37 results on '"Sutanto EN"'
Search Results
2. Efficient Restoration of dF508 CFTR Function in Primary Cystic Fibrosis Airway Epithelial Cells (AEC).
- Author
-
Stick, SM, primary, Sutanto, EN, additional, Scaffidi, AK, additional, Fischer, D, additional, and Kicic, A, additional
- Published
- 2009
- Full Text
- View/download PDF
3. Decreased fibronectin production significantly contributes to dysregulated repair of asthmatic epithelium.
- Author
-
Kicic A, Hallstrand TS, Sutanto EN, Stevens PT, Kobor MS, Taplin C, Paré PD, Beyer RP, Stick SM, Knight DA, Kicic, Anthony, Hallstrand, Teal S, Sutanto, Erika N, Stevens, Paul T, Kobor, Michael S, Taplin, Christopher, Paré, Peter D, Beyer, Richard P, Stick, Stephen M, and Knight, Darryl A
- Subjects
ALLERGIES ,ASTHMA ,CARCINOGENS ,CELLS ,ANALYTICAL chemistry ,DNA ,ENZYME inhibitors ,ENZYME-linked immunosorbent assay ,EPITHELIAL cells ,FIBRONECTINS ,GROWTH factors ,PIPERIDINE ,RESEARCH funding ,RESPIRATORY mucosa ,HYDROXY acids ,DEXAMETHASONE ,PHARMACODYNAMICS ,CELL physiology - Abstract
Rationale: Damage to airway epithelium is followed by deposition of extracellular matrix (ECM) and migration of adjacent epithelial cells. We have shown that epithelial cells from children with asthma fail to heal a wound in vitro.Objectives: To determine whether dysregulated ECM production by the epithelium plays a role in aberrant repair in asthma.Methods: Airway epithelial cells (AEC) from children with asthma (n = 36), healthy atopic control subjects (n = 23), and healthy nonatopic control subjects (n = 53) were investigated by microarray, gene expression and silencing, transcript regulation analysis, and ability to close mechanical wounds.Measurements and Main Results: Time to repair a mechanical wound in vitro by AEC from healthy and atopic children was not significantly different and both were faster than AEC from children with asthma. Microarray analysis revealed differential expression of multiple gene sets associated with repair and remodeling in asthmatic AEC. Fibronectin (FN) was the only ECM component whose expression was significantly lower in asthmatic AEC. Expression differences were verified by quantitative polymerase chain reaction and ELISA, and reduced FN expression persisted in asthmatic cells over passage. Silencing of FN expression in nonasthmatic AEC inhibited wound repair, whereas addition of FN to asthmatic AEC restored reparative capacity. Asthmatic AEC failed to synthesize FN in response to wounding or cytokine/growth factor stimulation. Exposure to 5', 2'deoxyazacytidine had no effect on FN expression and subsequent analysis of the FN promoter did not show evidence of DNA methylation.Conclusions: These data show that the reduced capacity of asthmatic epithelial cells to secrete FN is an important contributor to the dysregulated AEC repair observed in these cells. [ABSTRACT FROM AUTHOR]- Published
- 2010
- Full Text
- View/download PDF
4. Intrinsic biochemical and functional differences in bronchial epithelial cells of children with asthma.
- Author
-
Kicic A, Sutanto EN, Stevens PT, Knight DA, and Stick SM
- Abstract
RATIONALE: Convincing evidence of epithelial damage and aberrant repair exists in adult asthmatic airways, even in the absence of inflammation. However, comparable studies in children have been limited by access and availability of clinical samples. OBJECTIVES: To determine whether bronchial epithelial cells from children with asthma are inherently distinct from those obtained from children without asthma. METHODS: Epithelial cells were obtained by nonbronchoscopic bronchial brushing of children with mild asthma (n = 7), atopic children without asthma (n = 9), and healthy children (n = 12). Cells were subject to morphologic, biochemical, molecular, and functional assessment. Responses were also compared with commercially available epithelial cultures and the transformed cell line 16HBE140. RESULTS: All epithelial cells exhibited a 'cobblestone' morphology, which was maintained throughout culture and repeated passage. Expression of cytokeratin 19 varied, with disease phenotype being greatest in healthy nonatopics and lowest in asthmatics. In contrast, expression of cytokeratin 5/14 was greatest in asthmatic samples and least in healthy nonatopic samples. Asthmatic epithelial cells also spontaneously produced significantly greater amounts of interleukin (IL)-6, prostaglandin E2, and epidermal growth factor, and equivalent amounts of IL-1beta and soluble intracellular adhesion molecule-1, but significantly lower amounts of transforming growth factor beta1. This profile was maintained through successive passages. Asthmatic epithelial cells also exhibited greater rates of proliferation than nonasthmatic cells. CONCLUSIONS: This study has shown that epithelial cells from children with mild asthma are intrinsically different both biochemically and functionally compared with epithelial cells from children without asthma. Importantly, these differences are maintained over successive passages, suggesting that they are not dependent on an in vivo environment. [ABSTRACT FROM AUTHOR]
- Published
- 2006
- Full Text
- View/download PDF
5. Dysregulated Notch Signaling in the Airway Epithelium of Children with Wheeze.
- Author
-
Iosifidis T, Sutanto EN, Montgomery ST, Agudelo-Romero P, Looi K, Ling KM, Shaw NC, Garratt LW, Hillas J, Martinovich KM, Kicic-Starcevich E, Vijayasekaran S, Lannigan FJ, Rigby PJ, Knight DA, Stick SM, and Kicic A
- Abstract
The airway epithelium of children with wheeze is characterized by defective repair that contributes to disease pathobiology. Dysregulation of developmental processes controlled by Notch has been identified in chronic asthma. However, its role in airway epithelial cells of young children with wheeze, particularly during repair, is yet to be determined. We hypothesized that Notch is dysregulated in primary airway epithelial cells (pAEC) of children with wheeze contributing to defective repair. This study investigated transcriptional and protein expression and function of Notch in pAEC isolated from children with and without wheeze. Primary AEC of children with and without wheeze were found to express all known Notch receptors and ligands, although pAEC from children with wheeze expressed significantly lower NOTCH2 (10-fold, p = 0.004) and higher JAG1 (3.5-fold, p = 0.002) mRNA levels. These dysregulations were maintained in vitro and cultures from children with wheeze displayed altered kinetics of both NOTCH2 and JAG1 expression during repair. Following Notch signaling inhibition, pAEC from children without wheeze failed to repair (wound closure rate of 76.9 ± 3.2%). Overexpression of NOTCH2 in pAEC from children with wheeze failed to rescue epithelial repair following wounding. This study illustrates the involvement of the Notch pathway in airway epithelial wound repair in health and disease, where its dysregulation may contribute to asthma development.
- Published
- 2021
- Full Text
- View/download PDF
6. Cystic Fibrosis Clinical Isolates of Aspergillus fumigatus Induce Similar Muco-inflammatory Responses in Primary Airway Epithelial Cells.
- Author
-
McLean SA, Cullen L, Gardam DJ, Schofield CJ, Laucirica DR, Sutanto EN, Ling KM, Stick SM, Peacock CS, Kicic A, Garratt LW, On Behalf Of Arest Cf, and Waerp
- Abstract
Aspergillus is increasingly associated with lung inflammation and mucus plugging in early cystic fibrosis (CF) disease during which conidia burden is low and strains appear to be highly diverse. It is unknown whether clinical Aspergillus strains vary in their capacity to induce epithelial inflammation and mucus production. We tested the hypothesis that individual colonising strains of Aspergillus fumigatus would induce different responses. Ten paediatric CF Aspergillus isolates were compared along with two systemically invasive clinical isolates and an ATCC reference strain. Isolates were first characterised by ITS gene sequencing and screened for antifungal susceptibility. Three clusters (A-C) of Aspergillus isolates were identified by ITS. Antifungal susceptibility was variable, particularly for itraconazole. Submerged CF and non-CF monolayers as well as differentiated primary airway epithelial cell cultures were incubated with conidia for 24 h to allow germination. None of the clinical isolates were found to significantly differ from one another in either IL-6 or IL-8 release or gene expression of secretory mucins. Clinical Aspergillus isolates appear to be largely homogenous in their mucostimulatory and immunostimulatory capacities and, therefore, only the antifungal resistance characteristics are likely to be clinically important.
- Published
- 2021
- Full Text
- View/download PDF
7. ACE2 expression is elevated in airway epithelial cells from older and male healthy individuals but reduced in asthma.
- Author
-
Wark PAB, Pathinayake PS, Kaiko G, Nichol K, Ali A, Chen L, Sutanto EN, Garratt LW, Sohal SS, Lu W, Eapen MS, Oldmeadow C, Bartlett N, Reid A, Veerati P, Hsu AC, Looi K, Iosifidis T, Stick SM, Hansbro PM, and Kicic A
- Subjects
- Asthma epidemiology, Asthma metabolism, Australia epidemiology, COVID-19 epidemiology, COVID-19 metabolism, Comorbidity, Female, Humans, Male, Middle Aged, Peptidyl-Dipeptidase A biosynthesis, Asthma genetics, COVID-19 genetics, Epithelial Cells metabolism, Gene Expression Regulation, Peptidyl-Dipeptidase A genetics, SARS-CoV-2
- Abstract
Background and Objective: COVID-19 is complicated by acute lung injury, and death in some individuals. It is caused by SARS-CoV-2 that requires the ACE2 receptor and serine proteases to enter AEC. We determined what factors are associated with ACE2 expression particularly in patients with asthma and COPD., Methods: We obtained lower AEC from 145 people from two independent cohorts, aged 2-89 years, Newcastle (n = 115) and Perth (n = 30), Australia. The Newcastle cohort was enriched with people with asthma (n = 37) and COPD (n = 38). Gene expression for ACE2 and other genes potentially associated with SARS-CoV-2 cell entry was assessed by qPCR, and protein expression was confirmed with immunohistochemistry on endobronchial biopsies and cultured AEC., Results: Increased gene expression of ACE2 was associated with older age (P = 0.03) and male sex (P = 0.03), but not with pack-years smoked. When we compared gene expression between adults with asthma, COPD and healthy controls, mean ACE2 expression was lower in asthma patients (P = 0.01). Gene expression of furin, a protease that facilitates viral endocytosis, was also lower in patients with asthma (P = 0.02), while ADAM-17, a disintegrin that cleaves ACE2 from the surface, was increased (P = 0.02). ACE2 protein expression was also reduced in endobronchial biopsies from asthma patients., Conclusion: Increased ACE2 expression occurs in older people and males. Asthma patients have reduced expression. Altered ACE2 expression in the lower airway may be an important factor in virus tropism and may in part explain susceptibility factors and why asthma patients are not over-represented in those with COVID-19 complications., (© 2021 Asian Pacific Society of Respirology.)
