Publications by authors named "Beate Illek"

41 Publications

Role of Dual Oxidases in Ventilator-induced Lung Injury.

Am J Respir Cell Mol Biol 2021 02;64(2):208-215

University of California Lung Center, University of California, Davis, Davis, California.

Positive-pressure ventilation results in ventilator-induced lung injury, and few therapeutic modalities have been successful at limiting the degree of injury to the lungs. Understanding the primary drivers of ventilator-induced lung injury will aid in the development of specific treatments to ameliorate the progression of this syndrome. There are conflicting data for the role of neutrophils in acute respiratory distress syndrome pathogenesis. Here, we specifically examined the importance of neutrophils as a primary driver of ventilator-induced lung injury in a mouse model known to have impaired ability to recruit neutrophils in previous models of inflammation. We exposed and mice to low- or high-tidal volume ventilation with or without positive end-expiratory pressure (PEEP) and recruitment maneuvers for 4 hours. Absolute neutrophils in BAL fluid were significantly reduced in mice compared with mice (6.7 cells/μl; 16.4 cells/μl;  = 0.003), consistent with our hypothesis that neutrophil translocation across the capillary endothelium is reduced in the absence of DUOX1 or DUOX2 in response to ventilator-induced lung injury. Reduced lung neutrophilia was not associated with a reduction in overall lung injury in this study, suggesting that neutrophils do not play an important role in early features of acute lung injury. Surprisingly, mice exhibited significant hypoxemia, as measured by the arterial oxygen tension/fraction of inspired oxygen ratio and arterial oxygen content, which was out of proportion with that seen in the mice (141, 257,  = 0.012). These findings suggest a role for dual oxidases to limit physiologic impairment during early ventilator-induced lung injury.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1165/rcmb.2020-0197OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7874397PMC
February 2021

Functional Profiling of CFTR-Directed Therapeutics Using Pediatric Patient-Derived Nasal Epithelial Cell Models.

Front Pediatr 2020 4;8:536. Epub 2020 Sep 4.

UCSF Benioff Children's Hospital Oakland, Children's Hospital Oakland Research Institute, Oakland, CA, United States.

Functional profiling of CFTR-directed therapeutics offers the potential to provide significant benefits to young people with cystic fibrosis (CF). However, the development of 2D airway epithelial cell models for individual response tests in CF children remains a central task. The objective of this study was to determine the utility of EpiX technology for expansion of nasal epithelial cells for use in electrophysiological CFTR function measurements. An initial harvest of as few as 20,000 cells was sufficient to expand up to 50 million cells that were used to generate air-liquid interface (ALI) cultures for ion transport studies with the Ussing assay. CFTR function was assessed by measuring responses to forskolin and the CFTR potentiator VX-770 (ivacaftor) in ALI cultures generated from passage 3 and 4 cells. Short-circuit current (Isc) measurements of blocked CFTR currents (ΔI) discriminated CFTR function between healthy control (wild type, WT) and patients with intermediate (F508del/R117H-7T: 56% WT) and severe (F508del/F508del: 12% WT) CF disease. For the mixed genotypes, CFTR activity for F508del/c.850dupA was 12% WT, R334W/406-1G>A was 24% WT, and CFTRdele2,3(21 kb)/CFTRdele2,3(21 kb) was 9% WT. The CFTR correctors VX-809 (lumacaftor) and VX-661 (tezacaftor) significantly increased CFTR currents for F508del/R117H to 73 and 67% WT, respectively. Cultures with the large deletion mutation CFTRdele2,3(21 kb) unexpectedly responded to VX-661 treatment (20% WT). Amiloride-sensitive sodium currents were robust and ranged between 20-80 μA/cm depending on the subject. In addition to characterizing the electrophysiological profile of mutant CFTR activity in cultures for five genotypes, our study exemplifies the promising paradigm of bed-to-bench side cooperation and personalized medicine.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fped.2020.00536DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7500161PMC
September 2020

Differential Chloride Secretory Capacity in Transepithelial Ion Transport Properties in Chronic Rhinosinusitis.

Am J Rhinol Allergy 2020 Nov 23;34(6):830-837. Epub 2020 Jun 23.

Department of Otolaryngology-Head & Neck Surgery, University of Alabama at Birmingham, Birmingham, Alabama.

Background: Epithelial ion transport regulates hydration of airway mucosal surfaces, and thus promotes effective mucociliary clearance (MCC). Decreased transepithelial Cl transport may contribute to epithelial dysfunction by abrogating MCC and increasing mucus viscosity in chronic rhinosinusitis (CRS). The objective of the current study is to evaluate Cl channel transport properties from cultures of human sinonasal epithelia.

Methods: Human sinonasal epithelia (HSNE) from patients undergoing sinus surgery were cultured at an air-liquid interface to confluence and full differentiation. The epithelial monolayers were mounted in Ussing Chambers to investigate pharmacological manipulation of ion transport. Epithelial Na channel (via Amiloride), CFTR (via forskolin), and Ca-activated Cl channel (CaCC, via UTP) transport were investigated among three different patient groups: Control, CRS and CRS with polyposis. CFTR mRNA levels were evaluated with quantitative RT-PCR.

Results: HSNE cultures from 18 patients (Control = 9, CRS = 6, CRS with polyposis = 3) were evaluated in 142 experiments. Summary data from the 18 patients demonstrated that stimulated CFTR-mediated anion transport (Δ I) was significantly lower with CRS (7.58+/-2.24 µA/cm) compared to control (25.86+/-3.44 µA/cm) and CRS with polyposis (20.16+/-4.0 µA/cm) (p = 0.004). No statistically significant difference was found for CaCC anion transport between groups (p = 0.39). Significantly decreased mRNA (relative expression) was noted in CRS cultures (CRS = 40.83+/-1.76 vs. control = 116.2+/-24.27, p = 0.03).

Conclusions: A substantial decrease in the Cl secretory capacity of HSNE monolayers was demonstrated in CRS subjects. Data suggest that CFTR may contribute more to abnormal ion transport in CRS than CaCC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1177/1945892420930975DOI Listing
November 2020

CFTR modulator theratyping: Current status, gaps and future directions.

J Cyst Fibros 2019 01 20;18(1):22-34. Epub 2018 Jun 20.

Cystic Fibrosis Foundation, United States.

Background: New drugs that improve the function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein with discreet disease-causing variants have been successfully developed for cystic fibrosis (CF) patients. Preclinical model systems have played a critical role in this process, and have the potential to inform researchers and CF healthcare providers regarding the nature of defects in rare CFTR variants, and to potentially support use of modulator therapies in new populations.

Methods: The Cystic Fibrosis Foundation (CFF) assembled a workshop of international experts to discuss the use of preclinical model systems to examine the nature of CF-causing variants in CFTR and the role of in vitro CFTR modulator testing to inform in vivo modulator use. The theme of the workshop was centered on CFTR theratyping, a term that encompasses the use of CFTR modulators to define defects in CFTR in vitro, with application to both common and rare CFTR variants.

Results: Several preclinical model systems were identified in various stages of maturity, ranging from the expression of CFTR variant cDNA in stable cell lines to examination of cells derived from CF patients, including the gastrointestinal tract, the respiratory tree, and the blood. Common themes included the ongoing need for standardization, validation, and defining the predictive capacity of data derived from model systems to estimate clinical outcomes from modulator-treated CF patients.

Conclusions: CFTR modulator theratyping is a novel and rapidly evolving field that has the potential to identify rare CFTR variants that are responsive to approved drugs or drugs in development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jcf.2018.05.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6301143PMC
January 2019

Assessment and management of diarrhea following VEGF receptor TKI treatment in patients with ovarian cancer.

