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Scott H Randell

Publications and source records attributed to Scott H Randell.

At least 19 recordsLinked to original sources
PubMed2025

TGF-β Receptor-dependent Tissue Factor Release and Proteomic Profiling of Extracellular Vesicles from Mechanically Compressed Human Bronchial Epithelial Cells.

In asthma, tissue factor (TF) concentrations are elevated in the lung. In our previous studies using mechanically compressed human bronchial epithelial (HBE) cells, which are a well-defined in vitro model of bronchoconstriction during asthma exacerbations, we detected TF within extracellular vesicles (EVs) released from compressed HBE cells. Here, to better characterize the potential role of this mechanism in asthma, we tested the extent to which the transcriptional regulation of epithelial cell-derived TF varied between donors with and without asthma. Using RNA in situ hybridization, we detected epithelial expression of F3, the TF protein-encoding gene, in human airways. Next, to determine the role of TGF-β receptor (TGF-βR) in the regulation of TF, we exposed well-differentiated HBE cells to mechanical compression in the presence or absence of a pharmacological inhibitor of TGF-βR. Furthermore, to identify the protein cargo of EVs released from HBE cells, we used tandem mass tag mass spectrometry. Our findings revealed significantly higher F3 expression in the airways of patients with asthma compared with healthy control subjects. However, we observed no differences in F3 expression or TF release between asthmatic and nonasthmatic HBE cells, both at baseline and after compression. Mechanistically, compression-induced F3 expression in HBE cells depended on TGF-βR. Our proteomic analysis identified 22 differentially released proteins in EVs, with higher concentrations in compressed cells compared with controls. Gene Ontology analysis indicates that these proteins are involved in diverse biological processes, highlighting a potential role for epithelial cell-derived EVs during asthma exacerbations.

Humans↗
PubMed2022

An epithelial-immune circuit amplifies inflammasome and IL-6 responses to SARS-CoV-2.

Elevated levels of cytokines IL-1β and IL-6 are associated with severe COVID-19. Investigating the underlying mechanisms, we find that while primary human airway epithelia (HAE) have functional inflammasomes and support SARS-CoV-2 replication, they are not the source of IL-1β released upon infection. In leukocytes, the SARS-CoV-2 E protein upregulates inflammasome gene transcription via TLR2 to prime, but not activate, inflammasomes. SARS-CoV-2-infected HAE supply a second signal, which includes genomic and mitochondrial DNA, to stimulate leukocyte IL-1β release. Nuclease treatment, STING, and caspase-1 inhibition but not NLRP3 inhibition blocked leukocyte IL-1β release. After release, IL-1β stimulates IL-6 secretion from HAE. Therefore, infection alone does not increase IL-1β secretion by either cell type. Rather, bi-directional interactions between the SARS-CoV-2-infected epithelium and immune bystanders stimulates both IL-1β and IL-6, creating a pro-inflammatory cytokine circuit. Consistent with these observations, patient autopsy lungs show elevated myeloid inflammasome gene signatures in severe COVID-19.

Humans↗
PubMed2006

Lung development and repair: contribution of the ciliated lineage.

The identity of the endogenous epithelial cells in the adult lung that are responsible for normal turnover and repair after injury is still controversial. In part, this is due to a paucity of highly specific genetic lineage tools to follow efficiently the fate of the major epithelial cell populations: the basal, secretory, ciliated, neuroendocrine, and alveolar cells. As part of a program to address this problem we have used a 1-kb FOXJ1 promoter to drive CreER in the ciliated cells of the embryonic and adult lung. Analysis of FOXJ1-GFP transgenic lungs shows that labeled cells appear in a proximal-distal pattern during embryogenesis and that the promoter drives expression in all ciliated cells. Using FOXJ1CreER adult mice, we have followed the fate of ciliated cells after epithelial injury by naphthalene or sulfur dioxide. From quantitative analysis and confocal microscopy we conclude that ciliated cells transiently change their morphology in response to lung injury but do not proliferate or transdifferentiate as part of the repair process.