- Published
- 2021
- Full Text
- View/download PDF
8. Ivacaftor or lumacaftor/ivacaftor treatment does not alter the core CF airway epithelial gene response to rhinovirus.
- Author
-
De Jong E, Garratt LW, Looi K, Lee AHY, Ling KM, Smith ML, Falsafi R, Sutanto EN, Hillas J, Iosifidis T, Martinovich KM, Shaw NC, Montgomery ST, Kicic-Starcevich E, Lannigan FJ, Vijayasekaran S, Hancock REW, Stick SM, Kicic A, and Arest CF
- Subjects
- Cells, Cultured, Common Cold complications, Cystic Fibrosis complications, Drug Combinations, Humans, Respiratory Mucosa cytology, Aminophenols pharmacology, Aminopyridines pharmacology, Benzodioxoles pharmacology, Common Cold virology, Cystic Fibrosis genetics, Quinolones pharmacology, Respiratory Mucosa drug effects, Respiratory Mucosa virology, Rhinovirus
- Abstract
Background: Aberrant responses by the cystic fibrosis airway epithelium during viral infection may underly the clinical observations. Whether CFTR modulators affect antiviral responses by CF epithelia is presently unknown. We tested the hypothesis that treatment of CF epithelial cells with ivacaftor (Iva) or ivacaftor/lumacaftor (Iva/Lum) would improve control of rhinovirus infection., Methods: Nineteen CF epithelial cultures (10 homozygous for p.Phe508del as CFTR Class 2, 9 p.Phe508del/p.Gly551Asp as Class 3) were infected with rhinovirus 1B at multiplicity of infection 12 for 24 h. Culture RNA and supernatants were harvested to assess gene and protein expression respectively., Results: RNA-seq analysis comparing rhinovirus infected cultures to control identified 796 and 629 differentially expressed genes for Class 2 and Class 3, respectively. This gene response was highly conserved when cells were treated with CFTR modulators and were predicted to be driven by the same interferon-pathway transcriptional regulators (IFNA, IFNL1, IFNG, IRF7, STAT1). Direct comparisons between treated and untreated infected cultures did not yield any differentially expressed genes for Class 3 and only 68 genes for Class 2. Changes were predominantly related to regulators of lipid metabolism and inflammation, aspects of epithelial biology known to be dysregulated in CF. In addition, CFTR modulators did not affect viral copy number, or levels of pro-inflammatory cytokines produced post-infection., Conclusions: Though long-term clinical data is not yet available, results presented here suggest that first generation CFTR modulators do not interfere with core airway epithelial responses to rhinovirus infection. Future work should investigate the latest triple modulation therapies., Competing Interests: Declaration of Competing Interest The authors declare that no conflict of interest exists., (Copyright © 2020. Published by Elsevier B.V.)
- Published
- 2021
- Full Text
- View/download PDF
9. Update on SLC6A14 in lung and gastrointestinal physiology and physiopathology: focus on cystic fibrosis.
- Author
-
Ruffin M, Mercier J, Calmel C, Mésinèle J, Bigot J, Sutanto EN, Kicic A, Corvol H, and Guillot L
- Subjects
- Amino Acid Transport Systems genetics, Colonic Diseases genetics, Colonic Diseases metabolism, Colonic Diseases pathology, Cystic Fibrosis genetics, Cystic Fibrosis metabolism, Genetic Variation, Humans, Linkage Disequilibrium, Neoplasms genetics, Neoplasms metabolism, Neoplasms pathology, Severity of Illness Index, Amino Acid Transport Systems metabolism, Cystic Fibrosis pathology, Gastrointestinal Tract metabolism, Lung metabolism
- Abstract
The solute carrier family 6 member 14 (SLC6A14) protein imports and concentrates all neutral amino acids as well as the two cationic acids lysine and arginine into the cytoplasm of different cell types. Primarily described as involved in several cancer and colonic diseases physiopathological mechanisms, the SLC6A14 gene has been more recently identified as a genetic modifier of cystic fibrosis (CF) disease severity. It was indeed shown to have a pleiotropic effect, modulating meconium ileus occurrence, lung disease severity, and precocity of P. aeruginosa airway infection. The biological mechanisms explaining the impact of SLC6A14 on intestinal and lung phenotypes of CF patients are starting to be elucidated. This review focuses on SLC6A14 in lung and gastrointestinal physiology and physiopathology, especially its involvement in the pathophysiology of CF disease.
- Published
- 2020
- Full Text
- View/download PDF
10. Rhinovirus Infection Drives Complex Host Airway Molecular Responses in Children With Cystic Fibrosis.
- Author
-
Ling KM, Garratt LW, Gill EE, Lee AHY, Agudelo-Romero P, Sutanto EN, Iosifidis T, Rosenow T, Turvey SE, Lassmann T, Hancock REW, Kicic A, and Stick SM
- Subjects
- Cells, Cultured, Child, Preschool, Cystic Fibrosis genetics, Cytokines immunology, Epithelial Cells virology, Female, Humans, Infant, Male, Picornaviridae Infections genetics, Protein Interaction Maps, RNA-Seq, Transcriptome, Bronchi cytology, Cystic Fibrosis immunology, Epithelial Cells immunology, Picornaviridae Infections immunology, Rhinovirus
- Abstract
Early-life viral infections are responsible for pulmonary exacerbations that can contribute to disease progression in young children with cystic fibrosis (CF). The most common respiratory viruses detected in the CF airway are human rhinoviruses (RV), and augmented airway inflammation in CF has been attributed to dysregulated airway epithelial responses although evidence has been conflicting. Here, we exposed airway epithelial cells from children with and without CF to RV in vitro . Using RNA-Seq, we profiled the transcriptomic differences of CF and non-CF airway epithelial cells at baseline and in response to RV. There were only modest differences between CF and non-CF cells at baseline. In response to RV, there were 1,442 and 896 differentially expressed genes in CF and non-CF airway epithelial cells, respectively. The core antiviral responses in CF and non-CF airway epithelial cells were mediated through interferon signaling although type 1 and 3 interferon signaling, when measured, were reduced in CF airway epithelial cells following viral challenge consistent with previous reports. The transcriptional responses in CF airway epithelial cells were more complex than in non-CF airway epithelial cells with diverse over-represented biological pathways, such as cytokine signaling and metabolic and biosynthetic pathways. Network analysis highlighted that the differentially expressed genes of CF airway epithelial cells' transcriptional responses were highly interconnected and formed a more complex network than observed in non-CF airway epithelial cells. We corroborate observations in fully differentiated air-liquid interface (ALI) cultures, identifying genes involved in IL-1 signaling and mucin glycosylation that are only dysregulated in the CF airway epithelial response to RV infection. These data provide novel insights into the CF airway epithelial cells' responses to RV infection and highlight potential pathways that could be targeted to improve antiviral and anti-inflammatory responses in CF., (Copyright © 2020 Ling, Garratt, Gill, Lee, Agudelo-Romero, Sutanto, Iosifidis, Rosenow, Turvey, Lassmann, Hancock, Kicic and Stick.)
- Published
- 2020
- Full Text
- View/download PDF
11. Azithromycin Partially Mitigates Dysregulated Repair of Lung Allograft Small Airway Epithelium.
- Author
-
Ling KM, Garratt LW, Banerjee B, Lavender MA, Wrobel JP, Musk M, Martinovich KM, Shaw NC, Iosifidis T, Looi K, Kicic-Starcevich E, Sutanto EN, Yerkovich ST, Chambers DC, Stick SM, and Kicic A
- Subjects
- Adolescent, Adult, Airway Remodeling drug effects, Allografts cytology, Allografts diagnostic imaging, Allografts pathology, Azithromycin therapeutic use, Bronchi cytology, Bronchi diagnostic imaging, Bronchi pathology, Bronchiolitis Obliterans diagnosis, Bronchiolitis Obliterans etiology, Bronchiolitis Obliterans pathology, Bronchoscopy, Case-Control Studies, Cells, Cultured, Child, Drug Evaluation, Preclinical, Epithelial Cells pathology, Female, Graft Rejection diagnosis, Graft Rejection etiology, Graft Rejection pathology, Humans, Male, Middle Aged, Primary Cell Culture, Regeneration drug effects, Transplantation, Homologous, Young Adult, Azithromycin pharmacology, Bronchiolitis Obliterans prevention & control, Epithelial Cells drug effects, Graft Rejection prevention & control, Lung Transplantation adverse effects
- Abstract
Background: Dysregulated airway epithelial repair following injury is a proposed mechanism driving posttransplant bronchiolitis obliterans (BO), and its clinical correlate bronchiolitis obliterans syndrome (BOS). This study compared gene and cellular characteristics of injury and repair in large (LAEC) and small (SAEC) airway epithelial cells of transplant patients., Methods: Subjects were recruited at the time of routine bronchoscopy posttransplantation and included patients with and without BOS. Airway epithelial cells were obtained from bronchial and bronchiolar brushing performed under radiological guidance from these patients. In addition, bronchial brushings were also obtained from healthy control subjects comprising of adolescents admitted for elective surgery for nonrespiratory-related conditions. Primary cultures were established, monolayers wounded, and repair assessed (±) azithromycin (1 µg/mL). In addition, proliferative capacity as well as markers of injury and dysregulated repair were also assessed., Results: SAEC had a significantly dysregulated repair process postinjury, despite having a higher proliferative capacity than large airway epithelial cells. Addition of azithromycin significantly induced repair in these cells; however, full restitution was not achieved. Expression of several genes associated with epithelial barrier repair (matrix metalloproteinase 7, matrix metalloproteinase 3, the integrins β6 and β8, and β-catenin) were significantly different in epithelial cells obtained from patients with BOS compared to transplant patients without BOS and controls, suggesting an intrinsic defect., Conclusions: Chronic airway injury and dysregulated repair programs are evident in airway epithelium obtained from patients with BOS, particularly with SAEC. We also show that azithromycin partially mitigates this pathology.
