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1.
Am J Respir Cell Mol Biol ; 49(3): 471-80, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23614789

ABSTRACT

Receptor-targeted nanocomplexes are nonviral vectors developed for gene delivery to the airway epithelium for the treatment of pulmonary disease associated with cystic fibrosis. The present study aimed to optimize the delivery of the nanocomplex by nebulization, and to monitor the in vivo deposition of radiolabeled vector in the airways of a large animal model by γ-camera scintigraphy. Large White weaner pigs were nebulized with nanocomplexes mixed with technetium-99m radiopharmaceuticals. The aerosol deposition scans suggested that the nebulized radiovectors were deposited mainly in the trachea-main bronchi and in the midregion of the lungs. The plasmid biodistribution, assessed by real-time PCR, correlated with the scintigraphy images. The highest plasmid copy numbers were found in the bronchial areas and in the tissues proximal to the main bronchi bifurcation. Immunohistochemistry detected transgene expression in the tracheal and bronchial ciliated epithelium. Histological analysis of lung tissue showed no evidence of inflammation, and no increase in inflammatory cytokines or inflammatory cells was detected in the bronchoalveolar lavage. The deposition of nebulized nanocomplexes coassociated with technetium-99m can be monitored by nuclear medicine techniques. The use of a noninvasive strategy to follow the delivery of the vector could improve the clinical management of patients undergoing cystic fibrosis gene therapy.


Subject(s)
Gene Transfer Techniques , Molecular Imaging/methods , Radiopharmaceuticals/pharmacokinetics , Respiratory Mucosa/diagnostic imaging , Respiratory System/diagnostic imaging , Technetium/pharmacokinetics , Animals , Cystic Fibrosis/diagnostic imaging , Female , Genetic Therapy , Humans , Injections, Intravenous , Male , Nanoconjugates/administration & dosage , Nanoconjugates/chemistry , Nebulizers and Vaporizers , Plasmids , Radionuclide Imaging , Radiopharmaceuticals/administration & dosage , Respiratory Mucosa/ultrastructure , Respiratory System/ultrastructure , Swine , Technetium/administration & dosage
2.
PLoS One ; 6(10): e26768, 2011.
Article in English | MEDLINE | ID: mdl-22046351

ABSTRACT

BACKGROUND: Gene therapy mediated by synthetic vectors may provide opportunities for new treatments for cystic fibrosis (CF) via aerosolisation. Vectors for CF must transfect the airway epithelium efficiently and not cause inflammation so they are suitable for repeated dosing. The inhaled aerosol should be deposited in the airways since the cystic fibrosis transmembrane conductance regulator gene (CFTR) is expressed predominantly in the epithelium of the submucosal glands and in the surface airway epithelium. The aim of this project was to develop an optimised aerosol delivery approach applicable to treatment of CF lung disease by gene therapy. METHODOLOGY: The vector suspension investigated in this study comprises receptor-targeting peptides, cationic liposomes and plasmid DNA that self-assemble by electrostatic interactions to form a receptor-targeted nanocomplex (RTN) of approximately 150 nm with a cationic surface charge of +50 mV. The aerodynamic properties of aerosolised nanocomplexes produced with three different nebulisers were compared by determining aerosol deposition in the different stages of a Next Generation Pharmaceutical Impactor (NGI). We also investigated the yield of intact plasmid DNA by agarose gel electrophoresis and densitometry, and transfection efficacies in vitro and in vivo. RESULTS: RTNs nebulised with the AeroEclipse II BAN were the most effective, compared to other nebulisers tested, for gene delivery both in vitro and in vivo. The biophysical properties of the nanocomplexes were unchanged after nebulisation while the deposition of RTNs suggested a range of aerosol aerodynamic sizes between 5.5 µm-1.4 µm cut off (NGI stages 3-6) compatible with deposition in the central and lower airways. CONCLUSIONS: RTNs showed their ability at delivering genes via nebulisation, thus suggesting their potential applications for therapeutic interventions of cystic fibrosis and other respiratory disorders.