Animals↗
PubMed2006

The lactoperoxidase system links anion transport to host defense in cystic fibrosis.

Chronic respiratory infections in cystic fibrosis result from CFTR channel mutations but how these impair antibacterial defense is less clear. Airway host defense depends on lactoperoxidase (LPO) that requires thiocyanate (SCN-) to function and epithelia use CFTR to concentrate SCN- at the apical surface. To test whether CFTR mutations result in impaired LPO-mediated host defense, CF epithelial SCN- transport was measured. CF epithelia had significantly lower transport rates and did not accumulate SCN- in the apical compartment. The lower CF [SCN-] did not support LPO antibacterial activity. Modeling of airway LPO activity suggested that reduced transport impairs LPO-mediated defense and cannot be compensated by LPO or H2O2 upregulation.

Anions↗
PubMed2006

Custom-designed wells and flow chamber for exposing air-liquid interface cultures to wall shear stress.

The effects of mechanical stimuli such as wall shear stresses (WSS) on cellular processes have been studied in vitro in numerous cell types. In order to study WSS effects on cells cultured under air-liquid interface (ALI) conditions, we developed a custom-designed well that can be disassembled into sub-units that allow installation of the cultured cells in a flow chamber, and then, re-assembled for further incubation or biological tests. Human nasal epithelial cells were cultured in the new wells under ALI conditions, and some of their biological characteristics were compared with those cultured in commercial Millicells. The cultured cells from both types of wells secreted the same amount of mucin and had similar cytoskeletal structures. Preliminary WSS experiments demonstrated the advantage of the new wells and provided initial indications that WSS affects the performance of ALI cultured respiratory epithelial cells.

Cell Culture Techniques↗
PubMed2006

Smoking is associated with increased telomerase activity in short-term cultures of human bronchial epithelial cells.

Telomerase plays an important role in the maintenance of telomere ends in normal and tumor cells and ectopic expression can immortalize human bronchial epithelial (HBE) cells. We assessed telomerase activation, growth properties and methylation status in the hTERT promoter in a panel of HBE cell cultures in relation to smoking and previous lung cancer history. HBE cells were obtained from a total of 26 subjects, six of whom were lifelong non-smokers, while 20 subjects had a smoking history, including seven who had lung carcinoma. Telomerase activity was determined using the telomeric repeat amplification protocol (TRAP). Maximum passage number and time to senescence were also determined through extended culturing. The distribution of the telomerase activity between ever-smokers and never-smokers was significantly different (P=0.03, F-test), and there was a strong correlation between telomerase activity and the number of pack-years smoked (P=0.0012, F-test for slope). A small difference in telomerase activity was observed according to lung cancer status (P=0.02, F-test). Telomerase activity was not correlated with maximum passage number after extended culturing or with time to senescence. None of the HBE cultures demonstrated methylation of the hTERT promoter. Our results indicate an association between tobacco carcinogen exposure and telomerase activity in normal bronchial epithelium, although a causative role of tobacco smoking in the (re)activation of telomerase can not be proven. An increase in telomerase activity in normal bronchial epithelium might extend the lifespan of cells at risk for malignant transformation, and thus contribute to lung carcinogenesis.

Adolescent↗
PubMed2006

Circulating progenitor epithelial cells traffic via CXCR4/CXCL12 in response to airway injury.