- Published
- 2020
- Full Text
- View/download PDF
12. Aberrant cell migration contributes to defective airway epithelial repair in childhood wheeze.
- Author
-
Iosifidis T, Sutanto EN, Buckley AG, Coleman L, Gill EE, Lee AH, Ling KM, Hillas J, Looi K, Garratt LW, Martinovich KM, Shaw NC, Montgomery ST, Kicic-Starcevich E, Karpievitch YV, Le Souëf P, Laing IA, Vijayasekaran S, Lannigan FJ, Rigby PJ, Hancock RE, Knight DA, Stick SM, and Kicic A
- Subjects
- Adolescent, Asthma pathology, Cell Line, Child, Child, Preschool, Female, Humans, Infant, Integrin alpha5beta1 metabolism, Male, Phosphatidylinositol 3-Kinases metabolism, Proto-Oncogene Proteins c-akt metabolism, Respiratory Mucosa pathology, Asthma metabolism, Cell Movement, Respiratory Mucosa metabolism, Respiratory Sounds, Signal Transduction
- Abstract
Abnormal wound repair has been observed in the airway epithelium of patients with chronic respiratory diseases, including asthma. Therapies focusing on repairing vulnerable airways, particularly in early life, present a potentially novel treatment strategy. We report defective lower airway epithelial cell repair to strongly associate with common pre-school-aged and school-aged wheezing phenotypes, characterized by aberrant migration patterns and reduced integrin α5β1 expression. Next generation sequencing identified the PI3K/Akt pathway as the top upstream transcriptional regulator of integrin α5β1, where Akt activation enhanced repair and integrin α5β1 expression in primary cultures from children with wheeze. Conversely, inhibition of PI3K/Akt signaling in primary cultures from children without wheeze reduced α5β1 expression and attenuated repair. Importantly, the FDA-approved drug celecoxib - and its non-COX2-inhibiting analogue, dimethyl-celecoxib - stimulated the PI3K/Akt-integrin α5β1 axis and restored airway epithelial repair in cells from children with wheeze. When compared with published clinical data sets, the identified transcriptomic signature was also associated with viral-induced wheeze exacerbations highlighting the clinical potential of such therapy. Collectively, these results identify airway epithelial restitution via targeting the PI3K-integrin α5β1 axis as a potentially novel therapeutic avenue for childhood wheeze and asthma. We propose that the next step in the therapeutic development process should be a proof-of-concept clinical trial, since relevant animal models to test the crucial underlying premise are unavailable.
- Published
- 2020
- Full Text
- View/download PDF
13. Effects of human rhinovirus on epithelial barrier integrity and function in children with asthma.
- Author
-
Looi K, Buckley AG, Rigby PJ, Garratt LW, Iosifidis T, Zosky GR, Larcombe AN, Lannigan FJ, Ling KM, Martinovich KM, Kicic-Starcevich E, Shaw NC, Sutanto EN, Knight DA, Kicic A, and Stick SM
- Subjects
- Adolescent, Child, Child, Preschool, Female, Humans, Infant, Male, Rhinovirus, Tight Junctions pathology, Tight Junctions virology, Asthma pathology, Asthma virology, Picornaviridae Infections pathology, Respiratory Mucosa pathology, Respiratory Mucosa virology
- Abstract
Background: Bronchial epithelial tight junctions (TJ) have been extensively assessed in healthy airway epithelium. However, no studies have yet assessed the effect of human rhinovirus (HRV) infection on the expression and resultant barrier function in epithelial tight junctions (TJ) in childhood asthma., Objectives: To investigate the impact of HRV infection on airway epithelial TJ expression and barrier function in airway epithelial cells (AECs) of children with and without asthma. Furthermore, to test the hypothesis that barrier integrity and function is compromised to a greater extent by HRV in AECs from asthmatic children., Methods: Primary AECs were obtained from children with and without asthma, differentiated into air-liquid interface (ALI) cultures and infected with rhinovirus. Expression of claudin-1, occludin and zonula occluden-1 (ZO-1) was assessed via qPCR, immunocytochemistry (ICC), in-cell western (ICW) and confocal microscopy. Barrier function was assessed by transepithelial electrical resistance (TER; R
T ) and permeability to fluorescent dextran., Results: Basal TJ gene expression of claudin-1 and occludin was significantly upregulated in asthmatic children compared to non-asthmatics; however, no difference was seen with ZO-1. Interestingly, claudin-1, occludin and ZO-1 protein expression was significantly reduced in AEC of asthmatic children compared to non-asthmatic controls suggesting possible post-transcriptional inherent differences. HRV infection resulted in a transient dissociation of TJ and airway barrier integrity in non-asthmatic children. Although similar dissociation of TJ was observed in asthmatic children, a significant and sustained reduction in TJ expression concurrent with both a significant decrease in TER and an increase in permeability in asthmatic children was observed., Conclusion: This study demonstrates novel intrinsic differences in TJ gene and protein expression between AEC of children with and without asthma. Furthermore, it correlates directly the relationship between HRV infection and the resultant dissociation of epithelial TJ that causes a continued altered barrier function in children with asthma., (© 2018 John Wiley & Sons Ltd.)- Published
- 2018
- Full Text
- View/download PDF
14. Visualisation of Multiple Tight Junctional Complexes in Human Airway Epithelial Cells.
- Author
-
Buckley AG, Looi K, Iosifidis T, Ling KM, Sutanto EN, Martinovich KM, Kicic-Starcevich E, Garratt LW, Shaw NC, Lannigan FJ, Larcombe AN, Zosky G, Knight DA, Rigby PJ, Kicic A, and Stick SM
- Abstract
Background: Apically located tight junctions in airway epithelium perform a fundamental role in controlling macromolecule migration through paracellular spaces. Alterations in their expression may lead to disruptions in barrier integrity, which subsequently facilitates entry of potential bacterial and other pathogens into the host. Furthermore, there is emerging evidence that the barrier integrity of the airway in certain airway inflammatory diseases may be altered. However, there is little consensus on the way this is assessed and measured and the type of cells used to achieve this., Methods: Here, we assessed four fixation methods including; (i) 4% ( v /v) paraformaldehyde; (ii) 100% methanol; (iii) acetone or; (iv) 1:1 methanol: acetone. Pre-extraction with Triton X-100 was also performed and assessed on cells prior to fixation with either methanol or paraformaldehyde. Cells were also permeabilized with 0.1% (v/v) Saponin in 1× TBS following fixation and subsequently stained for tight junction proteins. Confocal microscopy was then used to visualise, compare and evaluate staining intensity of the tight junctional complexes in order to determine a standardised workflow of reproducible staining., Results: Positive staining was observed following methanol fixation for claudin-1 and ZO-1 tight junction proteins but no staining was detected for occludin in 16HBE14o- cells. Combinatorial fixation with methanol and acetone also produced consistent positive staining for both occludin and ZO-1 tight junction proteins in these cells. When assessed using primary cells cultured at air-liquid interface, similar positive staining for claudin-1 and ZO-1 was observed following methanol fixation, while similar positive staining for occludin and ZO-1 was observed following the same combinatorial fixation with methanol and acetone., Conclusions: The present study demonstrates the importance of a personalised approach to optimise staining for the visualisation of different tight junction proteins. Of significance, the workflow, once optimised, can readily be translated into primary airway epithelial cell air-liquid interface cultures where it can be used to assess barrier integrity in chronic lung diseases., Competing Interests: This study was approved by the Human Ethics Committee for both Princess Margaret Hospital for Children (Reference Number 1402EP, 1903EP) and St John of God Hospital (Reference Number 452).Not applicable.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
- Published
- 2018
- Full Text
- View/download PDF
15. Assessment of p.Phe508del-CFTR functional restoration in pediatric primary cystic fibrosis airway epithelial cells.
- Author
-
Sutanto EN, Scaffidi A, Garratt LW, Looi K, Foo CJ, Tessari MA, Janssen RA, Fischer DF, Stick SM, and Kicic A
- Subjects
- Adenoviridae genetics, Bronchi cytology, Cells, Cultured, Child, Cystic Fibrosis pathology, Cystic Fibrosis Transmembrane Conductance Regulator chemistry, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Epithelial Cells metabolism, Genetic Vectors, Humans, Protein Transport, Trachea cytology, Transduction, Genetic, Bronchi metabolism, Cystic Fibrosis physiopathology, Cystic Fibrosis Transmembrane Conductance Regulator physiology, Phenylalanine chemistry, Trachea metabolism
- Abstract
Background: Mutations in the cystic fibrosis transmembrane regulator (CFTR) gene can reduce function of the CFTR ion channel activity and impair cellular chloride secretion. The gold standard method to assess CFTR function of ion transport using the Ussing chamber requires a high number of airway epithelial cells grown at air-liquid interface, limiting the application of this method for high throughput screening of potential therapeutic compounds in primary airway epithelial cells (pAECs) featuring less common CFTR mutations. This study assessed an alternative approach, using a small scale halide assay that can be adapted for a personalized high throughput setting to analyze CFTR function of pAEC., Methods: Pediatric pAECs derived from children with CF (pAECCF) were established and expanded as monolayer cultures, before seeding into 96-well plates for the halide assay. Cells were then transduced with an adenoviral construct containing yellow fluorescent protein (eYFP) reporter gene, alone or in combination with either wild-type CFTR (WT-CFTR) or p.Phe508del CFTR. Four days post transduction, cells were stimulated with forskolin and genistein, and assessed for quenching of the eYFP signal following injection of iodide solution into the assay media., Results: Data showed that pAECCF can express eYFP at high efficiency following transduction with the eYFP construct. The halide assay was able to discriminate functional restoration of CFTR in pAECCF treated with either WT-CFTR construct or the positive controls syntaxin 8 and B-cell receptor-associated protein 31 shRNAs., Significance: The current study demonstrates that the halide assay can be adapted for pediatric pAECCF to evaluate restoration of CFTR function. With the ongoing development of small molecules to modulate the folding and/or activity of various mutated CFTR proteins, this halide assay presents a small-scale personalized screening platform that could assess therapeutic potential of molecules across a broad range of CFTR mutations.