Gynecol Oncol 2018 07 5;150(1):173-179. Epub 2018 Apr 5.

Christie Hospital and Institute of Cancer Studies, University of Manchester, Manchester, UK. Electronic address:

Angiogenesis is a proven clinical target for the treatment of advanced epithelial ovarian cancer. Vascular endothelial growth factor receptor tyrosine kinase inhibitors (VEGFR-TKIs) offer patients potential new treatment regimens as they can be given as monotherapy, in combination with poly(ADP-ribose) polymerase (PARP) inhibitors, or with and following cytotoxic chemotherapy. If VEGFR-TKIs are licensed for use in ovarian cancer, patients will require prompt and effective management of adverse events, including diarrhea, to optimize compliance and benefit. As diarrhea is one of the most prevalent toxicities of this class of drug, it is important to consider the potential causes, be they disease related (bowel obstruction), treatment related (VEGFR-TKI-related or infective/neutropenic septic diarrhea when patients are receiving cytotoxic chemotherapy combined with VEGFR inhibitor treatment), or incurred through diet. Here, we provide an overview of the possible mechanisms responsible for VEGFR-TKI-induced diarrhea. We review potential interventions that can help in the management of diarrhea induced by VEGFR-TKIs, when used in combination or as single agents, and we provide a diarrhea treatment algorithm to serve as a clinical reference point for the management of diarrhea in patients with ovarian cancer treated with a VEGFR-TKI in combination with chemotherapy or PARP inhibitors, or as monotherapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ygyno.2018.03.058DOI Listing
July 2018

Impaired PGE2-stimulated Cl- and HCO3- secretion contributes to cystic fibrosis airway disease.

PLoS One 2017 27;12(12):e0189894. Epub 2017 Dec 27.

Cystic Fibrosis Research Laboratory, Stanford University, Palo Alto, CA, United States of America.

Background: Airway mucociliary clearance (MCC) is an important defense mechanism against pulmonary infections and is compromised in cystic fibrosis (CF). Cl- and HCO3- epithelial transport are integral to MCC. During pulmonary infections prostaglandin E2 (PGE2) production is abundant.

Aim: To determine the effect of PGE2 on airway Cl- and HCO3- secretion and MCC in normal and CF airways.

Methods: We examined PGE2 stimulated MCC, Cl- and HCO3- secretion using ferret trachea, human bronchial epithelial cell cultures (CFBE41o- with wildtype CFTR (CFBE41 WT) or homozygous F508del CFTR (CFBE41 CF) and human normal bronchial submucosal gland cell line (Calu-3) in Ussing chambers with or without pH-stat.

Results: PGE2 stimulated MCC in a dose-dependent manner and was partially impaired by CFTRinh-172. PGE2-stimulated Cl- current in ferret trachea was partially inhibited by CFTRinh-172, with niflumic acid eliminating the residual current. CFBE41 WT cell monolayers produced a robust Cl- and HCO3- secretory response to PGE2, both of which were completely inhibited by CFTRinh-172. CFBE41 CF cells exhibited no response to PGE2. In Calu-3 cells, PGE2 stimulated Cl- and HCO3- secretion. Cl- secretion was partially inhibited by CFTRinh-172, with additional inhibition by niflumic acid. HCO3- secretion was completely inhibited by CFTRinh-172.

Conclusions: PGE2 stimulates bronchotracheal MCC and this response is decreased in CF. In CF airway, PGE2-stimulated Cl- and HCO3- conductance is impaired and may contribute to decreased MCC. There remains a CFTR-independent Cl- current in submucosal glands, which if exploited, could represent a means of improving airway Cl- secretion and MCC in CF.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0189894PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5744969PMC
January 2018

Effect of Vaccine-Elicited Antibodies on Colonization of Neisseria meningitidis Serogroup B and C Strains in a Human Bronchial Epithelial Cell Culture Model.

Clin Vaccine Immunol 2017 Oct 5;24(10). Epub 2017 Oct 5.

Center for Immunobiology and Vaccine Development, UCSF Benioff Children's Hospital Oakland Research Institute, Oakland, California, USA

Capsular polysaccharide-protein conjugate vaccines protect individuals from invasive disease and decrease carriage, which reduces spread of the organism in the population. In contrast, antibodies elicited by plain polysaccharide or protein antigen-based meningococcal (Men) vaccines have little or no effect on decreasing carriage. In this study, we investigated the mechanism by which vaccine-induced human immunoglobulin G (IgG) antibodies affect colonization by meningococcal serogroup B (MenB) or C (MenC) strains using a human bronchial epithelial cell culture model (16HBE14o-). Fluorescence microscopy showed that bacteria colonizing the apical side of 16HBE14o- monolayers had decreased capsular polysaccharide on the bacterial surface that resulted from shedding the capsule and not decreased production of polysaccharide. Capsular polysaccharide shedding depended on the presence of 16HBE14o- cells and bacteria but not direct adherence of the bacteria to the cells. Treatment of bacteria and cells with postimmunization MenC-conjugate IgG or murine anti-MenB polysaccharide monoclonal antibodies (MAbs) inhibited capsule shedding, microcolony dispersal, and invasion of the 16HBE14o- cell monolayer. In contrast, the IgG responses elicited by immunization with MenC polysaccharide (PS), MenB outer membrane vesicle (OMV)-based, or factor H binding protein (FHbp)-based vaccines were not different than preimmune IgG or no-treatment response. The results provide new insights on the mechanism by which high-avidity anticapsular antibodies elicited by polysaccharide-conjugate vaccines affect meningococcal colonization. The data also suggest that any effect on colonization by IgG elicited by OMV- or FHbp-based vaccines may involve a different mechanism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/CVI.00188-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5629670PMC
October 2017

In vivo and in vitro ivacaftor response in cystic fibrosis patients with residual CFTR function: N-of-1 studies.

Pediatr Pulmonol 2017 04 9;52(4):472-479. Epub 2017 Jan 9.

Pediatric Pulmonology, Department of Pediatrics, University of California, San Francisco, California.

Rationale: Ivacaftor, a cystic fibrosis transmembrane conductance regulator (CFTR) potentiator, decreases sweat chloride concentration, and improves pulmonary function in 6% of cystic fibrosis (CF) patients with specific CFTR mutations. Ivacaftor increases chloride transport in many other CFTR mutations in non-human cells, if CFTR is in the epithelium. Some CF patients have CFTR in the epithelium with residual CFTR function. The effect of ivacaftor in these patients is unknown.

Methods: This was a series of randomized, crossover N-of-1 trials of ivacaftor and placebo in CF patients ≥8 years old with potential residual CFTR function (intermediate sweat chloride concentration, pancreatic sufficient, or mild bronchiectasis on chest CT). Human nasal epithelium (HNE) was obtained via nasal brushing and cultured. Sweat chloride concentration change was the in vivo outcome. Chloride current change in HNE cultures with ivacaftor was the in vitro outcome.

Results: Three subjects had decreased sweat chloride concentration (-14.8 to -40.8 mmol/L, P < 0.01). Two subjects had unchanged sweat chloride concentration. Two subjects had increased sweat chloride concentration (+23.8 and +27.3 mmol/L, P < 0.001); both were heterozygous for A455E and pancreatic sufficient. Only subjects with decreased sweat chloride concentration had increased chloride current in HNE cultures.

