately predict effects of chronic treatment. Methods CF (F508del homozygous) primary human bronchial epithelial (HBE) cells grown at air-liquid interface were treated for 14 days with ivacaftor plus lumacaftor or ivacaftor plus tezacaftor, followed by a 14-day washout period. At various intervals during treatment and washout phases, drug concentrations were measured via mass spectrometry, electrophysiological function was assessed in Ussing chambers, and mature CFTR protein was quantified by Western blotting. Results During treatment, ivacaftor accumulated in CF-HBEs to a much greater extent than either lumacaftor or tezacaftor and remained persistently elevated even after 14 days of washout. CFTR activity peaked at 7 days of treatment but diminished with further ivacaftor accumulation, though remained above baseline even after washout. Conclusions Intracellular accrual and persistence of CFTR modulators during and after chronic treatment suggest complex pharmacokinetic and pharmacodynamic properties within airway epithelia that are not predicted by serum pharmacokinetics. Direct measurement of drugs in target tissues may be needed to optimize dosing strategies, and the persistence of CFTR modulators after treatment cessation has implications for personalized medicine approaches.
- Published
- 2019
16. 616: Effects of VX-445 on CFTR channel function and stability
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Deborah M. Cholon, Martina Gentzsch, Nancy L. Quinney, A. Aleksandrov, and Luba A. Aleksandrov
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Pulmonary and Respiratory Medicine ,business.industry ,Pediatrics, Perinatology and Child Health ,Biophysics ,Medicine ,Function (mathematics) ,Channel (broadcasting) ,business ,Stability (probability) - Published
- 2021
17. Treatment of cystic fibrosis airway cells with CFTR modulators reverses aberrant mucus properties via hydration
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Matthew R. Markovetz, Jimmy Liao, Camille Ehre, Wanda K. O'Neal, Cameron B. Morrison, Kendall M. Shaffer, Nancy L. Quinney, Lisa C. Morton, Jason A. Wykoff, Alexis Flen, David B. Hill, M. Delion, Mehmet Kesimer, Martina Gentzsch, Mitchell L. Drumm, Shuyu Hao, and Kenza C Araba
- Subjects
Pulmonary and Respiratory Medicine ,Ion Transport ,Cystic Fibrosis ,business.industry ,Mucin ,Cell ,Cystic Fibrosis Transmembrane Conductance Regulator ,respiratory system ,medicine.disease ,Molecular biology ,Cystic fibrosis ,Mucus ,Article ,Blot ,Ivacaftor ,Bicarbonates ,medicine.anatomical_structure ,Cell culture ,Ultrastructure ,Humans ,Medicine ,business ,medicine.drug - Abstract
QuestionCystic fibrosis (CF) is characterised by the accumulation of viscous adherent mucus in the lungs. While several hypotheses invoke a direct relationship with cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction (i.e. acidic airway surface liquid (ASL) pH, low bicarbonate (HCO3−) concentration, airway dehydration), the dominant biochemical alteration of CF mucus remains unknown.Materials/methodsWe characterised a novel cell line (CFTR-KO Calu3 cells) and the responses of human bronchial epithelial (HBE) cells from subjects with G551D or F508del mutations to ivacaftor and elexacaftor-tezacaftor-ivacaftor. A spectrum of assays such as short-circuit currents, quantitative PCR, ASL pH, Western blotting, light scattering/refractometry (size-exclusion chromatography with inline multi-angle light scattering), scanning electron microscopy, percentage solids and particle tracking were performed to determine the impact of CFTR function on mucus properties.ResultsLoss of CFTR function in Calu3 cells resulted in ASL pH acidification and mucus hyperconcentration (dehydration). Modulation of CFTR in CF HBE cells did not affect ASL pH or mucin mRNA expression, but decreased mucus concentration, relaxed mucus network ultrastructure and improved mucus transport. In contrast with modulator-treated cells, a large fraction of airway mucins remained attached to naïve CF cells following short apical washes, as revealed by the use of reducing agents to remove residual mucus from the cell surfaces. Extended hydration, but not buffers alkalised with sodium hydroxide or HCO3−, normalised mucus recovery to modulator-treated cell levels.ConclusionThese results indicate that airway dehydration, not acidic pH and/or low [HCO3−], is responsible for abnormal mucus properties in CF airways and CFTR modulation predominantly restores normal mucin entanglement.