- Published
- 2018
- Full Text
- View/download PDF
16. Accumulation mode particles and LPS exposure induce TLR-4 dependent and independent inflammatory responses in the lung.
- Author
-
Fonceca AM, Zosky GR, Bozanich EM, Sutanto EN, Kicic A, McNamara PS, Knight DA, Sly PD, Turner DJ, and Stick SM
- Subjects
- Airway Resistance physiology, Animals, Inflammation chemically induced, Inflammation metabolism, Lung drug effects, Mice, Mice, Inbred C3H, Mice, Knockout, Inflammation Mediators metabolism, Lipopolysaccharides toxicity, Lung metabolism, Particulate Matter toxicity, Toll-Like Receptor 4 biosynthesis
- Abstract
Background: Accumulation mode particles (AMP) are formed from engine combustion and make up the inhalable vapour cloud of ambient particulate matter pollution. Their small size facilitates dispersal and subsequent exposure far from their original source, as well as the ability to penetrate alveolar spaces and capillary walls of the lung when inhaled. A significant immuno-stimulatory component of AMP is lipopolysaccharide (LPS), a product of Gram negative bacteria breakdown. As LPS is implicated in the onset and exacerbation of asthma, the presence or absence of LPS in ambient particulate matter (PM) may explain the onset of asthmatic exacerbations to PM exposure. This study aimed to delineate the effects of LPS and AMP on airway inflammation, and potential contribution to airways disease by measuring airway inflammatory responses induced via activation of the LPS cellular receptor, Toll-like receptor 4 (TLR-4)., Methods: The effects of nebulized AMP, LPS and AMP administered with LPS on lung function, cellular inflammatory infiltrate and cytokine responses were compared between wildtype mice and mice not expressing TLR-4., Results: The presence of LPS administered with AMP appeared to drive elevated airway resistance and sensitivity via TLR-4. Augmented TLR4 driven eosinophilia and greater TNF-α responses observed in AMP-LPS treated mice independent of TLR-4 expression, suggests activation of allergic responses by TLR4 and non-TLR4 pathways larger than those induced by LPS administered alone. Treatment with AMP induced macrophage recruitment independent of TLR-4 expression., Conclusions: These findings suggest AMP-LPS as a stronger stimulus for allergic inflammation in the airways then LPS alone.
- Published
- 2018
- Full Text
- View/download PDF
17. Conditionally reprogrammed primary airway epithelial cells maintain morphology, lineage and disease specific functional characteristics.
- Author
-
Martinovich KM, Iosifidis T, Buckley AG, Looi K, Ling KM, Sutanto EN, Kicic-Starcevich E, Garratt LW, Shaw NC, Montgomery S, Lannigan FJ, Knight DA, Kicic A, and Stick SM
- Subjects
- Animals, Asthma pathology, Asthma physiopathology, Cell Differentiation, Cell Lineage, Cells, Cultured, Cellular Reprogramming Techniques, Child, Preschool, Cystic Fibrosis pathology, Cystic Fibrosis physiopathology, Female, Fibroblasts, Humans, Male, Mice, Respiratory Mucosa pathology, Respiratory Mucosa physiopathology, Respiratory Mucosa cytology
- Abstract
Current limitations to primary cell expansion led us to test whether airway epithelial cells derived from healthy children and those with asthma and cystic fibrosis (CF), co-cultured with an irradiated fibroblast feeder cell in F-medium containing 10 µM ROCK inhibitor could maintain their lineage during expansion and whether this is influenced by underlying disease status. Here, we show that conditionally reprogrammed airway epithelial cells (CRAECs) can be established from both healthy and diseased phenotypes. CRAECs can be expanded, cryopreserved and maintain phenotypes over at least 5 passages. Population doublings of CRAEC cultures were significantly greater than standard cultures, but maintained their lineage characteristics. CRAECs from all phenotypes were also capable of fully differentiating at air-liquid interface (ALI) and maintained disease specific characteristics including; defective CFTR channel function cultures and the inability to repair wounds. Our findings indicate that CRAECs derived from children maintain lineage, phenotypic and importantly disease-specific functional characteristics over a specified passage range.
- Published
- 2017
- Full Text
- View/download PDF
18. Impaired airway epithelial cell responses from children with asthma to rhinoviral infection.
- Author
-
Kicic A, Stevens PT, Sutanto EN, Kicic-Starcevich E, Ling KM, Looi K, Martinovich KM, Garratt LW, Iosifidis T, Shaw NC, Buckley AG, Rigby PJ, Lannigan FJ, Knight DA, and Stick SM
- Subjects
- Adolescent, Allergens immunology, Apoptosis, Asthma diagnosis, Asthma metabolism, Cell Proliferation, Cell Survival, Child, Child, Preschool, Common Cold, Cytokines metabolism, Disease Progression, Female, Humans, Immunoglobulin E immunology, Inflammation Mediators metabolism, Male, Picornaviridae Infections metabolism, Picornaviridae Infections virology, Receptors, Virus genetics, Receptors, Virus metabolism, Respiratory Mucosa pathology, Viral Load, Virus Replication, Asthma complications, Asthma immunology, Picornaviridae Infections complications, Respiratory Mucosa immunology, Respiratory Mucosa virology, Rhinovirus classification
- Abstract
Background: The airway epithelium forms an effective immune and physical barrier that is essential for protecting the lung from potentially harmful inhaled stimuli including viruses. Human rhinovirus (HRV) infection is a known trigger of asthma exacerbations, although the mechanism by which this occurs is not fully understood., Objective: To explore the relationship between apoptotic, innate immune and inflammatory responses to HRV infection in airway epithelial cells (AECs) obtained from children with asthma and non-asthmatic controls. In addition, to test the hypothesis that aberrant repair of epithelium from asthmatics is further dysregulated by HRV infection., Methods: Airway epithelial brushings were obtained from 39 asthmatic and 36 non-asthmatic children. Primary cultures were established and exposed to HRV1b and HRV14. Virus receptor number, virus replication and progeny release were determined. Epithelial cell apoptosis, IFN-β production, inflammatory cytokine release and epithelial wound repair and proliferation were also measured., Results: Virus proliferation and release was greater in airway epithelial cells from asthmatics but this was not related to the number of virus receptors. In epithelial cells from asthmatic children, virus infection dampened apoptosis, reduced IFN-β production and increased inflammatory cytokine production. HRV1b infection also inhibited wound repair capacity of epithelial cells isolated from non-asthmatic children and exaggerated the defective repair response seen in epithelial cells from asthmatics. Addition of IFN-β restored apoptosis, suppressed virus replication and improved repair of airway epithelial cells from asthmatics but did not reduce inflammatory cytokine production., Conclusions: Collectively, HRV infection delays repair and inhibits apoptotic processes in epithelial cells from non-asthmatic and asthmatic children. The delayed repair is further exaggerated in cells from asthmatic children and is only partially reversed by exogenous IFN-β., (© 2016 John Wiley & Sons Ltd.)
- Published
- 2016
- Full Text
- View/download PDF
19. Reduced transforming growth factor β1 (TGF-β1) in the repair of airway epithelial cells of children with asthma.
- Author
-
Ling KM, Sutanto EN, Iosifidis T, Kicic-Starcevich E, Looi K, Garratt LW, Martinovich KM, Lannigan FJ, Knight DA, Stick SM, and Kicic A
- Subjects
- Alveolar Epithelial Cells pathology, Cell Proliferation, Child, Female, Humans, Male, Matrix Metalloproteinase 14 metabolism, RNA, Messenger metabolism, Re-Epithelialization physiology, Statistics as Topic, Airway Remodeling physiology, Alveolar Epithelial Cells metabolism, Asthma metabolism, Asthma pathology, Transforming Growth Factor beta1 metabolism
- Abstract
Background and Objective: Evidence into the role of TGF-β1 in airway epithelial repair in asthma is still controversial. This study tested the hypothesis that the reduced TGF-β1 levels previously observed in paediatric asthmatic airway epithelial cells directly contribute to the dysregulated repair seen in these cells., Methods: Primary airway epithelial cells (pAEC) from children with asthma (n = 16) and non-asthmatic subjects (n = 20) were isolated, and subcultured for investigation of TGF-β1 gene and protein via quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Expression of other associated genes such as integrins αvβ6, αvβ8 and MT1-MMP were also tested. Small interfering RNA (siRNA) was employed to assess the role of TGF-β1 during wound repair., Results: TGF-β1 gene and protein expression were significantly downregulated in asthmatic pAEC over the course of repair, compared with cells from non-asthmatic children. Messenger RNA (mRNA) expression of TGF-β1 was also directly implicated in non-asthmatic and asthmatic pAEC proliferation over their quiescent counterparts. Small interfering RNA-mediated knockdown of TGF-β1 compromised repair in non-asthmatic pAEC and exacerbated the dysregulated repair seen in asthmatic pAEC. Expression of major TGF-β1 activators of epithelial cells, integrin αvβ6 and αvβ8 was also measured and there was no difference in αvβ6 gene expression between the two cohorts. Although integrin αvβ8 gene expression was significantly higher in asthmatic pAEC, the expression of MT1-MMP (MMP14) which facilitates the αvβ8 mediated TGF-β1 activation was significantly downregulated., Conclusion: Our data has highlighted the importance of TGF-β1 in pAEC wound repair in vitro. The significantly lower levels seen in asthmatic pAEC subsequently contributes to the dysregulated repair observed in these cells., (© 2016 Asian Pacific Society of Respirology.)