Conclusions: Some CF patients with residual CFTR function have decreased sweat chloride concentration with ivacaftor. Increased chloride current in HNE cultures among subjects with decreased sweat chloride concentrations may predict clinical response to ivacaftor. Ivacaftor can increase sweat chloride concentration in certain mutations with unclear clinical effect. Pediatr Pulmonol. 2017;52:472-479. © 2017 Wiley Periodicals, Inc.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/ppul.23659DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5461115PMC
April 2017

TALENs Facilitate Single-step Seamless SDF Correction of F508del CFTR in Airway Epithelial Submucosal Gland Cell-derived CF-iPSCs.

Mol Ther Nucleic Acids 2016 Jan 5;5:e273. Epub 2016 Jan 5.

Department of Otolaryngology - Head and Neck Surgery, University of California-San Francisco, San Francisco, California, USA.

Cystic fibrosis (CF) is a recessive inherited disease associated with multiorgan damage that compromises epithelial and inflammatory cell function. Induced pluripotent stem cells (iPSCs) have significantly advanced the potential of developing a personalized cell-based therapy for diseases like CF by generating patient-specific stem cells that can be differentiated into cells that repair tissues damaged by disease pathology. The F508del mutation in airway epithelial cell-derived CF-iPSCs was corrected with small/short DNA fragments (SDFs) and sequence-specific TALENs. An allele-specific PCR, cyclic enrichment strategy gave ~100-fold enrichment of the corrected CF-iPSCs after six enrichment cycles that facilitated isolation of corrected clones. The seamless SDF-based gene modification strategy used to correct the CF-iPSCs resulted in pluripotent cells that, when differentiated into endoderm/airway-like epithelial cells showed wild-type (wt) airway epithelial cell cAMP-dependent Cl ion transport or showed the appropriate cell-type characteristics when differentiated along mesoderm/hematopoietic inflammatory cell lineage pathways.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/mtna.2015.43DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5012545PMC
January 2016

Expression and function of the epithelial sodium channel δ-subunit in human respiratory epithelial cells in vitro.

Pflugers Arch 2015 Nov 13;467(11):2257-73. Epub 2015 Feb 13.

School of Pharmacy and Pharmaceutical Sciences and Trinity Biomedical Sciences Institute, Trinity College Dublin, Panoz Institute, Dublin 2, Ireland.

Using human airway epithelial cell lines (i.e. NCI-H441 and Calu-3) as well as human alveolar epithelial type I-like (ATI) cells in primary culture, we studied the contribution of the epithelial sodium channel δ-subunit (δ-ENaC) to transepithelial sodium transport in human lung in vitro. Endogenous δ-ENaC protein was present in all three cell types tested; however, protein abundance was low, and no expression was detected in the apical cell membrane of these cells. Similarly, known modulators of δ-ENaC activity, such as capsazepine and icilin (activators) and Evans blue (inhibitor), did not show effects on short-circuit current (I SC), suggesting that δ-ENaC is not involved in the modulation of transcellular sodium absorption in NCI-H441 cell monolayers. Over-expression of δ-ENaC in NCI-H441 cells resulted in detectable protein expression in the apical cell membrane, as well as capsazepine and icilin-stimulated increases in I SC that were effectively blocked by Evans blue and that were consistent with δ-ENaC activation and inhibition, respectively. Consequently, these observations suggest that δ-ENaC expression is low in NCI-H441, Calu-3, and ATI cells and does not contribute to transepithelial sodium absorption.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00424-015-1693-5DOI Listing
November 2015

Air pollutants cause release of hydrogen peroxide and interleukin-8 in a human primary nasal tissue culture model.

Int Forum Allergy Rhinol 2014 Dec 14;4(12):966-71. Epub 2014 Nov 14.

Division of Otolaryngology-Head and Neck Surgery, Department of Surgery, University of Alabama at Birmingham, Birmingham, AL; Children's Hospital Oakland Research Institute, Oakland, CA.

Background: A component of primary innate defense of the nasal mucosa against inhaled pathogens includes continuous, low-level release of hydrogen peroxide (H2 O2 ) into luminal secretions. Epidemiologically, an association exists between poor air quality and increased prevalence of sinonasal disease. To understand the effects of particulate matter (PM) in nasal mucosa, we studied the release of H2 O2 and interleukin 8 (IL-8) after PM exposure.

Methods: Human nasal specimens were collected from surgery and cultured in serum-free growth medium. Cell integrity and recovery during culture was monitored by lactate dehydrogenase (LDH) release into the medium. Cultures were exposed to PM for 24 hours in the presence/absence of diphenyleneiodonium sulfate (DPI; a nicotinamide adenine dinucleotide phosphate [NADPH] oxidase inhibitor). Luminex cytokine and Amplex-Red H2 O2 assays were performed.

Results: LDH levels dropped rapidly within 2 days, indicative of stabilization and cell recovery after harvest. All cultures released H2 O2 into the medium. Exposure to PM (20 μg/cm(2) ) increased H2 O2 levels significantly (94.6 ± 7.7 nM) compared to untreated controls (55.8 ± 4.0 nM; p = 0.001). PM-induced H2 O2 production was partially inhibited by DPI (80.1 ± 3.8nM), indicating that cellular NADPH oxidase may be a primary source of H2 O2 production. Exposure to PM increased IL-8 levels in a dose-dependent fashion (control = 2301 ± 412 MFI; 20 μg/cm(2) = 5002 ± 1327 MFI; 40 μg/cm(2) = 8219 ± 1090 MFI; p = 0.022).

Conclusion: PM increases the quantity of H2 O2 released by nasal epithelial cells, indicating that PM can contribute to oxidative stress in part by activating a normal cellular defense mechanism. Exposure to PM resulted in elevated IL-8 levels and mucin production in explants. Efforts to reduce airborne PM may lead to reduced H2 O2 and mucin production in sinonasal epithelium.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/alr.21413DOI Listing
December 2014

Thapsigargin blocks Pseudomonas aeruginosa homoserine lactone-induced apoptosis in airway epithelia.

Am J Physiol Cell Physiol 2014 May 5;306(9):C844-55. Epub 2014 Mar 5.

Department of Molecular and Cell Biology, University of California, Berkeley, California; and.

Pseudomonas aeruginosa secretes N-(3-oxododecanoyl)-homoserine lactone (C12) as a quorum-sensing molecule to regulate gene expression. Micromolar concentrations are found in the airway surface liquid of infected lungs. Exposure of the airway surface to C12 caused a loss of transepithelial resistance within 1 h that was accompanied by disassembly of tight junctions, as indicated by relocation of the tight junction protein zonula occludens 1 from the apical to the basolateral pole and into the cytosol of polarized human airway epithelial cell cultures (Calu-3 and primary tracheal epithelial cells). These effects were blocked by carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone, a pan-caspase blocker, indicating that tight junction disassembly was an early event in C12-triggered apoptosis. Short-duration (10 min) pretreatment of airway epithelial (Calu-3 and JME) cells with 1 μM thapsigargin (Tg), an inhibitor of Ca(2+) uptake into the endoplasmic reticulum (ER), was found to be protective against the C12-induced airway epithelial barrier breakdown and also against other apoptosis-related effects, including shrinkage and fragmentation of nuclei, activation of caspase 3/7 (the executioner caspase in apoptosis), release of ER-targeted redox-sensitive green fluorescent protein into the cytosol, and depolarization of mitochondrial membrane potential. Pretreatment of Calu-3 airway cell monolayers with BAPTA-AM [to buffer cytosolic Ca(2+) concentration (Cacyto)] or Ca(2+)-free solution + BAPTA-AM reduced C12 activation of apoptotic events, suggesting that C12-triggered apoptosis may involve Ca(2+). Because C12 and Tg reduced Ca(2+) concentration in the ER and increased Cacyto, while Tg increased mitochondrial Ca(2+) concentration (Camito) and C12 reduced Camito, it is proposed that Tg may reduce C12-induced apoptosis in host cells not by raising Cacyto, but by preventing C12-induced decreases in Camito.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1152/ajpcell.00002.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4010806PMC
May 2014

Expression of dual oxidases and secreted cytokines in chronic rhinosinusitis.