Recipient airway epithelial cells are found in human sex-mismatched lung transplants, implying that circulating progenitor epithelial cells contribute to the repair of the airway epithelium. Markers of circulating progenitor epithelial cells and mechanisms for their trafficking remain to be elucidated. We demonstrate that a population of progenitor epithelial cells exists in the bone marrow and the circulation of mice that is positive for the early epithelial marker cytokeratin 5 (CK5) and the chemokine receptor CXCR4. We used a mouse model of sex-mismatched tracheal transplantation and found that CK5+ circulating progenitor epithelial cells contribute to re-epithelialization of the airway and re-establishment of the pseudostratified epithelium. The presence of CXCL12 in tracheal transplants provided a mechanism for CXCR4+ circulating progenitor epithelial cell recruitment to the airway. Depletion of CXCL12 resulted in the epithelium defaulting to squamous metaplasia, which was derived solely from the resident tissue progenitor epithelial cells. Our findings demonstrate that CK5+CXCR4+ cells are markers of circulating progenitor epithelial cells in the bone marrow and circulation and that CXCR4/CXCL12-mediated recruitment of circulating progenitor epithelial cells is necessary for the re-establishment of a normal pseudostratified epithelium after airway injury. These findings support a novel paradigm for the development of squamous metaplasia of the airway epithelium and for developing therapeutic strategies for circulating progenitor epithelial cells in airway diseases.

Animals↗
PubMed2006

Potential role of abnormal ion transport in the pathogenesis of chronic sinusitis.

OBJECTIVES: To create well-differentiated cultures of normal and chronic sinusitis paranasal sinus epithelial cells and to compare their electrophysiologic properties. DESIGN: In vitro investigation using primary sinus epithelial cells, initially cultured on plastic tissue culture dishes. Cells were characterized by means of immunocytochemical analysis and then passaged to air-liquid interface culture conditions. The morphologic features of air-liquid interface cultures were assessed using light and electron microscopy. Epithelial Na(+) channel, Na(+)-K(+)-2Cl(-) cotransporter, cystic fibrosis transmembrane conductance regulator, and Ca(2+)-activated Cl(-) channel function were investigated in Ussing chambers. SUBJECTS: Specimens were obtained from 15 patients undergoing transsphenoidal pituitary procedures, tumor removal, or trauma repair and from 9 patients with chronic sinusitis. RESULTS: After culture at an air-liquid interface for 21 days, the epithelium was pseudostratified and contained basal, mucous secretory, and ciliated cells. There were no detectable morphologic differences between normal and chronic sinusitis cells. In cultures of normal cells, median basal short circuit current was 4.7 microA/cm(2), and Na(+) transport, defined as the amiloride hydrochloride-sensitive component, was approximately 20% of the total. Basal and amiloride-sensitive short circuit currents were greater in cultures of chronic sinusitis cells. Basal short circuit currents in both types of cultures were insensitive to the Cl(-) transport inhibitor bumetanide, but all responded to forskolin or uridine triphosphate. After amiloride pretreatment, forskolin and uridine triphosphate responses were greater in chronic sinusitis cells. CONCLUSIONS: We established methods for well-differentiated sinus epithelial cultures. The cells exhibited Na(+) absorption and Cl(-) secretion, and elevated rates of ion transport may be pathophysiologically relevant in chronic sinusitis.

Cells, Cultured↗
PubMed2006

Airway epithelial stem cells and the pathophysiology of chronic obstructive pulmonary disease.

Characteristic pathologic changes in chronic obstructive pulmonary disease (COPD) include an increased fractional volume of bronchiolar epithelial cells, fibrous thickening of the airway wall, and luminal inflammatory mucus exudates, which are positively correlated with airflow limitation and disease severity. The mechanisms driving general epithelial expansion, mucous secretory cell hyperplasia, and mucus accumulation must relate to the effects of initial toxic exposures on patterns of epithelial stem and progenitor cell proliferation and differentiation, eventually resulting in a self-perpetuating, and difficult to reverse, cycle of injury and repair. In this review, current concepts in stem cell biology and progenitor-progeny relationships related to COPD are discussed, focusing on the factors, pathways, and mechanisms leading to mucous secretory cell hyperplasia and mucus accumulation in the airways. A better understanding of alterations in airway epithelial phenotype in COPD will provide a logical basis for novel therapeutic approaches.

Bronchi↗
PubMed2005

Trends in lung pH and PO2 after circulatory arrest: implications for non-heart-beating donors and cell culture models of lung ischemia-reperfusion injury.