- Published
- 2016
- Full Text
- View/download PDF
20. Alpha-1 Antitrypsin Mitigates the Inhibition of Airway Epithelial Cell Repair by Neutrophil Elastase.
- Author
-
Garratt LW, Sutanto EN, Ling KM, Looi K, Iosifidis T, Martinovich KM, Shaw NC, Buckley AG, Kicic-Starcevich E, Lannigan FJ, Knight DA, Stick SM, and Kicic A
- Subjects
- Apoptosis drug effects, Case-Control Studies, Cell Adhesion drug effects, Cell Movement drug effects, Cell Proliferation drug effects, Cell Survival drug effects, Cells, Cultured, Child, Child, Preschool, Cystic Fibrosis pathology, Cytokines metabolism, Dose-Response Relationship, Drug, Epithelial Cells enzymology, Epithelial Cells pathology, Female, Humans, Infant, Infant, Newborn, Inflammation Mediators metabolism, Male, Phenotype, Respiratory Mucosa enzymology, Respiratory Mucosa pathology, Time Factors, Cystic Fibrosis enzymology, Epithelial Cells drug effects, Leukocyte Elastase pharmacology, Regeneration drug effects, Respiratory Mucosa drug effects, alpha 1-Antitrypsin pharmacology
- Abstract
Neutrophil elastase (NE) activity is associated with many destructive lung diseases and is a predictor for structural lung damage in early cystic fibrosis (CF), which suggests normal maintenance of airway epithelium is prevented by uninhibited NE. However, limited data exist on how the NE activity in airways of very young children with CF affects function of the epithelia. The aim of this study was to determine if NE activity could inhibit epithelial homeostasis and repair and whether any functional effect was reversible by antiprotease alpha-1 antitrypsin (α1AT) treatment. Viability, inflammation, apoptosis, and proliferation were assessed in healthy non-CF and CF pediatric primary airway epithelial cells (pAECnon-CF and pAECCF, respectively) during exposure to physiologically relevant NE. The effect of NE activity on pAECCF wound repair was also assessed. We report that viability after 48 hours was significantly decreased by 100 nM NE in pAECnon-CF and pAECCF owing to rapid cellular detachment that was accompanied by inflammatory cytokine release. Furthermore, both phenotypes initiated an apoptotic response to 100 nM NE, whereas ≥ 50 nM NE activity significantly inhibited the proliferative capacity of cultures. Similar concentrations of NE also significantly inhibited wound repair of pAECCF, but this effect was reversed by the addition of α1AT. Collectively, our results demonstrate free NE activity is deleterious for epithelial homeostasis and support the hypothesis that proteases in the airway contribute directly to CF structural lung disease. Our results also highlight the need to investigate antiprotease therapies in early CF disease in more detail.
- Published
- 2016
- Full Text
- View/download PDF
21. Effect of human rhinovirus infection on airway epithelium tight junction protein disassembly and transepithelial permeability.
- Author
-
Looi K, Troy NM, Garratt LW, Iosifidis T, Bosco A, Buckley AG, Ling KM, Martinovich KM, Kicic-Starcevich E, Shaw NC, Sutanto EN, Zosky GR, Rigby PJ, Larcombe AN, Knight DA, Kicic A, and Stick SM
- Abstract
Rationale: No studies have assessed the effects of human rhinovirus (HRV) infection on epithelial tight junctions (TJs) and resultant barrier function., Aim of the Study: To correlate viral infection with TJ disassembly, epithelial barrier integrity, and function., Materials and Methods: Human airway epithelial cells were infected with HRV minor serotype 1B (HRV-1B) at various 50% tissue culture infectivity doses (TCID
50 ) over 72 hours. HRV replication was assessed by quantitative-polymerase chain reaction (qPCR) while cell viability and apoptosis were assessed by proliferation and apoptotic assays, respectively. Protein expression of claudin-1, occludin, and zonula occludens protein-1 (ZO-1) was assessed using In-Cell™ Western assays. Transepithelial permeability assays were performed to assess effects on barrier functionality. RT2 Profiler focused qPCR arrays and pathway analysis evaluating associations between human TJ and antiviral response were performed to identify potential interactions and pathways between genes of interests., Results: HRV-1B infection affected viability that was both time and TCID50 dependent. Significant increases in apoptosis and viral replication post-infection correlated with viral titer. Viral infection significantly decreased claudin-1 protein expression at the lower TCID50 , while a significant decrease in all three TJ protein expressions occurred at higher TCID50 . Decrease in protein expression was concomitant with significant increases in epithelial permeability of fluorescein isothiocynate labeled-dextran 4 and 20 kDa. Analysis of focused qPCR arrays demonstrated a significant decrease in ZO-1 gene expression. Furthermore, network analysis between human TJ and antiviral response genes revealed possible interactions and regulation of TJ genes via interleukin (IL)-15 in response to HRV-1B infection., Conclusion: HRV-1B infection directly alters human airway epithelial TJ expression leading to increased epithelial permeability potentially via an antiviral response of IL-15.- Published
- 2016
- Full Text
- View/download PDF
22. Matrix metalloproteinase activation by free neutrophil elastase contributes to bronchiectasis progression in early cystic fibrosis.
- Author
-
Garratt LW, Sutanto EN, Ling KM, Looi K, Iosifidis T, Martinovich KM, Shaw NC, Kicic-Starcevich E, Knight DA, Ranganathan S, Stick SM, and Kicic A
- Subjects
- Bronchiectasis complications, Bronchoalveolar Lavage Fluid chemistry, Child, Child, Preschool, Cystic Fibrosis enzymology, Disease Progression, Female, Humans, Infant, Male, Tomography, X-Ray Computed, Bronchiectasis enzymology, Cystic Fibrosis complications, Leukocyte Elastase metabolism, Matrix Metalloproteinases metabolism, Tissue Inhibitor of Metalloproteinase-1 metabolism, Tissue Inhibitor of Metalloproteinase-2 metabolism
- Abstract
Neutrophil elastase is the most significant predictor of bronchiectasis in early-life cystic fibrosis; however, the causal link between neutrophil elastase and airway damage is not well understood. Matrix metalloproteinases (MMPs) play a crucial role in extracellular matrix modelling and are activated by neutrophil elastase. The aim of this study was to assess if MMP activation positively correlates with neutrophil elastase activity, disease severity and bronchiectasis in young children with cystic fibrosis.Total MMP-1, MMP-2, MMP-7, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-2 and TIMP-1 levels were measured in bronchoalveolar lavage fluid collected from young children with cystic fibrosis during annual clinical assessment. Active/pro-enzyme ratio of MMP-9 was determined by gelatin zymography. Annual chest computed tomography imaging was scored for bronchiectasis.A higher MMP-9/TIMP-1 ratio was associated with free neutrophil elastase activity. In contrast, MMP-2/TIMP-2 ratio decreased and MMP-1 and MMP-7 were not detected in the majority of samples. Ratio of active/pro-enzyme MMP-9 was also higher in the presence of free neutrophil elastase activity, but not infection. Across the study cohort, both MMP-9/TIMP-1 and active MMP-9 were associated with progression of bronchiectasis.Both MMP-9/TIMP-1 and active MMP-9 increased with free neutrophil elastase and were associated with bronchiectasis, further demonstrating that free neutrophil elastase activity should be considered an important precursor to cystic fibrosis structural disease., (Copyright ©ERS 2015.)
- Published
- 2015
- Full Text
- View/download PDF
23. Determinants of culture success in an airway epithelium sampling program of young children with cystic fibrosis.
- Author
-
Garratt LW, Sutanto EN, Foo CJ, Ling KM, Looi K, Kicic-Starcevich E, Iosifidis T, Martinovich KM, Lannigan FJ, Stick SM, and Kicic A
- Subjects
- Bronchoalveolar Lavage Fluid chemistry, Bronchoalveolar Lavage Fluid microbiology, Child, Child, Preschool, Cystic Fibrosis enzymology, Cystic Fibrosis genetics, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Cytological Techniques, Female, Humans, Infant, Inflammation enzymology, Interleukin-8 metabolism, Leukocyte Elastase metabolism, Male, Mutation, Retrospective Studies, Specimen Handling, Cell Culture Techniques statistics & numerical data, Cystic Fibrosis pathology, Respiratory Mucosa pathology
- Abstract
Aim of the Study: The bronchial brushing technique presents an opportunity to establish a gold standard in vitro model of Cystic Fibrosis (CF) airway disease. However, unique obstacles exist when establishing CF airway epithelial cells (pAECCF). We aimed to identify determinants of culture success through retrospective analysis of a program of routinely brushing children with CF., Materials and Methods: Anaesthetised children (CF and non-CF) had airway samples taken which were immediately processed for cell culture. Airway data for the CF cohort was obtained from clinical records and the AREST CF database., Results: Of 260 brushings processed for culture, 114 (43.8%) pAECCF successfully cultured to passage one (P1) and 63 (24.2% of total) progressed to passage two (P2). However, >80% of non-CF specimens (pAECnon-CF) cultured to P2 from similar cell numbers. Within the CF cohort, specimens successfully cultured to P2 had a higher initial cell count and lower proportion of severe CF mutation phenotype than those that did not proliferate beyond initial seeding. Elevated airway IL-8 concentration was also negatively associated with culture establishment. Contamination by opportunistic pathogens was observed in 81 (31.2% of total) cultures and brushings from children with lower respiratory tract infections were more likely to co-culture contaminating flora., Conclusions: Lower passage rates of pAECCF cultures uniquely contrasts with pAECnon-CF despite similar cell numbers. An equivalent establishment rate of CF nasal epithelium reported elsewhere, significant associations to CFTR mutation phenotype, elevated airway IL-8 and opportunistic pathogens all suggest this is likely related to the CF disease milieu.