Subject(s)
Gene Transfer Techniques , Genetic Therapy/methods , Nebulizers and Vaporizers , Respiratory Mucosa/metabolism , Cystic Fibrosis/therapy , Epithelium/metabolism , Molecular Targeted Therapy , Nanocomposites/administration & dosage , Plasmids/administration & dosage , Transfection
3.
PLoS One ; 5(3): e9674, 2010 Mar 12.
Article in English | MEDLINE | ID: mdl-20300191

ABSTRACT

The TGF-beta family of mediators are thought to play important roles in the regulation of inflammation and airway remodelling in asthma. All three mammalian isoforms of TGF-beta, TGF-beta(1-3), are expressed in the airways and TGF-beta(1) and -beta(2) are increased in asthma. However, there is little information on the specific roles of individual TGF-beta isoforms. In this study we assess the roles of TGF-beta(1) and TGF-beta(2) in the regulation of allergen-induced airway inflammation and remodelling associated with asthma, using a validated murine model of ovalbumin sensitization and challenge, and isoform specific TGF-beta neutralising antibodies. Antibodies to both isoforms inhibited TGF-beta mediated Smad signalling. Anti-TGF-beta(1) and anti-TGF-beta(2) inhibited ovalbumin-induced sub-epithelial collagen deposition but anti-TGF-beta(1) also specifically regulated airway and fibroblast decorin deposition by TGF-beta(1). Neither antibody affected the allergen-induced increase in sub-epithelial fibroblast-like cells. Anti- TGF-beta(1) also specifically inhibited ovalbumin-induced increases in monocyte/macrophage recruitment. Whereas, both TGF-beta(1) and TGF-beta(2) were involved in regulating allergen-induced increases in eosinophil and lymphocyte numbers. These data show that TGF-beta(1) and TGF-beta(2) exhibit a combination of specific and shared roles in the regulation of allergen-induced airway inflammation and remodelling. They also provide evidence in support of the potential for therapeutic regulation of specific subsets of cells and extracellular matrix proteins associated with inflammation and remodelling in airway diseases such as asthma and COPD, as well as other fibroproliferative diseases.


Subject(s)
Asthma/metabolism , Transforming Growth Factor beta/metabolism , Animals , Bronchoalveolar Lavage Fluid , Collagen/chemistry , Eosinophils/metabolism , Extracellular Matrix/metabolism , Image Processing, Computer-Assisted , Immunohistochemistry , Inflammation , Lung/metabolism , Lymphocytes/cytology , Lymphocytes/metabolism , Mice , Mice, Inbred C57BL , Protein Isoforms
4.
Am J Respir Crit Care Med ; 182(1): 73-82, 2010 Jul 01.
Article in English | MEDLINE | ID: mdl-20203246

ABSTRACT

RATIONALE: Patients with idiopathic pulmonary fibrosis (IPF), a progressive disease with a dismal prognosis, exhibit an unexplained disparity of increased alveolar epithelial cell (AEC) apoptosis but reduced fibroblast apoptosis. OBJECTIVES: To examine whether the failure of patients with IPF to up-regulate cyclooxygenase (COX)-2, and thus the antifibrotic mediator prostaglandin (PG)E(2), accounts for this imbalance. METHODS: Fibroblasts and primary type II AECs were isolated from control and fibrotic human lung tissue. The effects of COX-2 inhibition and exogenous PGE(2) on fibroblast and AEC sensitivity to Fas ligand (FasL)-induced apoptosis were assessed. MEASUREMENTS AND MAIN RESULTS: IPF lung fibroblasts are resistant to FasL-induced apoptosis compared with control lung fibroblasts. Inhibition of COX-2 in control lung fibroblasts resulted in an apoptosis-resistant phenotype. Administration of PGE(2) almost doubled the rate of FasL-induced apoptosis in fibrotic lung fibroblasts compared with FasL alone. Conversely, in primary fibrotic lung type II AECs, PGE(2) protected against FasL-induced apoptosis. In human control and, to a greater extent, fibrotic lung fibroblasts, PGE(2) inhibits the phosphorylation of Akt, suggesting that regulation of this prosurvival protein kinase is an important mechanism by which PGE(2) modulates cellular apoptotic responses. CONCLUSIONS: The observation that PGE(2) deficiency results in increased AEC but reduced fibroblast sensitivity to apoptosis provides a novel pathogenic insight into the mechanisms driving persistent fibroproliferation in IPF.


Subject(s)
Apoptosis/physiology , Cyclooxygenase 2/physiology , Dinoprostone/physiology , Fibroblasts/physiology , Idiopathic Pulmonary Fibrosis/physiopathology , Adult , Aged , Aged, 80 and over , Case-Control Studies , Cells, Cultured , Epithelial Cells/physiology , Female , Humans , Male , Middle Aged , Pulmonary Alveoli/physiology , Wound Healing/physiology
5.
Mol Ther ; 16(5): 907-15, 2008 May.
Article in English | MEDLINE | ID: mdl-18388925