BACKGROUND: A better understanding of lung tissue environment after circulatory arrest would allow more accurate cell culture models to study ischemia-reperfusion lung injury and facilitate retrieval of lungs from non-heart-beating donors. METHODS: To establish the time course of changes in pH and PO2 in lung tissue after circulatory arrest, 12 Sprague-Dawley rats were sacrificed. After sternotomy, pH and PO2 microelectrodes were inserted into the lungs and sealed by application of Focal Seal. Rats were maintained at normothermia (37 degrees C). Two groups of rats (n = 6 atelectatic, n = 6 room air-inflated) were followed for 4 hours after arrest, when lung tissue adenine nucleotide levels were measured by chromatography and cell death was quantified by trypan blue exclusion. Human umbilical vein endothelial cells underwent simulated ischemia and 6 hours of cold storage by replacement of culture medium with cold Perfadex. Interleukin (IL)-6 and IL-8 were measured in medium 21 hours later by enzyme-linked immunosorbent assay (ELISA). RESULTS: In both groups of rats, lung [H+] increased linearly with time. In atelectatic lungs, PO2 fell precipitously, but in inflated lungs, PO2 decreased linearly for 60 to 75 minutes post-mortem and then became stable. After 4 hours at 37 degrees C, most parenchymal lung cells were dead in both groups. IL-6 and IL-8 levels increased significantly in medium of cultured endothelial cells subjected to cold storage without hypoxia. CONCLUSIONS: In room-air-inflated lungs maintained at 37 degrees C, oxygen consumption continues for at least 1 hour after circulatory arrest. Warm atelectasis is poorly tolerated. Hypothermic storage can induce elaboration of cytokines by endothelial cells in the absence of hypoxia.

Animals↗
PubMed2005

SERCA pump inhibitors do not correct biosynthetic arrest of deltaF508 CFTR in cystic fibrosis.

Deletion of phenylalanine 508 (deltaF508) accounts for nearly 70% of all mutations that occur in the cystic fibrosis transmembrane conductance regulator (CFTR). The deltaF508 mutation is a class II processing mutation that results in very little or no mature CFTR protein reaching the apical membrane and thus no cAMP-mediated Cl- conductance. Therapeutic strategies have been developed to enhance processing of the defective deltaF508 CFTR molecule so that a functional cAMP-regulated Cl- channel targets to the apical membrane. Sarcoplasmic/endoplasmic reticulum calcium (SERCA) inhibitors, curcumin and thapsigargin, have been reported to effectively correct the CF ion transport defects observed in the deltaF508 CF mice. We investigated the effect of these compounds in human airway epithelial cells to determine if they could induce deltaF508 CFTR maturation, and Cl- secretion. We also used Baby Hamster Kidney cells, heterologously expressing deltaF508 CFTR, to determine if SERCA inhibitors could interfere with the interaction between calnexin and CFTR and thereby correct the deltaF508 CFTR misfolding defect. Finally, at the whole animal level, we tested the ability of curcumin and thapsigargin to (1) induce Cl- secretion and reduce hyperabsorption of Na+ in the nasal epithelia of the deltaF508 mouse in vivo, and (2) induce Cl- secretion in intestine (jejunum and distal colon) and the gallbladder of the deltaF508 CF mouse. We conclude that curcumin and thapsigargin failed to induce maturation of deltaF508 CFTR, or induce Cl- secretion, as measured by biochemical and electrophysiologic techniques in a variety of model systems ranging from cultured cells to in vivo studies.

Animals↗
PubMed2005

Reduced three-dimensional motility in dehydrated airway mucus prevents neutrophil capture and killing bacteria on airway epithelial surfaces.