- Published
- 2021
18. WS07.2 A PI3Kγ-peptide promotes Cl− secretion through activation of both CFTR - dependent and independent currents
- Author
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Valentina Sala, A. Ghigo, Emanuela Caci, Nancy L. Quinney, Ambra Gianotti, E. Hirsch, A. Murabito, Nicoletta Pedemonte, and Martina Gentzsch
- Subjects
Pulmonary and Respiratory Medicine ,chemistry.chemical_classification ,chemistry ,business.industry ,Pediatrics, Perinatology and Child Health ,Medicine ,Peptide ,Chloride secretion ,business ,medicine.disease ,Cystic fibrosis ,Molecular biology - Published
- 2021
19. Restoration of R117H CFTR folding and function in human airway cells through combination treatment with VX-809 and VX-770
- Author
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Martina Gentzsch, Nancy L. Quinney, Imron G. Chaudhry, Deborah M. Cholon, Scott A. Houck, Scott H. Randell, Hong Yu Ren, Pattarawut Sopha, Jhuma Das, Douglas M. Cyr, and Nikolay V. Dokholyan
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0301 basic medicine ,Pulmonary and Respiratory Medicine ,Protein Folding ,medicine.medical_specialty ,Cystic Fibrosis ,Physiology ,Drug Evaluation, Preclinical ,Mutation, Missense ,Aminopyridines ,Cystic Fibrosis Transmembrane Conductance Regulator ,Gating ,Quinolones ,Biology ,Aminophenols ,medicine.disease_cause ,Cystic fibrosis ,Cell Line ,03 medical and health sciences ,0302 clinical medicine ,Physiology (medical) ,Internal medicine ,medicine ,Humans ,Benzodioxoles ,Allele ,Sequence Deletion ,Mutation ,Heterozygote advantage ,Cell Biology ,Potentiator ,medicine.disease ,Cystic fibrosis transmembrane conductance regulator ,030104 developmental biology ,Endocrinology ,030228 respiratory system ,Cell culture ,Call for Papers ,Cancer research ,biology.protein - Abstract
Cystic fibrosis (CF) is a lethal recessive genetic disease caused primarily by the F508del mutation in the CF transmembrane conductance regulator (CFTR). The potentiator VX-770 was the first CFTR modulator approved by the FDA for treatment of CF patients with the gating mutation G551D. Orkambi is a drug containing VX-770 and corrector VX809 and is approved for treatment of CF patients homozygous for F508del, which has folding and gating defects. At least 30% of CF patients are heterozygous for the F508del mutation with the other allele encoding for one of many different rare CFTR mutations. Treatment of heterozygous F508del patients with VX-809 and VX-770 has had limited success, so it is important to identify heterozygous patients that respond to CFTR modulator therapy. R117H is a more prevalent rare mutation found in over 2,000 CF patients. In this study we investigated the effectiveness of VX-809/VX-770 therapy on restoring CFTR function in human bronchial epithelial (HBE) cells from R117H/F508del CF patients. We found that VX-809 stimulated more CFTR activity in R117H/F508del HBEs than in F508del/F508del HBEs. R117H expressed exclusively in immortalized HBEs exhibited a folding defect, was retained in the ER, and degraded prematurely. VX-809 corrected the R117H folding defect and restored channel function. Because R117 is involved in ion conductance, VX-770 acted additively with VX-809 to restore CFTR function in chronically treated R117H/F508del cells. Although treatment of R117H patients with VX-770 has been approved, our studies indicate that Orkambi may be more beneficial for rescue of CFTR function in these patients.