- Published
- 2014
- Full Text
- View/download PDF
24. Suppression of adrenomedullin contributes to vascular leakage and altered epithelial repair during asthma.
- Author
-
Hagner S, Welz H, Kicic A, Alrifai M, Marsh LM, Sutanto EN, Ling KM, Stick SM, Müller B, Weissmann N, and Renz H
- Subjects
- Administration, Intranasal, Adrenomedullin pharmacology, Allergens immunology, Animals, Asthma immunology, Capillary Permeability drug effects, Cytokines biosynthesis, Cytokines immunology, Disease Models, Animal, Endothelial Cells metabolism, Epithelial Cells drug effects, Gene Expression Regulation, Humans, Inflammation immunology, Inflammation metabolism, Macrophages, Alveolar immunology, Macrophages, Alveolar metabolism, Mice, Mice, Inbred BALB C, Adrenomedullin genetics, Adrenomedullin metabolism, Asthma genetics, Asthma metabolism, Capillary Permeability immunology, Epithelial Cells metabolism
- Abstract
Background: The anti-inflammatory peptide, adrenomedullin (AM), and its cognate receptor are expressed in lung tissue, but its pathophysiological significance in airway inflammation is unknown., Objectives: This study investigated whether allergen-induced airway inflammation involves an impaired local AM response., Methods: Airway AM expression was measured in acute and chronically sensitized mice following allergen inhalation and in airway epithelial cells of asthmatic and nonasthmatic patients. The effects of AM on experimental allergen-induced airway inflammation and of AM on lung epithelial repair in vitro were investigated., Results: Adrenomedullin mRNA levels were significantly (P < 0.05) reduced in acute ovalbumin (OVA)-sensitized mice after OVA challenge, by over 60% at 24 h and for up to 6 days. Similarly, reduced AM expression was observed in two models of chronic allergen-induced inflammation, OVA- and house dust mite-sensitized mice. The reduced AM expression was restricted to airway epithelial and endothelial cells, while AM expression in alveolar macrophages was unaltered. Intranasal AM completely attenuated the OVA-induced airway hyperresponsiveness and mucosal plasma leakage but had no effect on inflammatory cells or cytokines. The effects of inhaled AM were reversed by pre-inhalation of the putative AM receptor antagonist, AM ((22-52)) . AM mRNA levels were significantly (P < 0.05) lower in human asthmatic airway epithelial samples than in nonasthmatic controls. In vitro, AM dose-dependently (10(-11) -10(-7) M) accelerated experimental wound healing in human and mouse lung epithelial cell monolayers and stimulated epithelial cell migration., Conclusion: Adrenomedullin suppression in T(H) 2-related inflammation is of pathophysiological significance and represents loss of a factor that maintains tissue integrity during inflammation., (© 2012 John Wiley & Sons A/S.)
- Published
- 2012
- Full Text
- View/download PDF
25. DNA methylation profiles of airway epithelial cells and PBMCs from healthy, atopic and asthmatic children.
- Author
-
Stefanowicz D, Hackett TL, Garmaroudi FS, Günther OP, Neumann S, Sutanto EN, Ling KM, Kobor MS, Kicic A, Stick SM, Paré PD, and Knight DA
- Subjects
- Adolescent, Asthma pathology, Carrier Proteins genetics, Carrier Proteins metabolism, Case-Control Studies, Child, Child, Preschool, Cohort Studies, CpG Islands genetics, Demography, Epithelial Cells pathology, Female, Gene Expression Regulation, Humans, Hypersensitivity, Immediate pathology, LIM Domain Proteins genetics, LIM Domain Proteins metabolism, Male, Phenotype, STAT5 Transcription Factor genetics, STAT5 Transcription Factor metabolism, Tumor Suppressor Proteins genetics, Tumor Suppressor Proteins metabolism, Asthma genetics, Bronchi pathology, DNA Methylation genetics, Epithelial Cells metabolism, Health, Hypersensitivity, Immediate genetics, Leukocytes, Mononuclear metabolism
- Abstract
Background: Allergic inflammation is commonly observed in a number of conditions that are associated with atopy including asthma, eczema and rhinitis. However, the genetic, environmental or epigenetic factors involved in these conditions are likely to be different. Epigenetic modifications, such as DNA methylation, can be influenced by the environment and result in changes to gene expression., Objectives: To characterize the DNA methylation pattern of airway epithelial cells (AECs) compared to peripheral blood mononuclear cells (PBMCs) and to discern differences in methylation within each cell type amongst healthy, atopic and asthmatic subjects., Methods: PBMCs and AECs from bronchial brushings were obtained from children undergoing elective surgery for non-respiratory conditions. The children were categorized as atopic, atopic asthmatic, non-atopic asthmatic or healthy controls. Extracted DNA was bisulfite treated and 1505 CpG loci across 807 genes were analyzed using the Illumina GoldenGate Methylation Cancer Panel I. Gene expression for a subset of genes was performed using RT-PCR., Results: We demonstrate a signature set of CpG sites that are differentially methylated in AECs as compared to PBMCs regardless of disease phenotype. Of these, 13 CpG sites were specific to healthy controls, 8 sites were only found in atopics, and 6 CpGs were unique to asthmatics. We found no differences in the methylation status of PBMCs between disease phenotypes. In AECs derived from asthmatics compared to atopics, 8 differentially methylated sites were identified including CpGs in STAT5A and CRIP1. We demonstrate STAT5A gene expression is decreased whereas CRIP1 gene expression is elevated in the AECs from asthmatic compared to both healthy and atopic subjects., Discussion: We characterized a cell specific DNA methylation signature for AECs compared to PBMCs regardless of asthmatic or atopic status. Our data highlight the importance of understanding DNA methylation in the epithelium when studying the epithelial contribution to asthma.
- Published
- 2012
- Full Text
- View/download PDF
26. Regional differences in susceptibiity of bronchial epithelium to mesenchymal transition and inhibition by the macrolide antibiotic azithromycin.
- Author
-
Banerjee B, Musk M, Sutanto EN, Yerkovich ST, Hopkins P, Knight DA, Lindsey-Temple S, Stick SM, Kicic A, and Chambers DC
- Subjects
- Adult, Aged, Bronchi metabolism, Cells, Cultured, Female, Humans, Lung Transplantation, Male, Middle Aged, Receptors, Transforming Growth Factor beta metabolism, Smad3 Protein metabolism, Transforming Growth Factor beta1 pharmacology, Anti-Bacterial Agents pharmacology, Azithromycin pharmacology, Bronchi cytology, Bronchi pathology, Epithelial-Mesenchymal Transition drug effects
- Abstract
Objective: Dysregulated repair following epithelial injury is a key forerunner of disease in many organs, and the acquisition of a mesenchymal phenotype by the injured epithelial cells (epithelial to mesenchymal transition, EMT) may serve as a source of fibrosis. The macrolide antibiotic azithromycin and the DNA synthesis inhibitor mycophenolate are in clinical use but their mechanism of action remains unknown in post-transplant bronchiolitis obliterans syndrome (BOS). Here we determined if regional variation in the EMT response to TGFβ1 underlies the bronchiolocentric fibrosis leading to BOS and whether EMT could be inhibited by azithromycin or mycophenolate., Methods/results: We found that small and large airway epithelial cells from stable lung transplant patients underwent EMT when stimulated with TGFβ1, however mesenchymal protein expression was higher and loss of epithelial protein expression more complete in small airway epithelial cells. This regional difference was not mediated by changes in expression of the TGFβRII or Smad3 activation. Azithromycin potentially inhibited EMT in both small and large airway epithelial cells by inhibiting Smad3 expression, but not activation., Conclusion: Collectively, these observations provide a biologic basis for a previously unexplained but widely observed clinical phenomena, and a platform for the development of new approaches to fibrotic diseases.
- Published
- 2012
- Full Text
- View/download PDF
27. The airway epithelium is a direct source of matrix degrading enzymes in bronchiolitis obliterans syndrome.
- Author
-
Banerjee B, Ling KM, Sutanto EN, Musk M, Yerkovich ST, Hopkins PM, Stick SM, Kicic A, and Chambers DC
- Subjects
- Adolescent, Adult, Bronchoalveolar Lavage, Bronchoscopy, Epithelium metabolism, Female, Gene Expression, Humans, Idiopathic Pulmonary Fibrosis surgery, Immunohistochemistry, Male, Matrix Metalloproteinase 2 metabolism, Matrix Metalloproteinase 9 metabolism, Middle Aged, Pulmonary Disease, Chronic Obstructive surgery, Pulmonary Emphysema surgery, Real-Time Polymerase Chain Reaction, Tissue Inhibitor of Metalloproteinase-2 metabolism, Young Adult, Bronchi cytology, Bronchiolitis Obliterans enzymology, Lung Transplantation
- Abstract
Background: Long-term survival after lung transplantation is hindered by the development of bronchiolitis obliterans syndrome (BOS), and recent evidence suggests that dysregulated epithelial repair may underlie its development. Because matrix metalloproteinase (MMP) -2 and MMP-9 secretion is integral to repair, we hypothesized that airway epithelial cells from patients with BOS would over-express these matrix-degrading enzymes., Methods: Cells obtained from bronchial and bronchiolar brushings from patients with and without BOS (without acute rejection or infection) were analyzed via quantitative polymerase chain reaction and immunocytochemistry for MMP-2, and MMP-9 gene and protein expression. The expression of tissue inhibitor of metalloproteinase (TIMP)2 and TIMP1 was also assessed. MMP activity in bronchoalveolar lavage was determined via gelatin zymography., Results: MMP-2 and MMP-9 production was significantly higher in bronchoalveolar lavage (3.85- and 11.59-fold, p < 0.001) and airway epithelium (MMP-2 bronchial: 6.33-fold, bronchiolar: 3.57-fold, both p < 0.001; MMP-9 bronchial: 32.55-fold, p < 0.001; bronchiolar: 8.60-fold, p = 0.01) in patients with BOS, but expression in patients without BOS was not different from healthy controls. TIMP expression was similar in patients with and without BOS. Immunostaining confirmed that the airway epithelium was a direct source of MMP-2 and MMP-9 expression in patients with BOS., Conclusion: In patients with BOS, the airway epithelium over-expresses MMPs, even in the absence of acute rejection or infection. Dysregulated epithelial repair may be a key feature of BOS., (Copyright © 2011 International Society for Heart and Lung Transplantation. All rights reserved.)