Int Forum Allergy Rhinol 2013 May 21;3(5):376-83. Epub 2012 Dec 21.

Division of Rhinology, Department of Otolaryngology-Head and Neck Surgery, Stanford University School of Medicine, Stanford, CA, USA.

Background: The airway epithelium generates reactive oxygen species (ROS) as a first line of defense. Dual oxidases (DUOX1 and DUOX2) are the H2 O2 -producing isoforms of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family in the airway epithelium. The purpose of this study was to explore the molecular expression, function, and regulation of DUOXs in chronic rhinosinusitis (CRS).

Methods: Human nasal tissue samples and nasal secretions were collected from 3 groups of patients undergoing sinus surgery (normal, n = 7; CRS with polyposis [CRSwP], n = 6; CRS without polyposis [CRSsP], n = 6). Nasal secretions were studied for cytokine and H2 O2 content. Tissue samples were used to determine DUOX mRNA and protein expression.

Results: DUOX1 mRNA level (80.7 ± 60.5) was significantly increased in CRSwP compared to normal (2.7 ± 1.2) and CRSsP (2.3 ± 0.5, p = 0.042). DUOX2 mRNA levels were increased in both CRSwP (18.6 ± 9.9) and CRSsP (4.0 ± 1.3) compared to normal (1.1 ± 0.3; p = 0.008). DUOX protein was found in the apical portion of the nasal epithelium and protein expression was increased in CRSwP and CRSsP. H2 O2 production was significantly higher in CRSwP (160.9 ± 59.4 nM) and CRSsP (81.7 ± 5.6 nM) compared to normal (53.5 ± 11.5 nM, p = 0.032). H2 O2 content of nasal secretions correlated tightly with DUOX expression (p < 0.001). Cytokines (eotaxin, monokine-induced by interferon γ [MIG], tumor necrosis factor [TNF]-α, interleukin [IL]-8) showed significantly higher levels in nasal secretions from CRSwP compared to normal (p < 0.05). Levels of eotaxin, MIG, and TNF-α correlated closely with DUOX expression.

Conclusion: DUOX1 and DUOX2 were identified as factors upregulated in CRS. Close correlations between DUOX expression and H2 O2 release, and correlation between key inflammatory cytokines and DUOX expression, indicate DUOX in the inflammatory response in CRS.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/alr.21133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4028033PMC
May 2013

Pseudomonas aeruginosa biofilm-associated homoserine lactone C12 rapidly activates apoptosis in airway epithelia.

Cell Microbiol 2012 May 9;14(5):698-709. Epub 2012 Feb 9.

Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3200, USA.

Pseudomonas aeruginosa (PA) forms biofilms in lungs of cystic fibrosis (CF) patients, a process regulated by quorum-sensing molecules including N-(3-oxododecanoyl)-l-homoserine lactone (C12). C12 (10-100 µM) rapidly triggered events commonly associated with the intrinsic apoptotic pathway in JME (CF ΔF508CFTR, nasal surface) epithelial cells: depolarization of mitochondrial (mito) membrane potential (Δψ(mito)) and release of cytochrome C (cytoC) from mitos into cytosol and activation of caspases 3/7, 8 and 9. C12 also had novel effects on the endoplasmic reticulum (release of both Ca(2+) and ER-targeted GFP and oxidized contents into the cytosol). Effects began within 5 min and were complete in 1-2 h. C12 caused similar activation of caspases and release of cytoC from mitos in Calu-3 (wtCFTR, bronchial gland) cells, showing that C12-triggered responses occurred similarly in different airway epithelial types. C12 had nearly identical effects on three key aspects of the apoptosis response (caspase 3/7, depolarization of Δψ(mito) and reduction of redox potential in the ER) in JME and CFTR-corrected JME cells (adenoviral expression), showing that CFTR was likely not an important regulator of C12-triggered apoptosis in airway epithelia. Exposure of airway cultures to biofilms from PAO1wt caused depolarization of Δψ(mito) and increases in Ca(cyto) like 10-50 µM C12. In contrast, biofilms from PAO1ΔlasI (C12 deficient) had no effect, suggesting that C12 from P. aeruginosa biofilms may contribute to accumulation of apoptotic cells that cannot be cleared from CF lungs. A model to explain the effects of C12 is proposed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1462-5822.2012.01753.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4112999PMC
May 2012

Acid and base secretion in freshly excised nasal tissue from cystic fibrosis patients with ΔF508 mutation.

Int Forum Allergy Rhinol 2011 Mar-Apr;1(2):123-7

Division of Rhinology, Department of Otolaryngology–Head and Neck Surgery, Stanford University School of Medicine, Stanford, CA, USA.

Background: Cystic fibrosis (CF) is caused by a misfunctional CF transmembrane conductance regulator (CFTR) protein, which is believed to contributes to the regulation of the airway surface liquid (ASL) pH. This study investigated acid and base secretion in freshly excised human nasal tissues from CF patients homozygous for the ΔF508 mutation.

Methods: Human nasal mucosa was collected during sinus surgery and investigated in Ussing chambers. Mucosal equilibrium pH values and rate of acid and base secretion were determined using the pH-stat technique.

Results: The equilibrium pH of nasal epithelia from ΔF508 CF patients with chronic rhinosinusitis (CRS) was pH = 7.08 ± 0.09 and was significantly lower compared to nasal epithelia from CRS patients without CF (pH = 7.33 ± 0.06) and normal subjects (pH = 7.34 ± 0.08, n = 6). The rate of base secretion in CF nasal tissues was 11.8 ± 2.4 nmol · min(−1) · cm(−2), which was significantly lower than normal (57.2 ± 9.2 nmol · min(−1) · cm(−2)). The HCO3(−) secretory rate was further increased by forskolin by 16.1% in normal, but not in CF tissues.

Conclusion: Our data suggests that CF patients exhibited significantly lower base secretion by the nasal airway epithelium. It is possible that improper regulation of ASL pH in CF may negatively impact the innate host defense system.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/alr.20028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3199580PMC
May 2012

Vitamin E forms inhibit IL-13/STAT6-induced eotaxin-3 secretion by up-regulation of PAR4, an endogenous inhibitor of atypical PKC in human lung epithelial cells.

J Nutr Biochem 2012 Jun 20;23(6):602-8. Epub 2011 Jul 20.

Department of Foods and Nutrition, Purdue University, Stone Hall, West Lafayette, IN 47907, USA.