- Published
- 2016
20. Nasospheroids permit measurements of CFTR-dependent fluid transport
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Martina Gentzsch, Jennifer S. Guimbellot, Inmaculada Aban, Nancy L. Quinney, Susan E. Boyles, Justin M. Leach, Imron G. Chaudhry, Michael Chua, and Ilona Jaspers
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0301 basic medicine ,congenital, hereditary, and neonatal diseases and abnormalities ,Cystic Fibrosis ,Aminopyridines ,Cystic Fibrosis Transmembrane Conductance Regulator ,Mucous membrane of nose ,Pharmacology ,Quinolones ,Aminophenols ,Cystic fibrosis ,law.invention ,Ivacaftor ,03 medical and health sciences ,chemistry.chemical_compound ,Confocal microscopy ,law ,Spheroids, Cellular ,medicine ,Humans ,Benzodioxoles ,Particle Size ,Precision Medicine ,biology ,Chemistry ,Lumacaftor ,Colforsin ,Biological Transport ,General Medicine ,respiratory system ,medicine.disease ,Fluid transport ,Cystic fibrosis transmembrane conductance regulator ,digestive system diseases ,respiratory tract diseases ,Drug Combinations ,Nasal Mucosa ,030104 developmental biology ,Cell culture ,Mutation ,biology.protein ,medicine.drug ,Research Article - Abstract
Expansion of novel therapeutics to all patients with cystic fibrosis (CF) requires personalized CFTR modulator therapy. We have developed nasospheroids, a primary cell culture-based model derived from individual CF patients and healthy subjects by a minimally invasive nasal biopsy. Confocal microscopy was utilized to measure CFTR activity by analyzing changes in cross-sectional area over time that resulted from CFTR-mediated ion and fluid movement. Both the rate of change over time and AUC were calculated. Non-CF nasospheroids with active CFTR-mediated ion and fluid movement showed a reduction in cross-sectional area, whereas no changes were observed in CF spheroids. Non-CF spheroids treated with CFTR inhibitor lost responsiveness for CFTR activation. However, nasospheroids from F508del CF homozygotes that were treated with lumacaftor and ivacaftor showed a significant reduction in cross-sectional area, indicating pharmacologic rescue of CFTR function. This model employs a simple measurement of size corresponding to changes in CFTR activity and is applicable for detection of small changes in CFTR activity from individual patients in vitro. Advancements of this technique will provide a robust model for individualized prediction of CFTR modulator efficacy.
- Published
- 2017
21. Pharmacological rescue of conditionally reprogrammed cystic fibrosis bronchial epithelial cells
- Author
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Scott H. Randell, Imron G. Chaudhry, Richard Schlegel, Nancy L. Quinney, Susan E. Boyles, Xuefeng Liu, Martina Gentzsch, Hong Dang, Crescentia Cho, and Chaitra Cheluvaraju
- Subjects
0301 basic medicine ,Pulmonary and Respiratory Medicine ,Pathology ,medicine.medical_specialty ,RHOA ,Cystic Fibrosis ,Clinical Biochemistry ,Cell ,Bronchi ,Cystic fibrosis ,Cell Line ,03 medical and health sciences ,Mice ,medicine ,Animals ,Humans ,Fibroblast ,Molecular Biology ,Cell Shape ,Cell Proliferation ,biology ,Ussing chamber ,Cell growth ,Epithelial Cells ,Cell Biology ,medicine.disease ,Cellular Reprogramming ,Molecular biology ,Cystic fibrosis transmembrane conductance regulator ,digestive system diseases ,Electrophysiological Phenomena ,Major Technical Advances ,030104 developmental biology ,medicine.anatomical_structure ,Cell culture ,biology.protein - Abstract