Cystic fibrosis (CF) lung disease is characterized by persistent lung infection. Thickened (concentrated) mucus in the CF lung impairs airway mucus clearance, which initiates bacterial infection. However, airways have other mechanisms to prevent bacterial infection, including neutrophil-mediated killing. Therefore, we examined whether neutrophil motility and bacterial capture and killing functions are impaired in thickened mucus. Mucus of three concentrations, representative of the range of normal (1.5 and 2.5% dry weight) and CF-like thickened (6.5%) mucus, was obtained from well-differentiated human bronchial epithelial cultures and prepared for three-dimensional studies of neutrophil migration. Neutrophil chemotaxis in the direction of gravity was optimal in 1.5% mucus, whereas 2.5% mucus best supported neutrophil chemotaxis against gravity. Lateral chemokinetic movement was fastest on airway epithelial surfaces covered with 1.5% mucus. In contrast, neutrophils exhibited little motility in any direction in thickened (6.5%) mucus. In in vivo models of airway mucus plugs, neutrophil migration was inhibited by thickened mucus (CF model) but not by normal concentrations of mucus ("normal" model). Paralleling the decreased neutrophil motility in thickened mucus, bacterial capture and killing capacity were decreased in CF-like thickened mucus. Similar results with each mucus concentration were obtained with mucus from CF cultures, indicating that inhibition of neutrophil functions was mucus concentration dependent not CF source dependent. We conclude that concentrated ("thick") mucus inhibits neutrophil migration and killing and is a key component in the failure of defense against chronic airways infection in CF.

Bronchi↗
PubMed2005

Airway epithelial cell tolerance to Pseudomonas aeruginosa.

BACKGROUND: The respiratory tract epithelium is a critical environmental interface that regulates inflammation. In chronic infectious airway diseases, pathogens may permanently colonize normally sterile luminal environments. Host-pathogen interactions determine the intensity of inflammation and thus, rates of tissue injury. Although many cells become refractory to stimulation by pathogen products, it is unknown whether the airway epithelium becomes either tolerant or hypersensitive in the setting of chronic infection. Our goals were to characterize the response of well-differentiated primary human tracheobronchial epithelial cells to Pseudomonas aeruginosa, to understand whether repeated exposure induced tolerance and, if so, to explore the mechanism(s). METHODS: The apical surface of well-differentiated primary human tracheobronchial epithelial cell cultures was repetitively challenged with Pseudomonas aeruginosa culture filtrates or the bacterial media control. Toxicity, cytokine production, signal transduction events and specific effects of dominant negative forms of signaling molecules were examined. Additional experiments included using IL-1beta and TNFalpha as challenge agents, and performing comparative studies with a novel airway epithelial cell line. RESULTS: An initial challenge of the apical surface of polarized human airway epithelial cells with Pseudomonas aeruginosa culture filtrates induced phosphorylation of IRAK1, JNK, p38, and ERK, caused degradation of IkappaBalpha, generation of NF-kappaB and AP-1 transcription factor activity, and resulted in IL-8 secretion, consistent with activation of the Toll-like receptor signal transduction pathway. These responses were strongly attenuated following a second Pseudomonas aeruginosa, or IL-1beta, but not TNFalpha, challenge. Tolerance was associated with decreased IRAK1 protein content and kinase activity and dominant negative IRAK1 inhibited Pseudomonas aeruginosa--stimulated NF-kappaB transcriptional activity. CONCLUSION: The airway epithelial cell response to Pseudomonas aeruginosa entails adaptation and tolerance likely mediated, in part, by down-regulation of IRAK1.

Cell Line↗
PubMed2005

Chromosomal abnormalities in bronchial epithelium from smokers, nonsmokers, and lung cancer patients.

The identification of individuals who are at greatest risk of developing lung cancer would greatly improve diagnosis and possibly lead to early treatment. To study the use of karyotypes for this purpose, we used short-term human bronchial epithelial (hBE) cell cultures from nonsmokers, smokers, and lung cancer patients. Twenty-five metaphases were scored for hBE cell cultures obtained from 32 patients: 8 were nonsmokers, and 24 had a history of smoking (of whom 11 had had lung cancer surgery). The number of abnormal metaphases ranged from 0 to 4 per cell culture. No overall differences in the number of abnormal metaphases were observed between nonsmokers and smokers or between lung cancer patients and non-lung cancer patients. The most commonly observed abnormalities were structural changes in chromosome 1 (six cultures), loss of chromosome 17 (six cultures), and trisomy of chromosome 20 (three cultures). These specific alterations were found almost exclusively in patients with a history of tobacco smoking. The results did not indicate that general chromosomal abnormalities are a useful marker for tobacco smoke exposure or cancer risk.