Eotaxin-3 (CCL-26), a potent chemokine for eosinophil recruitment and contributing significantly to the pathogenesis of asthma, is secreted by lung epithelial cells in response to T helper 2 cytokines including interleukin 13 (IL-13). Here we showed that vitamin E forms, but not their metabolites, differentially inhibited IL-13-stimulated generation of eotaxin-3 in human lung epithelial A549 cells. The relative inhibitory potency was γ-tocotrienol (γ-TE) (IC50 ~15 μM)>γ-tocopherol, δ-tocopherol (IC50 ~25-50 μM)>α-tocopherol. Consistent with suppression of eotaxin, γ-TE treatment impaired IL-13-induced phosphorylation of STAT6, the key transcription factor for activation of eotaxin expression, and consequently blocked IL-13-stimulated DNA-binding activity of STAT6. In search of the upstream target of γTE by using inhibitor and siRNA approaches, we discovered that the atypical protein kinase C (aPKC) signaling, instead of classical PKC, p38 MAPK, JNK or ERK, played a critical role in IL-13-stimulated eotaxin generation and STAT6 activation. While showing no obvious effect on aPKC expression or phosphorylation, γ-TE treatment resulted in increased expression of prostate-apoptosis-response 4 (PAR4), an endogenous negative regulator of aPKCs. Importantly, γ-TE treatment led to enhanced formation of aPKC/PAR4 complex that is known to reduce aPKC activity via protein-protein crosstalk. Our study demonstrated that γ-TE inhibited IL-13/STAT6-activated eotaxin secretion via up-regulation of PAR4 expression and enhancement of aPKC-PAR4 complex formation. These results support the notion that specific vitamin E forms may be useful anti-asthmatic agents.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jnutbio.2011.03.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3201713PMC
June 2012

Sensitivity of chloride efflux vs. transepithelial measurements in mixed CF and normal airway epithelial cell populations.

Cell Physiol Biochem 2010 4;26(6):983-90. Epub 2011 Jan 4.

Children's Hospital Oakland Research Institute, Oakland, USA.

Background/aims: While the Cl(-) efflux assays are relatively straightforward, their ability to assess the efficacy of phenotypic correction in cystic fibrosis (CF) tissue or cells may be limited. Accurate assessment of therapeutic efficacy, i.e., correlating wild type CF transmembrane conductance regulator (CFTR) levels with phenotypic correction in tissue or individual cells, requires a sensitive assay.

Methods: Radioactive chloride ((36)Cl) efflux was compared to Ussing chamber analysis for measuring cAMP-dependent Cl(-) transport in mixtures of human normal (16HBE14o-) and cystic fibrosis (CF) (CFTE29o- or CFBE41o-, respectively) airway epithelial cells. Cell mixtures with decreasing amounts of 16HBE14o- cells were evaluated.

Results: Efflux and Ussing chamber studies on mixed populations of normal and CF airway epithelial cells showed that, as the number of CF cells within the population was progressively increased, the cAMP-dependent Cl(-) decreased. The (36)Cl efflux assay was effective for measuring Cl(-) transport when ≥ 25% of the cells were normal. If < 25% of the cells were phenotypically wild-type (wt), the (36)Cl efflux assay was no longer reliable. Polarized CFBE41o- cells, also homozygous for the ΔF508 mutation, were used in the Ussing chamber studies. Ussing analysis detected cAMP-dependent Cl(-) currents in mixtures with ≥1% wild-type cells indicating that Ussing analysis is more sensitive than (36)Cl efflux analysis for detection of functional CFTR.

Conclusions: Assessment of CFTR function by Ussing analysis is more sensitive than (36)Cl efflux analysis. Ussing analysis indicates that cell mixtures containing 10% 16HBE14o- cells showed 40-50% of normal cAMP-dependent Cl(-) transport that drops off exponentially between 10-1% wild-type cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1159/000324011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3221260PMC
April 2011

Pseudomonas aeruginosa Homoserine lactone activates store-operated cAMP and cystic fibrosis transmembrane regulator-dependent Cl- secretion by human airway epithelia.

J Biol Chem 2010 Nov 25;285(45):34850-63. Epub 2010 Aug 25.

Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3200, USA.

The ubiquitous bacterium Pseudomonas aeruginosa frequently causes hospital-acquired infections. P. aeruginosa also infects the lungs of cystic fibrosis (CF) patients and secretes N-(3-oxo-dodecanoyl)-S-homoserine lactone (3O-C12) to regulate bacterial gene expression critical for P. aeruginosa persistence. In addition to its effects as a quorum-sensing gene regulator in P. aeruginosa, 3O-C12 elicits cross-kingdom effects on host cell signaling leading to both pro- or anti-inflammatory effects. We find that in addition to these slow effects mediated through changes in gene expression, 3O-C12 also rapidly increases Cl(-) and fluid secretion in the cystic fibrosis transmembrane regulator (CFTR)-expressing airway epithelia. 3O-C12 does not stimulate Cl(-) secretion in CF cells, suggesting that lactone activates the CFTR. 3O-C12 also appears to directly activate the inositol trisphosphate receptor and release Ca(2+) from the endoplasmic reticulum (ER), lowering [Ca(2+)] in the ER and thereby activating the Ca(2+)-sensitive ER signaling protein STIM1. 3O-C12 increases cytosolic [Ca(2+)] and, strikingly, also cytosolic [cAMP], the known activator of CFTR. Activation of Cl(-) current by 3O-C12 was inhibited by a cAMP antagonist and increased by a phosphodiesterase inhibitor. Finally, a Ca(2+) buffer that lowers [Ca(2+)] in the ER similar to the effect of 3O-C12 also increased cAMP and I(Cl). The results suggest that 3O-C12 stimulates CFTR-dependent Cl(-) and fluid secretion in airway epithelial cells by activating the inositol trisphosphate receptor, thus lowering [Ca(2+)] in the ER and activating STIM1 and store-operated cAMP production. In CF airways, where CFTR is absent, the adaptive ability to rapidly flush the bacteria away is compromised because the lactone cannot affect Cl(-) and fluid secretion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M110.167668DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2966100PMC
November 2010

A phospholipid-apolipoprotein A-I nanoparticle containing amphotericin B as a drug delivery platform with cell membrane protective properties.

Int J Pharm 2010 Oct 7;399(1-2):148-55. Epub 2010 Aug 7.

Children's Hospital Oakland Research Institute, Oakland, CA 94609, USA.

Amphotericin B (AMB), a potent antifungal agent, has been employed as an inhalable therapy for pulmonary fungal infections. We recently described a novel nano-sized delivery vehicle composed of phospholipid (PL) and apolipoprotein A-I, NanoDisk (ND), to which we added AMB as a payload (ND-AMB). The goal of the present study was to evaluate whether ND-AMB, compared to other formulations, preserves lung cell integrity in vitro, as AMB can be toxic to mammalian cells and reduce lung function when inhaled. Epithelial integrity was assessed by measuring K(+) ion flux across a model airway epithelium, Calu-3 cells. In this assay ND-AMB was at least 8-fold less disruptive than AMB/deoxycholate (DOC). Cell viability studies confirmed this observation. Unexpectedly, the ND vehicle restored the integrity of a membrane compromised by prior exposure to AMB. An alternative formulation of ND-AMB containing a high load of AMB per ND was not protective, suggesting that ND with a low ratio of AMB to PL can sequester additional AMB from membranes. ND-AMB also protected HepG2 cells from the cytotoxicity of AMB, as determined by cellular viability and lactate dehydrogenase (LDH) levels. This study suggests that ND-AMB may be safe for administration via inhalation and reveals a unique activity whereby ND-AMB protects lung epithelial membranes from AMB toxicity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ijpharm.2010.07.057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2946961PMC
October 2010

CFTR and calcium-activated chloride channels in primary cultures of human airway gland cells of serous or mucous phenotype.

Am J Physiol Lung Cell Mol Physiol 2010 Oct 30;299(4):L585-94. Epub 2010 Jul 30.

Children’s Hospital Oakland Research Institute, Oakland, California, USA.