Well-differentiated primary human bronchial epithelial (HBE) cell cultures are vital for cystic fibrosis (CF) research, particularly for the development of cystic fibrosis transmembrane conductance regulator (CFTR) modulator drugs. Culturing of epithelial cells with irradiated 3T3 fibroblast feeder cells plus the RhoA kinase inhibitor Y-27632 (Y), termed conditionally reprogrammed cell (CRC) technology, enhances cell growth and lifespan while preserving cell-of-origin functionality. We initially determined the electrophysiological and morphological characteristics of conventional versus CRC-expanded non-CF HBE cells. On the basis of these findings, we then created six CF cell CRC populations, three from sequentially obtained CF lungs and three from F508 del homozygous donors previously obtained and cryopreserved using conventional culture methods. Growth curves were plotted, and cells were subcultured, without irradiated feeders plus Y, into air-liquid interface conditions in nonproprietary and proprietary Ultroser G-containing media and were allowed to differentiate. Ussing chamber studies were performed after treatment of F508 del homozygous CF cells with the CFTR modulator VX-809. Bronchial epithelial cells grew exponentially in feeders plus Y, dramatically surpassing the numbers of conventionally grown cells. Passage 5 and 10 CRC HBE cells formed confluent mucociliary air-liquid interface cultures. There were differences in cell morphology and current magnitude as a function of extended passage, but the effect of VX-809 in increasing CFTR function was significant in CRC-expanded F508 del HBE cells. Thus, CRC technology expands the supply of functional primary CF HBE cells for testing CFTR modulators in Ussing chambers.
- Published
- 2017
- Full Text
- View/download PDF
22. The cystic fibrosis airway milieu enhances rescue of F508del in a pre-clinical model
- Author
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Martina Gentzsch, Susan E. Boyles, Nancy L. Quinney, Carla Ribeiro, Deborah M. Cholon, and Mary E. B. Martino
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0301 basic medicine ,Pulmonary and Respiratory Medicine ,medicine.medical_specialty ,Cystic Fibrosis ,Respiratory System ,Aminopyridines ,Cystic Fibrosis Transmembrane Conductance Regulator ,Pulmonary disease ,Bronchi ,Disease ,Quinolones ,Aminophenols ,Cystic fibrosis ,Article ,Cell Line ,03 medical and health sciences ,Nothing ,medicine ,Humans ,Benzodioxoles ,Intensive care medicine ,Inflammation ,biology ,business.industry ,Colforsin ,Conflict of interest ,Epithelial Cells ,Airway obstruction ,medicine.disease ,Cystic fibrosis transmembrane conductance regulator ,030104 developmental biology ,Mutation ,biology.protein ,Airway ,business - Abstract
Cystic Fibrosis (CF) is a life-shortening genetic disease with autosomal recessive inheritance. Most CF morbidity and mortality is associated with pulmonary disease. The cystic fibrosis transmembrane conductance regulator (CFTR) gene, which codes for a chloride channel required for proper hydration of airway epithelial surfaces, is mutated in CF, resulting in airway dehydration. As a result, CF patients suffer from chronic airway infection, inflammation, and overproduction of mucus, which leads to airway obstruction. During the last decade, basic and clinical research has led to strategies for targeted therapies to successfully restore CFTR function in CF patients. Footnotes This manuscript has recently been accepted for publication in the European Respiratory Journal . It is published here in its accepted form prior to copyediting and typesetting by our production team. After these production processes are complete and the authors have approved the resulting proofs, the article will move to the latest issue of the ERJ online. Please open or download the PDF to view this article. Conflict of interest: Dr. Gentzsch has nothing to disclose. Conflict of interest: Dr. Cholon has nothing to disclose. Conflict of interest: Dr. Quinney has nothing to disclose. Conflict of interest: Dr. Boyles has nothing to disclose. Conflict of interest: Dr. Martino has nothing to disclose. Conflict of interest: Dr. Ribeiro has nothing to disclose.