- Published
- 2011
- Full Text
- View/download PDF
28. Bronchial brushings for investigating airway inflammation and remodelling.
- Author
-
Looi K, Sutanto EN, Banerjee B, Garratt L, Ling KM, Foo CJ, Stick SM, and Kicic A
- Subjects
- Asthma diagnosis, Australia, Biopsy, Bronchoalveolar Lavage, Epithelial Cells pathology, Humans, Airway Remodeling, Asthma pathology, Bronchi pathology, Bronchoscopy methods
- Abstract
Asthma is the commonest medical cause for hospital admission for children in Australia, affects more than 300 million people worldwide, and is incurable, severe in large number and refractory to treatment in many. However, there have been no new significant treatments despite intense research and billions of dollars. The advancement in our understanding in this disease has been limited due to its heterogeneity, genetic complexity and has severely been hampered particularly in children by the difficulty in obtaining relevant target organ tissue. This review attempts to provide an overview of the currently used and recently developed/adapted techniques used to obtain lung tissue with specific reference to the airway epithelium., (© 2011 The Authors; Respirology © 2011 Asian Pacific Society of Respirology.)
- Published
- 2011
- Full Text
- View/download PDF
29. Innate inflammatory responses of pediatric cystic fibrosis airway epithelial cells: effects of nonviral and viral stimulation.
- Author
-
Sutanto EN, Kicic A, Foo CJ, Stevens PT, Mullane D, Knight DA, and Stick SM
- Subjects
- Apoptosis, Child, Child, Preschool, Cystic Fibrosis virology, Cytokines metabolism, Epithelial Cells virology, Female, Homozygote, Humans, Infant, Inflammation, Interferon-gamma metabolism, Interleukin-1beta metabolism, Lipopolysaccharides metabolism, Male, Rhinovirus metabolism, Tumor Necrosis Factor-alpha metabolism, Cystic Fibrosis metabolism, Epithelial Cells metabolism
- Abstract
There is controversy regarding whether cystic fibrosis (CF) airway epithelial cells (AECs) are intrinsically proinflammatory. The objective of the current study was to characterize the inflammatory profiles of AECs from children with CF compared with cells from healthy control subjects. We obtained AECs from healthy children (12) and children with CF (27). Biochemical and functional characteristics were assessed by stimulating cells with IFNγ, LPS, a cocktail referred to as cytomix, which consists of IFNγ, IL-1β, TNF-α, and LPS, or with human rhinovirus (HRV). Cytokine production was assessed using ELISA. Apoptotic responses to HRV infection were measured via production of single-stranded DNA. Our results indicated that CF and healthy cells exhibited similar morphology in monolayer culture. CF cells constitutively produced greater amounts of IL-6, IL-1β, and prostaglandin E(2), but similar levels of IL-8 and soluble intracellular adhesion molecule-1 compared with healthy cells, and this profile was maintained through repeated passage. Stimulation with LPS or cytomix elicited similar levels of IL-8 in CF and non-CF cells. In contrast, exposure to HRV1b resulted in a marked increase in IL-8 production from CF compared with non-CF cells. CF cells also exhibited reduced apoptosis and increased viral replication compared with non-CF cells after exposure to HRV1b. We conclude that CF and healthy AECs have similar basal and stimulated expression of IL-8 in response to proinflammatory stimuli, but elevated IL-8 release in response to HRV infection. The elevated IL-8, together with dampened apoptotic responses by CF cells to HRV, could contribute to augmented airway inflammation in the setting of recurrent viral infections early in life.
- Published
- 2011
- Full Text
- View/download PDF
30. Identifying peroxidases and their oxidants in the early pathology of cystic fibrosis.
- Author
-
Thomson E, Brennan S, Senthilmohan R, Gangell CL, Chapman AL, Sly PD, Kettle AJ, Balding E, Berry LJ, Carlin JB, Carzino R, de Klerk N, Douglas T, Foo C, Garratt LW, Hall GL, Harrison J, Kicic A, Laing IA, Logie KM, Massie J, Mott LS, Murray C, Parsons F, Pillarisetti N, Poreddy SR, Ranganathan SC, Robertson CF, Robins-Browne R, Robinson PJ, Skoric B, Stick SM, Sutanto EN, and Williamson E
- Subjects
- Bronchoalveolar Lavage Fluid cytology, Child, Child, Preschool, Cystic Fibrosis complications, Cystic Fibrosis physiopathology, Disease Progression, Female, Humans, Infant, Inflammation, Male, Neutrophils pathology, Oxidation-Reduction, Pseudomonas aeruginosa pathogenicity, Respiratory Tract Infections complications, Respiratory Tract Infections physiopathology, Tyrosine analogs & derivatives, Tyrosine analysis, Bronchoalveolar Lavage Fluid chemistry, Cystic Fibrosis enzymology, Neutrophils metabolism, Peroxidase metabolism, Pseudomonas aeruginosa immunology, Respiratory Tract Infections enzymology
- Abstract
We aimed to determine whether myeloperoxidase (MPO) is the main peroxidase present in the airways of children with cystic fibrosis (CF) and to assess which oxidants it produces and whether they are associated with clinical features of CF. Children with CF (n=54) and without CF (n=16) underwent bronchoscopy and bronchoalveolar lavage (BAL) for assessment of pulmonary infection and inflammation. BAL fluid was analyzed for MPO, halogenated tyrosines as markers of hypohalous acids, thiocyanate, and protein carbonyls. MPO was the only peroxidase detected in BAL samples from children with CF and its concentration was markedly higher than in controls. Levels of 3-chlorotyrosine and 3-bromotyrosine in proteins were higher in the CF group. They correlated with neutrophils and MPO. The concentration of thiocyanate in BAL samples was below 1μM. Protein carbonyl levels correlated with MPO and halogenated tyrosines in patients with CF. Levels of MPO and halogenated tyrosines were higher in children with infections, especially Pseudomonas aeruginosa, and in the presence of respiratory symptoms. They also correlated with the Kanga clinical score. Our findings suggest that MPO produces hypobromous acid as well as hypochlorous acid in the airways of children with CF and that these oxidants are involved in the early pathogenesis of CF., (Copyright © 2010 Elsevier Inc. All rights reserved.)
- Published
- 2010
- Full Text
- View/download PDF
31. Successful establishment of primary small airway cell cultures in human lung transplantation.
- Author
-
Banerjee B, Kicic A, Musk M, Sutanto EN, Stick SM, and Chambers DC
- Subjects
- Adolescent, Adult, Bronchiolitis Obliterans etiology, Bronchoscopy, Cell Lineage, Cell Proliferation, Cells, Cultured, Epithelial Cells metabolism, Female, Humans, Lung pathology, Male, Middle Aged, Odds Ratio, Uteroglobin metabolism, Bronchiolitis Obliterans pathology, Cell Culture Techniques, Epithelial Cells pathology, Lung surgery, Lung Transplantation adverse effects
- Abstract
Background: The study of small airway diseases such as post-transplant bronchiolitis obliterans syndrome (BOS) is hampered by the difficulty in assessing peripheral airway function either physiologically or directly. Our aims were to develop robust methods for sampling small airway epithelial cells (SAEC) and to establish submerged SAEC cultures for downstream experimentation., Methods: SAEC were obtained at 62 post-transplant bronchoscopies in 26 patients using radiologically guided bronchial brushings. Submerged cell cultures were established and SAEC lineage was confirmed using expression of clara cell secretory protein (CCSP)., Results: The cell yield for SAEC (0.956 +/- 0.063 x 106) was lower than for large airway cells (1.306 +/- 0.077 x 106) but did not significantly impact on the culture establishment rate (79.0 +/- 5.2% vs. 83.8 +/- 4.7% p = 0.49). The presence of BOS significantly compromised culture success (independent of cell yield) for SAEC (odds ratio (95%CI) 0.067 (0.01-0.40)) but not LAEC (0.3 (0.05-1.9)). Established cultures were successfully passaged and expanded., Conclusion: Primary SAEC can be successfully obtained from human lung transplant recipients and maintained in culture for downstream experimentation. This technique will facilitate the development of primary in vitro models for BOS and other diseases with a small airway component such as asthma, cystic fibrosis and COPD.
- Published
- 2009
- Full Text
- View/download PDF
32. Dysregulated repair in asthmatic paediatric airway epithelial cells: the role of plasminogen activator inhibitor-1.
- Author
-
Stevens PT, Kicic A, Sutanto EN, Knight DA, and Stick SM
- Subjects
- Asthma metabolism, Bronchi metabolism, Cells, Cultured, Child, Child, Preschool, Female, Gene Silencing, Humans, Male, Plasminogen Activator Inhibitor 1 genetics, Asthma pathology, Bronchi pathology, Plasminogen Activator Inhibitor 1 physiology, Respiratory Mucosa pathology, Respiratory Mucosa physiology, Wound Healing
- Abstract
Background: Asthma is associated with structural changes to airways such as extracellular matrix deposition and epithelial damage. Evidence suggests that asthmatic airway epithelial repair is abnormal and that elevated plasminogen activator inhibitor-1 levels observed in asthma may be involved in the epithelial repair process and in excessive matrix accumulation., Objective: To assess the ability of asthmatic airway epithelial cells (AECs) to repair mechanically induced wounds and to investigate the role that plasminogen activator inhibitor-1 plays in the repair process., Methods: AECs were isolated from atopic asthmatic and healthy non-atopic children by bronchial brushing, subcultured and wound repair experiments were performed. Plasminogen activator inhibitor-1 gene expression was assessed using real-time PCR while protein activity was measured in cell lysates as well as plasma. The role of plasminogen activator inhibitor-1 in epithelial proliferation and wound repair was investigated using siRNA., Results: Cells from asthmatic children have a significantly longer repair time in comparison with cells from otherwise healthy donors. Plasminogen activator inhibitor-1 mRNA expression was up-regulated 68-fold in freshly isolated asthmatic cells compared with normal cells, and protein levels were also significantly elevated in the asthmatic cell lysates, but plasma levels were similar in both groups. Plasminogen activator inhibitor-1 cells expression increased in both cohorts during culture. Gene silencing substantially reduced the rate of proliferation in asthmatic and healthy cells. Mechanical wounding of epithelial monolayers induced plasminogen activator inhibitor-1 expression in asthmatic and non-asthmatic cohorts, while gene silencing delayed wound repair of healthy cell, with minimal effect on those from asthmatics., Conclusion: Asthmatic AECs are inherently dysfunctional in their ability to repair wounds; plasminogen activator inhibitor-1 mRNA and protein activity are constitutively up-regulated in asthmatic epithelium and play functional roles in both proliferation and repair of healthy cells. In asthmatic cells, elevated plasminogen activator inhibitors-1 levels fail to stimulate epithelial repair.