Using cell culture models, we have investigated the relative importance of cystic fibrosis transmembrane conductance regulator (CFTR) and calcium-activated chloride channels (CaCC) in Cl secretion by mucous and serous cells of human airway glands. In transepithelial recordings in Ussing chambers, the CFTR inhibitor CFTR(inh)-172 abolished 60% of baseline Cl secretion in serous cells and 70% in mucous. Flufenamic acid (FFA), an inhibitor of CaCC, reduced baseline Cl secretion by ∼20% in both cell types. Methacholine and ATP stimulated Cl secretion in both cell types, which was largely blocked by treatment with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and partially by mucosal FFA or CFTR(inh)-172 with the exception of methacholine responses in mucous cells, which were not blocked by FFA and partially (∼60%) by CFTR(inh)-172. The effects of ionomycin on short-circuit current (I(sc)) were less than those of ATP or methacholine. Forskolin stimulated Cl secretion only if Cl in the mucosal medium was replaced by gluconate. In whole cell patch-clamp studies of single isolated cells, cAMP-induced Cl currents were ∼3-fold greater in serous than mucous cells. Ionomycin-induced Cl currents were 13 times (serous) or 26 times (mucous) greater than those generated by cAMP and were blocked by FFA. In serous cells, mRNA for transmembrane protein 16A (TMEM16A) was ∼10 times more abundant than mRNA for CFTR. In mucous cells it was ∼100 times more abundant. We conclude: 1) serous and mucous cells both make significant contributions to gland fluid secretion; 2) baseline Cl secretion in both cell types is mediated predominantly by CFTR, but CaCC becomes increasingly important after mediator-induced elevations of intracellular Ca; and 3) the high CaCC currents seen in patch-clamp studies and the high TMEM16A expression in intact polarized cells sheets are not reflected in transepithelial current recordings.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1152/ajplung.00421.2009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2957417PMC
October 2010

Characteristics of chloride transport in nasal mucosa from patients with primary ciliary dyskinesia.

Laryngoscope 2010 Jul;120(7):1460-4

Division of Rhinology, Department of Otolaryngology-Head and Neck Surgery, Stanford University School of Medicine, Stanford, California, USA.

Objectives/hypothesis: Primary ciliary dyskinesia (PCD) is an inherited disorder that produces lifelong difficulties with chronic airway inflammation. Little is known about the role of chronic airway inflammation on chloride ion transport properties in PCD. This study assessed the cyclic adenosine monophosphate (cAMP)-regulated chloride (Cl) ion transport properties of freshly excised nasal mucosa from PCD compared with normal and chronic rhinosinusitis (CRS).

Study Design: Electrophysiology study utilizing Ussing type hemi-chamber technique with three different types of nasal tissue (normal, CRS, PCD) obtained from patients during endoscopic surgery at a tertiary referral center.

Methods: Nasal tissues were examined under short-circuit conditions, and gradient-driven Cl currents were continuously recorded. The cAMP elevating agonist (forskolin) was added to stimulate cystic fibrosis transmembrane conductance regulator-mediated Cl secretion. To prevent misinterpretation of flux measurement, Cl transport inhibitors were used at the end of all experiments. Basal Cl currents (I(Cl)) and changes in I(Cl) to forskolin (DeltaI(Cl)) were compared between normal, CRS, and PCD nasal tissues.

Results: Forskolin stimulated Cl currents across all different types of nasal epithelia. The Cl secretory response was effectively blocked by the Cl ion transport inhibitors. I(Cl) were significantly higher in normals (155.0 +/- 9.3 microA/cm(2)) compared to CRS (79.1 +/- 15.0 microA/cm(2)) and PCD (70.9 +/- 20.4 microA/cm(2)) (P = .005). DeltaI(Cl) in CRS (14.8 +/- 2.3 microA/cm(2)) and PCD (12.2 +/- 2.4 microA/cm(2)) were markedly diminished compared to normals (28.3 +/- 4.7 microA/cm(2)) (P = .024).

Conclusions: PCD tissues were characterized by impaired I(Cl) and DeltaI(Cl). Both parameters were reduced by 54.3% and 56.9% in PCD when compared to normals.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/lary.20928DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3196355PMC
July 2010

Function of the HVCN1 proton channel in airway epithelia and a naturally occurring mutation, M91T.

J Gen Physiol 2010 Jul 14;136(1):35-46. Epub 2010 Jun 14.

Children's Hospital Oakland Research Institute, Oakland, CA 94609, USA.

Airways secrete considerable amounts of acid. In this study, we investigated the identity and the pH-dependent function of the apical H(+) channel in the airway epithelium. In pH stat recordings of confluent JME airway epithelia in Ussing chambers, Zn-sensitive acid secretion was activated at a mucosal threshold pH of approximately 7, above which it increased pH-dependently at a rate of 339 +/- 34 nmol x h(-1) x cm(-2) per pH unit. Similarly, H(+) currents measured in JME cells in patch clamp recordings were readily blocked by Zn and activated by an alkaline outside pH. Small interfering RNA-mediated knockdown of HVCN1 mRNA expression in JME cells resulted in a loss of H(+) currents in patch clamp recordings. Cloning of the open reading frame of HVCN1 from primary human airway epithelia resulted in a wild-type clone and a clone characterized by two sequential base exchanges (452T>C and 453G>A) resulting in a novel missense mutation, M91T HVCN1. Out of 95 human genomic DNA samples that were tested, we found one HVCN1 allele that was heterozygous for the M91T mutation. The activation of acid secretion in epithelia that natively expressed M91T HVCN1 required approximately 0.5 pH units more alkaline mucosal pH values compared with wild-type epithelia. Similarly, activation of H(+) currents across recombinantly expressed M91T HVCN1 required significantly larger pH gradients compared with wild-type HVCN1. This study provides both functional and molecular indications that the HVCN1 H(+) channel mediates pH-regulated acid secretion by the airway epithelium. These data indicate that apical HVCN1 represents a mechanism to acidify an alkaline airway surface liquid.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1085/jgp.200910379DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2894549PMC
July 2010

Regulated gene expression in cultured type II cells of adult human lung.

Am J Physiol Lung Cell Mol Physiol 2010 Jul 9;299(1):L36-50. Epub 2010 Apr 9.

Department of Pediatrics, University of California San Francisco, San Francisco, USA.

Alveolar type II cells have multiple functions, including surfactant production and fluid clearance, which are critical for lung function. Differentiation of type II cells occurs in cultured fetal lung epithelial cells treated with dexamethasone plus cAMP and isobutylmethylxanthine (DCI) and involves increased expression of 388 genes. In this study, type II cells of human adult lung were isolated at approximately 95% purity, and gene expression was determined (Affymetrix) before and after culturing 5 days on collagen-coated dishes with or without DCI for the final 3 days. In freshly isolated cells, highly expressed genes included SFTPA/B/C, SCGB1A, IL8, CXCL2, and SFN in addition to ubiquitously expressed genes. Transcript abundance was correlated between fetal and adult cells (r = 0.88), with a subset of 187 genes primarily related to inflammation and immunity that were expressed >10-fold higher in adult cells. During control culture, expression increased for 8.1% of expressed genes and decreased for approximately 4% including 118 immune response and 10 surfactant-related genes. DCI treatment promoted lamellar body production and increased expression of approximately 3% of probed genes by > or =1.5-fold; 40% of these were also induced in fetal cells. Highly induced genes (> or =10-fold) included PGC, ZBTB16, DUOX1, PLUNC, CIT, and CRTAC1. Twenty-five induced genes, including six genes related to surfactant (SFTPA/B/C, PGC, CEBPD, and ADFP), also had decreased expression during control culture and thus are candidates for hormonal regulation in vivo. Our results further define the adult human type II cell molecular phenotype and demonstrate that a subset of genes remains hormone responsive in cultured adult cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1152/ajplung.00427.2009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2904098PMC
July 2010

Proton secretion in freshly excised sinonasal mucosa from asthma and sinusitis patients.