- Published
- 2018
23. Potentiator ivacaftor abrogates pharmacological correction of ΔF508 CFTR in cystic fibrosis
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Nancy L. Quinney, Jhuma Das, Martina Gentzsch, Nikolay V. Dokholyan, Deborah M. Cholon, Charles R. Esther, Scott H. Randell, Richard C. Boucher, and M. Leslie Fulcher
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Models, Molecular ,congenital, hereditary, and neonatal diseases and abnormalities ,Cystic Fibrosis ,Cystic Fibrosis Transmembrane Conductance Regulator ,Bronchi ,Pharmacology ,Biology ,Quinolones ,medicine.disease_cause ,Aminophenols ,Cystic fibrosis ,Article ,Ivacaftor ,chemistry.chemical_compound ,medicine ,Humans ,ΔF508 ,Mutation ,Dose-Response Relationship, Drug ,Protein Stability ,Lumacaftor ,Epithelial Cells ,General Medicine ,Potentiator ,medicine.disease ,In vitro ,Cystic fibrosis transmembrane conductance regulator ,chemistry ,biology.protein ,Mutant Proteins ,medicine.drug - Abstract
Cystic Fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR). Newly developed “correctors” such as lumacaftor (VX-809) that improve CFTR maturation and trafficking and “potentiators” such as ivacaftor (VX-770) that enhance channel activity may provide important advances in CF therapy. Although VX-770 has demonstrated substantial clinical efficacy in the small subset of patients with a mutation (G551D) that affects only channel activity, a single compound is not sufficient to treat patients with the more common CFTR mutation, ΔF508. Thus, patients with ΔF508 will likely require treatment with both correctors and potentiators to achieve clinical benefit. However, whereas the effectiveness of acute treatment with this drug combination has been demonstrated in vitro, the impact of chronic therapy has not been established. In studies of human primary airway epithelial cells, we found that both acute and chronic treatment with VX-770 improved CFTR function in cells with the G551D mutation, consistent with clinical studies. In contrast, chronic VX-770 administration caused a dose-dependent reversal of VX-809-mediated CFTR correction in ΔF508 homozygous cultures. This result reflected the destabilization of corrected ΔF508 CFTR by VX-770, dramatically increasing its turnover rate. Chronic VX-770 treatment also reduced mature wild-type CFTR levels and function. These findings demonstrate that chronic treatment with CFTR potentiators and correctors may have unexpected effects that cannot be predicted from short-term studies. Combining of these drugs to maximize rescue of ΔF508 CFTR may require changes in dosing and/or development of new potentiator compounds that do not interfere with CFTR stability.
- Published
- 2014
24. Bestrophin‐mediated Ca2+ activated Cl‐ conductance of the airway epithelium
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Sherif E. Gabriel, Joshua McDaniel, Silvia M. Kreda, Tarra Wasilchen, and Nancy L. Quinney
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Bestrophin 1 ,biology ,Chemistry ,Genetics ,biology.protein ,Biophysics ,Respiratory epithelium ,Molecular Biology ,Biochemistry ,Cl conductance ,Biotechnology - Published
- 2006
25. Telomere looping in P. sativum (common garden pea)
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Nancy L. Quinney, Anthony J. Cesare, Jack D. Griffith, Deepa Subramanian, and Smaranda Willcox
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DNA, Plant ,Size-exclusion chromatography ,Plant Science ,Biology ,Oxytricha ,Chromosomes, Plant ,Restriction fragment ,Pisum ,chemistry.chemical_compound ,Sativum ,Genetics ,Image Processing, Computer-Assisted ,Oligonucleotide Array Sequence Analysis ,Gel electrophoresis ,Base Sequence ,Peas ,food and beverages ,Cell Biology ,Telomere ,biology.organism_classification ,Molecular biology ,chemistry ,biology.protein ,Nucleic Acid Conformation ,DNA - Abstract