Adolescent↗
PubMed2005

Cytokine amplification by respiratory syncytial virus infection in human nasal epithelial cells.

OBJECTIVES: Respiratory syncytial virus (RSV) is an important cause of upper respiratory infections and is known to play a causal role in the pathogenesis of rhinitis, sinusitis, acute otitis media, and pneumonia. RSV appears to prime the respiratory tract to secondary inciting events, such as bacterial or antigen challenges. To study the proinflammatory priming effects of RSV infection, cytokine expression was measured in well-differentiated human nasal epithelial cells (WD-NE) after RSV infection alone or after subsequent tumor necrosis factor (TNF)-alpha stimulation. STUDY DESIGN: In vitro investigation. METHODS: Human nasal epithelial cells were obtained from surgical specimens and allowed to differentiate in air-liquid interface cultures until ciliation and mucus production were evident. Two experimental paradigms were used. First, accumulation of cytokines in the media was measured by real-time, quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay after RSV infection alone. In the second set of experiments, cytokines were also measured after TNF-alpha stimulation in both RSV-infected and uninfected cultures. RESULTS: RSV infection of WD-NE resulted in significant accumulations of interleukin (IL)-6, IL-8, and RANTES when compared with findings in control samples. Real-time, quantitative RT-PCR demonstrated significant increases in IL-8 gene expression following RSV infection when compared to controls. Secondary TNF-alpha stimulation following well-established (i.e., 72 h) RSV infection induced marked increases in IL-6, IL-8, and RANTES when compared with both RSV infection alone and TNF-alpha stimulation alone. CONCLUSIONS: These findings suggest that RSV infection primes nasal epithelial cells to secondary proinflammatory challenge, resulting in a hyperimmune response. RSV-induced priming of a hyperimmune response may be important in the pathogenesis of sinusitis, acute otitis media, and pneumonia.

Chemokine CCL5↗
PubMed2005

Calcineurin inhibitor effects on growth and phenotype of human airway epithelial cells in vitro.

Calcineurin inhibitors (CIs) cyclosporin and tacrolimus form the basis for immunosuppression in lung transplantation, yet also exert biological effects on nonlymphoid tissue. With the advent of inhaled cyclosporin, we hypothesize that the airway epithelium is also subject to CI effects at high doses. The aim of this study was to identify human tracheobronchial epithelial cell (hTBEC) calcineurin gene expression and quantify effects of CIs on hTBEC growth, interleukin-1-beta stimulated IL-8 production and hTBEC phenotype. Cyclophillin B and FK-associated binding protein, calcineurin A (alpha and beta), and NFATC3 and NFAT5 were detected in hTBEC cultures by RT-PCR. Acute and chronic cyclosporine treatment 1000 ng/mL significantly inhibited hTBEC proliferation, while tacrolimus did not (range of 10 ng/mL to 1000 ng/mL for acute treatment, 50 ng/mL for chronic treatment). Cyclosporin at 10,000 ng/mL significantly increased LDH release by well-differentiated hTBEC cultures (n = 6) and trended towards significance at 1000 ng/mL. IL1-beta stimulated IL-8 production was significantly increased in rapidly growing hTBEC cultures (n = 8) treated with cyclosporin (p = 0.049). Prolonged treatment of well-differentiated hTBECs at air-liquid-interface (ALI) with cyclosporin 1000 ng/mL significantly reduced intact multilayered mucociliary epithelium (p = 0.009). Inhibition of hTBEC growth, stimulation of IL-8 production and long-term effects on mucociliary phenotype and intact multi-layered epithelium suggest that cyclosporin may have a direct toxic effect on airway epithelium after transplantation.

Bronchi↗