Am J Rhinol Allergy 2009 Nov-Dec;23(6):e10-3

Division of Rhinology, Department of Otolaryngology-Head and Neck Surgery, Stanford University School of Medicine, Stanford, California, USA.

Background: Proton (H+) secretion and the HVCN1 H+ channel are part of the innate host defense mechanism of the airways. The objective of this study was to determine H+ secretion in asthmatic and nonasthmatic patients with chronic rhinosinusitis (CRS) in freshly excised human sinonasal tissue.

Methods: Nasal or sinus mucosa from subjects with three different conditions (normal, CRS, and CRS with asthma) was harvested during sinus surgery. The equilibrium pH and the rate of H+ secretion were measured in an Ussing chamber using the pH-stat titration technique.

Results: Nasal epithelia isolated from subjects with CRS and asthma had a mucosal equilibrium pH = 6.95 (n = 5), which was significantly lower than in normal subjects (7.35 +/- 0.21; n = 5) or from subjects with CRS without asthma (7.33 +/- 0.15 In = 5). Nasal epithelia from CRS with asthma (n = 5) secreted H+ at a rate of 135 +/- 46 nmol x min(-1) x cm(-2). This rate was significantly higher compared with normal (73 +/- 39 nmol x min(-1) x cm(-2); n = 8) or CRS without asthma (51 +/- 28 nmol x min(-1) x cm(-2); n = 7). Mucosal addition of the HVCN1 blocker ZnCl2 blocked H+ secretion by 70% in normal, 53% in CRS without asthma, and by 51% in CRS with asthma. In contrast, measures in sinus tissues were unaffected by the disease condition.

Conclusion: Freshly excised human nasal and sinus epithelia secrete acid. Nasal (but not sinus) tissues from asthmatic CRS patients showed lower mucosal pH values and higher rates of H+ secretion than CRS and normal subjects. The increased acid secretion might contribute to epithelial injury in CRS patients with asthma.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2500/ajra.2009.23.3389DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2888960PMC
June 2010

Effect of L-ascorbate on chloride transport in freshly excised sinonasal epithelia.

Am J Rhinol Allergy 2009 May-Jun;23(3):294-9

Division of Rhinology, Department of Otolaryngology-Head and Neck Surgery, Stanford University School of Medicine, Stanford, California, USA.

Background: Chronic rhinosinusitis (CRS) occurs at high frequency in patients with cystic fibrosis, suggesting that the cystic fibrosis transmembrane conductance regulator (CFTR) chloride (Cl) ion channel might be involved in the development of chronic sinusitis in the general population. CFTR Cl ion transport controls the hydration of mucosal surfaces and promotes effective mucociliary clearance. Altered ion transport and, hence, disrupted mucociliary function, could play a role in the pathogenesis of sinus disease. L-ascorbate is a metabolically active component of the nasal and tracheobronchial airway lining fluids and appears to serve as an important biological effector of CFTR-mediated chloride secretion. The purpose of this study was to determine the effects of L-ascorbate on Cl ion transport in freshly excised sinonasal epithelia from normal controls and patients with CRS.

Methods: Four different types of sinonasal tissue (normal sinus mucosa, sinus mucosa from CRS, normal nasal mucosa, nasal mucosa from CRS) were obtained during endoscopic sinus surgery and mounted on sliders with open areas of 0.03-0.71 cm2 between Ussing hemichambers. Short-circuit current (Isc) was continuously recorded, and a serosa-to-mucosa-directed Cl gradient was applied to increase the electrochemical driving force.

Results: L-ascorbate (500 microM) stimulated Cl currents (DeltaI(Cl), microA/cm2) across sinonasal epithelia from normal and CRS patients. The Cl secretory response to L-ascorbate was effectively blocked by the Cl ion transport inhibitors glibenclamide and bumetanide. A maximal dose of L-ascorbate (at 1 mM) stimulated 53-70% of Cl currents elicited by the cAMP agonist forskolin. CRS sinonasal tissue was characterized by impaired Cl secretory responses to L-ascorbate that were reduced by 33% in sinus epithelial tissue and by 70% in nasal epithelial tissue when compared with normal subjects. In nasal epithelial tissue from normal subjects, Cl secretion was approximately twofold increased when compared with sinus epithelial tissue. In contrast, nasal versus sinus epithelial tissue from CRS patients showed no differences.

Conclusion: Topical administration of L-ascorbate to freshly excised sinus and nasal mucosa enhances chloride secretion. Given that decreased CFTR-mediated Cl secretion may contribute to the development of CRS, L-ascorbate may offer potential as a therapeutic agent for the improvement of mucociliary clearance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2500/ajra.2009.23.3316DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3196350PMC
July 2009

Gamma-tocopherol attenuates ozone-induced exacerbation of allergic rhinosinusitis in rats.

Toxicol Pathol 2009 Jun 23;37(4):481-91. Epub 2009 Apr 23.

Department of Pathobiology and Diagnostic Investigation, Michigan State University, East Lansing, MI 48824, USA.

Compared to healthy subjects, individuals with allergic airway disease (e.g., asthma, allergic rhinitis) have enhanced inflammatory responses to inhaled ozone. We created a rodent model of ozone-enhanced allergic nasal responses in Brown Norway rats to test the therapeutic effects of the dietary supplement gamma-tocopherol (gammaT). Ovalbumin (OVA)-sensitized rats were intranasally challenged with 0% or 0.5% OVA (in saline) on Days 1 and 2, and then exposed to 0 or 1 ppm ozone (eight hours/day) on Days 4 and 5. Rats were also given 0 or 100 mg/kg gammaT (p.o., in corn oil) on days 2 through 5, beginning twelve hours after the last OVA challenge. On Day 6, nasal tissues were collected for histological evaluation and morphometric analyses of intraepithelial mucosubstances (IM) and eosinophilic inflammation. Nasal septal tissue was microdissected and analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) for mucin glycoprotein 5AC (MUC5AC) expression levels. Histological analysis revealed mild to moderate eosinophil influx in the mucosa lining the nasal airways and maxillary sinus of OVA-challenged rats (eosinophilic rhinosinusitis). Ozone exposure of allergic rats further increased eosinophils in the maxillary sinus (400%), nasolacrimal duct (250%), and proximal midseptum (150%). Storage of intraepithelial mucosubstances (IM) was not significantly affected by OVA challenge in filtered air-exposed rats, but it was increased by ozone in the septum (45%) and maxillary sinus (55%) of allergic compared to control rats. Treatment with gammaT attenuated the ozone/ OVA-induced synergistic increases in IM and mucosal eosinophils in both nasal and paranasal airways. gamma-Tocopherol also blocked OVA and ozone-induced MUC5AC gene expression. Together, these data describe a unique model of ozone enhancement of allergic rhinosinusitis and the novel therapeutic efficacy of a common supplement, gammaT, to inhibit ozone exacerbation of allergic airway responses.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1177/0192623309335630DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4465783PMC
June 2009

Activation of the CFTR Cl- channel by trimethoxyflavone in vitro and in vivo.

Cell Physiol Biochem 2008 9;22(5-6):685-92. Epub 2008 Dec 9.