Summary Telomeres vary greatly in size among plants and, in most higher plants, consist of a long array of 5′-TTTAGGG-3′/3′-AAATCCC-5′ (TTTAGGG) repeats. Recently, telomeric DNA in human, mouse, oxytricha, and trypanosome chromosomes have been found arranged into loops (t-loops), proposed to sequester the telomere from unwanted repair events and prevent activation of DNA damage checkpoints. We have asked whether t-loops exist in the higher order plant Pisum sativum (garden pea). DNA was isolated from the shoots and root tips of germinating seeds. Analysis of the telomeric restriction fragments showed that DNA hybridizing to a (TTTAGGG)n probe migrated as a smear centering around 25 kb, and direct sequencing verified the repeat to be (TTTAGGG)n. Total DNA in isolated nuclei was photo-cross-linked, and the telomeric restriction fragments were purified by gel filtration. Electron microscopic (EM) analysis revealed DNA molecules arranged as t-loops with a size distribution consistent with that seen by gel electrophoresis. Some molecules had loops as large as 75 kb. These results show that the arrangement of telomeric DNA into loops occurs in higher plants.
- Published
- 2003
26. Multicenter Intestinal Current Measurements in Rectal Biopsies from CF and Non-CF Subjects to Monitor CFTR Function
- Author
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Amir Rezayat, Umer Khan, Philip H. Karp, Sherif E. Gabriel, Sarah E. Ernst, Nancy L. Quinney, Shajan P. Sugandha, Rhonda D. Szczesniak, James Lymp, Steven M. Rowe, Melissa A. Ashlock, Douglas B. Hornick, Hongtao Sun, Timothy D. Starner, John P. Clancy, Alicia J. Ostmann, Scott H. Donaldson, and Lijuan Fan
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Adult ,Male ,medicine.medical_specialty ,IBMX ,Carbachol ,Cystic Fibrosis ,Biopsy ,Urology ,Cystic Fibrosis Transmembrane Conductance Regulator ,lcsh:Medicine ,Cold storage ,Cystic fibrosis ,Young Adult ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Chlorides ,Internal medicine ,Cyclic AMP ,medicine ,Humans ,lcsh:Science ,Aged ,030304 developmental biology ,0303 health sciences ,Multidisciplinary ,Forskolin ,biology ,business.industry ,Sodium ,lcsh:R ,Rectum ,Area under the curve ,Middle Aged ,medicine.disease ,Cystic fibrosis transmembrane conductance regulator ,Endocrinology ,ROC Curve ,030228 respiratory system ,chemistry ,biology.protein ,Female ,lcsh:Q ,business ,Bumetanide ,Research Article ,medicine.drug - Abstract
Intestinal current measurements (ICM) from rectal biopsies are a sensitive means to detect cystic fibrosis transmembrane conductance regulator (CFTR) function, but have not been optimized for multicenter use. We piloted multicenter standard operating procedures (SOPs) to detect CFTR activity by ICM and examined key questions for use in clinical trials. SOPs for ICM using human rectal biopsies were developed across three centers and used to characterize ion transport from non-CF and CF subjects (two severe CFTR mutations). All data were centrally evaluated by a blinded interpreter. SOPs were then used across four centers to examine the effect of cold storage on CFTR currents and compare CFTR currents in biopsies from one subject studied simultaneously either at two sites (24 hours post-biopsy) or when biopsies were obtained by either forceps or suction. Rectal biopsies from 44 non-CF and 17 CF subjects were analyzed. Mean differences (µA/cm(2); 95% confidence intervals) between CF and non-CF were forskolin/IBMX=102.6(128.0 to 81.1), carbachol=96.3(118.7 to 73.9), forskolin/IBMX+carbachol=200.9(243.1 to 158.6), and bumetanide=-44.6 (-33.7 to -55.6) (P
- Published
- 2013
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