- Published
- 2008
- Full Text
- View/download PDF
33. Comparison of techniques for obtaining lower airway epithelial cells from children.
- Author
-
McNamara PS, Kicic A, Sutanto EN, Stevens PT, and Stick SM
- Subjects
- Adolescent, Bronchi cytology, Cell Count, Cells, Cultured, Child, Child, Preschool, Humans, Male, Asthma, Biopsy methods, Bronchoscopy methods, Epithelial Cells
- Abstract
Airway epithelial cells (AECs) are important in asthma as they are the first cells to encounter pathogens/allergens. In children, AECs can be obtained using a "blind" nonbronchoscopic technique through an endotracheal tube. However, due to the increasing use of laryngeal masks the number of children in whom this technique is applicable has become limited. Recently, the present authors began to use a portable "bronchoscope-directed" technique to sample AECs. The current study compares both techniques in both asthmatic and nonasthmatic children. A total of 81 children undergoing elective surgery, were grouped according to atopic status and respiratory symptoms. Cellular yield of blind and bronchoscope-directed brushings were compared and immunocytochemistry performed. AECs were cultured and cytokine analysis of culture supernatant undertaken. Both techniques were equally well-tolerated, with the only adverse effect being a cough in 10% of the subjects. The mean+/-SD cell yield was higher in bronchoscope-directed than blind brushings (5.1+/-2.4 versus 3.1+/-1.4x10(6) cells). Immunocytochemistry confirmed an epithelial cell lineage. Culture supernatant cytokine concentrations were similar regardless of sampling technique with patterns preserved between asthmatic and healthy nonatopic phenotypes. Compared with blind brushing portable bronchoscope-directed brushing is well-tolerated, yields significantly more cells and is a potentially quick and useful technique for obtaining airway epithelial cells for research into childhood respiratory disease, specifically asthma.
- Published
- 2008
- Full Text
- View/download PDF
34. Potential use of cellular promoter(s) to target RPE in AAV-mediated delivery. Cellular promoters and RPE-targeting.
- Author
-
Sutanto EN, Zhang D, Lai YK, Shen WY, and Rakoczy PE
- Subjects
- Cell Line, Cell Separation, Flow Cytometry, Genetic Vectors, Green Fluorescent Proteins chemistry, Humans, Immunohistochemistry, Models, Genetic, Plasmids metabolism, Promoter Regions, Genetic, Transcriptional Activation, Dependovirus genetics, Gene Transfer Techniques, Genetic Therapy methods, Pigment Epithelium of Eye metabolism
- Published
- 2006
- Full Text
- View/download PDF
35. Development and evaluation of the specificity of a cathepsin D proximal promoter in the eye.
- Author
-
Sutanto EN, Zhang D, Lai YK, Shen WY, and Rakoczy EP
- Subjects
- Animals, Cell Culture Techniques, Cytomegalovirus genetics, Dependovirus genetics, Flow Cytometry, Genetic Therapy, Green Fluorescent Proteins metabolism, Humans, Microscopy, Fluorescence, Middle Aged, Plasmids, Rats, Rats, Mutant Strains, Retina metabolism, Sensitivity and Specificity, Transduction, Genetic, Transfection, Cathepsin D genetics, Gene Expression physiology, Green Fluorescent Proteins genetics, Pigment Epithelium of Eye metabolism, Promoter Regions, Genetic, Retinitis Pigmentosa metabolism
- Abstract
Purpose: Recombinant adeno-associated virus (rAAV)-mediated gene delivery has emerged as a valuable tool for alternative treatment of ocular diseases. Cellular specificity of transgene expression could be influenced by either the viral capsid or the choice of promoter. The use of cellular promoter, cathepsin D (CatD) proximal promoter, and its potential for application in rAAV-based gene therapy are evaluated in this study., Materials and Methods: Different sizes of CatD proximal promoter fragments -769 to -1 (CD768), -366 to -1 (CD365), -253 to -1 (CD252), and -124 to -1 (CD123) were subcloned upstream of the green fluorescent protein (GFP) gene. The specificity and activity of the promoter were tested in vitro using human retinal pigment epithelium (RPE) cell lines (RPE51, D407), with the human fibroblast cell line (F2000) used as control. The promoter fragment that showed higher activity in RPE cells was chosen to generate rAAV vector based on AAV serotype 2. The ability of CatD promoter to target transgene expression to RPE in vivo was determined following subretinal delivery of rAAV particles into nonpigmented RCS/rdy+ rats., Results: In vitro studies showed that the proximal promoter fragment CD365 targeted high GFP expression in RPE cells. This fragment was then used to generate the AAV.CD365.gfp construct. It was shown in vivo that following subretinal injection, the CD365 fragment in AAV.CD365.gfp directed GFP expression preferentially into RPE cells. Relatively lower level of GFP expression was detected in the neuroretina. In contrast, injection of control virus (AAV.CMV.gfp) resulted in equal levels of transduction and fluorescence signal intensity in both the RPE and photoreceptor cells., Conclusions: The results of our study demonstrate that the promoter fragment CD365 has the potential to target preferential gene expression in the RPE following subretinal injection in rAAV-mediated gene therapy.
- Published
- 2005
- Full Text
- View/download PDF
36. Concurrent enhancement of transcriptional activity and specificity of a retinal pigment epithelial cell-preferential promoter.
- Author
-
Zhang D, Sutanto EN, and Rakoczy PE
- Subjects
- Animals, Carrier Proteins, Cell Line, DNA-Binding Proteins genetics, Eye Proteins, Humans, Luciferases genetics, Middle Aged, RNA, Messenger metabolism, Trans-Activators genetics, Transcription, Genetic, Transfection, Transgenes, cis-trans-Isomerases, Pigment Epithelium of Eye metabolism, Promoter Regions, Genetic genetics, Proteins genetics, Transcriptional Activation
- Abstract
Purpose: To develop a transgene expression system in retinal pigment epithelial cells with the aim of enhancing the transcriptional activity of a weak RPE-specific/preferential promoter., Methods: The transgene expression system was established by introducing a chimeric transcriptional activator (GAL4-VP16) and its DNA binding sequence and using truncated human and mouse RPE65 promoters in combination with a luciferase reporter gene. Two groups of expression plasmids were constructed for transfection. The group for co-transfection contained two DNA constructs where the reporter and GAL4-VP16 were separately expressed in pLuc and pGV series. The other group, pLuc-GV series, was prepared as single DNA constructs expressing both the reporter and GAL4-VP16. The transcriptional activities of the DNA constructs were assayed by transfection of human RPE cells (RPE51 and D407) and other cell lines (HEK293, COS-1, Hela, HepG2, and F2000)., Results: We found that the transcriptional activity of the human RPE65 promoter was dramatically enhanced 10-13 fold in RPE cells co-transfected with DNA constructs phR65luc and phR65GV when compared to the human RPE65 promoter alone. A comparatively lower, 4-5 fold, increase was observed following transfection with the single DNA construct phR65luc-GV. In RPE cells, when the transcriptional responses to GAL4-VP16 expression were compared between the RPE65 promoter of phR65luc and the minimal promoter of pLuc, the increase in transcriptional activity was about 10 fold higher in phR65luc constructs. Low or non-significant enhancement of promoter activity was observed with these constructs following transfection of the non-RPE cell lines., Conclusions: Our results indicate that the current transgene expression system dramatically amplifies transcriptional activity of weak and cell-specific/preferential promoters (e.g., the hRPE65 promoter) whilst retaining relative cell specificity.
- Published
- 2004
37. Practical considerations of recombinant adeno-associated virus-mediated gene transfer for treatment of retinal degenerations.
- Author
-
Shen WY, Lai CM, Lai YK, Zhang D, Zaknich T, Sutanto EN, Constable IJ, and Rakoczy PE
- Subjects
- Animals, Brain metabolism, Cell Line, Defective Viruses genetics, Genetic Vectors, Green Fluorescent Proteins, Helper Viruses genetics, Heparin genetics, Humans, Luminescent Proteins, Mice, Mice, Mutant Strains, Photoreceptor Cells metabolism, Pigment Epithelium of Eye metabolism, Rats, Recombinant Fusion Proteins metabolism, Transgenes, Dependovirus genetics, Gene Transfer Techniques, Genetic Therapy, Retinal Degeneration therapy
- Abstract
Background: Photoreceptor (PR) and retinal pigment epithelium (RPE) are the principal cell targets in retinal gene therapy. Recombinant adeno-associated virus (rAAV) has emerged as a very promising vector for gene therapy in hereditary retinal diseases. Gene transfer at different stages of the disease is a practical consideration for future clinical application., Methods: A rAAV carrying the enhanced green fluorescent protein gene driven by a cytomegalovirus promoter was produced by either co-infecting the 293 cell line with E1-defective adenovirus and purified by CsCl(2) density gradient (CsCl(2)-rAAV), or by transfecting with an adenoviral helper plasmid and purified by iodixanol density gradient followed by heparin column chromatography (heparin-rAAV). The impact of different virus preparations on the patterns of transgene expression was investigated after subretinal injection. Furthermore, rAAV-mediated gene transfer was evaluated at both early and advanced stages of retinal degeneration in four disease models including the RCS rat, rd, RPE(65) (-)/(-) and cathepsin D mutant mice that are associated with PR- or RPE-related gene defects., Results: CsCl(2)-rAAV predominantly transduced RPE and with less efficiency in PR. In contrast, heparin-rAAV predominantly transduced PR but with much less efficiency in RPE. Subretinal injection of either rAAV preparation induced no changes to retinal morphology and retinal-choroidal vasculature. The product of transgene, however, could be observed in multiple tracts in the brain. In the four disease models, target cells were efficiently transduced not only at the early stage, but also at the late stage of disease as long as the target cells were present., Conclusions: Different preparations of rAAV have an impact on the patterns of transgene expression after subretinal injection. Patients at advanced stages of retinal degeneration may still benefit from rAAV-mediated gene therapy. The possible side effects of transgenic products on the central nervous system should be carefully monitored once therapeutic genes are employed., (Copyright 2003 John Wiley & Sons, Ltd.)
- Published
- 2003
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.