Children's Hospital Oakland Research Institute, Oakland, CA 94609, USA.

The flavone apigenin has been previously selected as a potent pharmacological activator of the CFTR Cl(-) channel, however, its utility for the activation of CFTR in vivo is expected to be limited because flavonoids are readily metabolized. We therefore investigated the poorly metabolizable methylether of apigenin, 5,7,4'-trimethoxyflavone (TMF) as a CFTR activator using transepithelial short-circuit current measurements, whole cell and single cell patch clamp techniques, and nasal potential difference (PD) measurements. Transepithelial Cl(-) secretion by Calu-3 epithelia was stimulated by TMF with a halfmaximal concentration of 64+/-5 microM to 55+/-15% of maximal currents achieved by subsequent addition of cAMP agonist forskolin (10 microM). In forskolin-prestimulated tissues, TMF showed small effects and stimulated Cl(-) secretion by an additional 6%. Single channel and whole cell patch clamp techniques were used to verify these effects and identify CFTR as the target of TMF. TMF increased the open probability of silent CFTR (to 0.31+/-0.06) but showed small effects once CFTR had been prestimulated with forskolin. In nasal PD measurements in humans, perfusion of TMF onto the nasal mucosa activated nasal PD by -9.5+/-1.1 mV, which was 69% of the effect of TMF+isoproterenol (-13.8+/-3.9 mV). These data show that TMF is an activator of CFTR in both in vitro and in vivo assays that targets mainly the unstimulated CFTR.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1159/000185552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2820299PMC
February 2009

Oxidative stress caused by pyocyanin impairs CFTR Cl(-) transport in human bronchial epithelial cells.

Free Radic Biol Med 2008 Dec 23;45(12):1653-62. Epub 2008 Sep 23.

Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA 94720, USA.

Pyocyanin (N-methyl-1-hydroxyphenazine), a redox-active virulence factor produced by the human pathogen Pseudomonas aeruginosa, is known to compromise mucociliary clearance. Exposure of human bronchial epithelial cells to pyocyanin increased the rate of cellular release of H(2)O(2) threefold above the endogenous H(2)O(2) production. Real-time measurements of the redox potential of the cytosolic compartment using the redox sensor roGFP1 showed that pyocyanin (100 microM) oxidized the cytosol from a resting value of -318+/-5 mV by 48.0+/-4.6 mV within 2 h; a comparable oxidation was induced by 100 microM H(2)O(2). Whereas resting Cl(-) secretion was slightly activated by pyocyanin (to 10% of maximal currents), forskolin-stimulated Cl(-) secretion was inhibited by 86%. The decline was linearly related to the cytosolic redox potential (1.8% inhibition/mV oxidation). Cystic fibrosis bronchial epithelial cells homozygous for DeltaF508 CFTR failed to secrete Cl(-) in response to pyocyanin or H(2)O(2), indicating that these oxidants specifically target the CFTR and not other Cl(-) conductances. Treatment with pyocyanin also decreased total cellular glutathione levels to 62% and cellular ATP levels to 46% after 24 h. We conclude that pyocyanin is a key factor that redox cycles in the cytosol, generates H(2)O(2), depletes glutathione and ATP, and impairs CFTR function in Pseudomonas-infected lungs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.freeradbiomed.2008.09.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2628806PMC
December 2008

Cl transport in complemented CF bronchial epithelial cells correlates with CFTR mRNA expression levels.

Cell Physiol Biochem 2008 25;22(1-4):57-68. Epub 2008 Jul 25.

Children's Hospital Oakland Research Institute, Oakland, USA.

Little is known about the relationship between CF transmembrane conductance regulator (CFTR) gene expression and the corresponding transport of Cl. The phenotypic characteristics of polarized DeltaF508 homozygote CF bronchial epithelial (CFBE41o-) cells were evaluated following transfection with episomal expression vector containing either full-length (6.2kb) wild type (wt) and (4.7kb) DeltaF508CFTR cDNA. Forskolin-stimulated Cl secretion in two clones expressing the full-length wild type CFTR was assessed; clone c7-6.2wt gave 13.4+/-2.5 microA/cm(2) and clone c10-6.2wt showed 41.3+/-25.3 microA/cm(2). Another clone (c4-4.7DeltaF) complemented with the DeltaF508 CFTR cDNA showed high and stable expression of vector-derived DeltaF508 CFTR mRNA and a small cAMP-stimulated Cl current (4.7+/-0.7 microA/cm(2)) indicating DeltaF508CFTR trafficking to the plasma membrane at physiological temperatures. Vector-driven CFTR mRNA levels were 5-fold (c7-6.2wt), 14-fold (c10-6.2wt), and 27-fold (c7-4.7DeltaF) higher than observed in normal bronchial epithelial cells (16HBE14o-) endogenously expressing wtCFTR. Assessment of CFTR mRNA levels and CFTR function showed that cAMP-stimulated CFTR Cl currents were 33%, 167% and 24%, respectively, of those in 16HBE14o- cells. The data suggest that transgene expression needs to be significantly higher than endogenously expressed CFTR to restore functional wtCFTR Cl transport to levels sufficient to reverse CF pathology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1159/000149783DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2927120PMC
November 2008

Flagellin-stimulated Cl- secretion and innate immune responses in airway epithelia: role for p38.

Am J Physiol Lung Cell Mol Physiol 2008 Oct 25;295(4):L531-42. Epub 2008 Jul 25.

Dept. of Molecular and Cell Biology, Univ. of California-Berkeley, Berkeley, CA 94720-3200, USA.

Activation of an innate immune response in airway epithelia by the human pathogen Pseudomonas aeruginosa requires bacterial expression of flagellin. Addition of flagellin (10(-7) M) to airway epithelial cell monolayers (Calu-3, airway serous cell-like) increased Cl(-) secretion (I(Cl)) beginning after 3-10 min, reaching a plateau after 20-45 min at DeltaI(Cl) = 15-50 microA/cm(2). Similar, although 10-fold smaller, responses were observed in well-differentiated bronchial epithelial cultures. Flagellin stimulated I(Cl) in the presence of maximally stimulating doses of the purinergic agonist ATP, but had no effects following forskolin. IL-1beta (produced by both epithelia and neutrophils during infections) stimulated I(Cl) similar to flagellin. Flagellin-, IL-1beta-, ATP-, and forskolin-stimulated I(Cl) were inhibited by cystic fibrosis transmembrane conductance regulator (CFTR) blockers GlyH101, CFTRinh172, and glibenclamide. Neither flagellin nor IL-1beta altered transepithelial fluxes of membrane-impermeant dextran (10 kDa) or lucifer yellow (mol wt = 457), but both activated p38, NF-kappaB, and IL-8 secretion. Blockers of p38 (SB-202190 and SB-203580) reduced flagellin- and IL-1beta-stimulated I(Cl) by 33-50% but had smaller effects on IL-8 and NF-kappaB. It is concluded that: 1) flagellin and IL-1beta activated p38, NF-kappaB, IL-8, and CFTR-dependent anion secretion without altering tight junction permeability; 2) p38 played a role in regulating I(Cl) and IL-8 but not NF-kappaB; and 3) p38 was more important in flagellin- than IL-1beta-stimulated responses. During P. aeruginosa infections, flagellin and IL-1beta are expected to increase CFTR-dependent ion and fluid flow into and bacterial clearance from the airways. In cystic fibrosis, the secretory response would be absent, but activation of p38, NF-kappaB, and IL-8 would persist.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1152/ajplung.90292.2008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2575945PMC
October 2008
-->