ABSTRACT

Synthetic vectors for cystic fibrosis (CF) gene therapy are required that efficiently and safely transfect airway epithelial cells, rather than alveolar epithelial cells or macrophages, and that are nonimmunogenic, thus allowing for repeated delivery. We have compared several vector systems against these criteria including GL67, polyethylenimine (PEI) 22 and 25 kd and two new, synthetic vector formulations, comprising a cationic, receptor-targeting peptide K(16)GACSERSMNFCG (E), and the cationic liposomes (L) DHDTMA/DOPE or DOSEP3/DOPE. The lipid and peptide formulations self assemble into receptor-targeted nanocomplexes (RTNs) LED-1 and LED-2, respectively, on mixing with plasmid (D). LED-1 transfected airway epithelium efficiently, while LED-2 and GL67 preferentially transfected alveolar cells. PEI transfected airway epithelial cells with high efficiency, but was more toxic to the mice than the other formulations. On repeat dosing, LED-1 was equally as effective as the single dose, while GL67 was 30% less effective and PEI 22 kd displayed a 90% reduction of efficiency on repeated delivery. LED-1 thus was the only formulation that fulfilled the criteria for a CF gene therapy vector while GL67 and LED-2 may be appropriate for other respiratory diseases. Opportunities for PEI depend on a solution to its toxicity problems. LED-1 formulations were stable to nebulization, the most appropriate delivery method for CF.


Subject(s)
Cystic Fibrosis/therapy , Gene Transfer Techniques , Genetic Therapy/methods , Nanotechnology/methods , Animals , Caspase 3/metabolism , Cations , Female , Genetic Therapy/instrumentation , Genetic Vectors , Humans , Lung/metabolism , Macrophages/metabolism , Mice , Mice, Inbred C57BL , Models, Biological
6.
PLoS Pathog ; 3(9): 1208-19, 2007 Sep 28.
Article in English | MEDLINE | ID: mdl-17845072

ABSTRACT

The physiological functions of the acute phase protein serum amyloid P (SAP) component are not well defined, although they are likely to be important, as no natural state of SAP deficiency has been reported. We have investigated the role of SAP for innate immunity to the important human pathogen Streptococcus pneumoniae. Using flow cytometry assays, we show that SAP binds to S. pneumoniae, increases classical pathway-dependent deposition of complement on the bacteria, and improves the efficiency of phagocytosis. As a consequence, in mouse models of infection, mice genetically engineered to be SAP-deficient had an impaired early inflammatory response to S. pneumoniae pneumonia and were unable to control bacterial replication, leading to the rapid development of fatal infection. Complement deposition, phagocytosis, and control of S. pneumoniae pneumonia were all improved by complementation with human SAP. These results demonstrate a novel and physiologically significant role for SAP for complement-mediated immunity against an important bacterial pathogen, and provide further evidence for the importance of the classical complement pathway for innate immunity.


Subject(s)
Complement System Proteins/physiology , Immunity, Innate/physiology , Serum Amyloid P-Component/physiology , Streptococcus pneumoniae/immunology , Animals , Complement Activation/physiology , Complement C3b/physiology , Disease Models, Animal , Immunity, Innate/immunology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Pneumococcal Infections/immunology , Serum Amyloid P-Component/genetics , Streptococcus pneumoniae/pathogenicity
7.
Respir Res ; 6: 30, 2005 Apr 08.
Article in English | MEDLINE | ID: mdl-15819978

ABSTRACT

BACKGROUND: Sub-epithelial extracellular matrix deposition is a feature of asthmatic airway remodelling associated with severity of disease, decline in lung function and airway hyperresponsiveness. The composition of, and mechanisms leading to, this increase in subepithelial matrix, and its importance in the pathogenesis of asthma are unclear. This is partly due to limitations of the current models and techniques to assess airway remodelling. METHODS: In this study we used a modified murine model of ovalbumin sensitisation and challenge to reproduce features of airway remodelling, including a sustained increase in sub-epithelial matrix deposition. In addition, we have established techniques to accurately and specifically measure changes in sub-epithelial matrix deposition, using histochemical and immunohistochemical staining in conjunction with digital image analysis, and applied these to the measurement of collagen and proteoglycans. RESULTS: 24 hours after final ovalbumin challenge, changes similar to those associated with acute asthma were observed, including inflammatory cell infiltration, epithelial cell shedding and goblet cell hyperplasia. Effects were restricted to the bronchial and peribronchial regions with parenchymal lung of ovalbumin sensitised and challenged mice appearing histologically normal. By 12 days, the acute inflammatory changes had largely resolved and increased sub-epithelial staining for collagen and proteoglycans was observed. Quantitative digital image analysis confirmed the increased deposition of sub-epithelial collagen (33%, p < 0.01) and proteoglycans (32%, p < 0.05), including decorin (66%, p < 0.01). In addition, the increase in sub-epithelial collagen deposition was maintained for at least 28 days (48%, p < 0.001). CONCLUSION: This animal model reproduces many of the features of airway remodelling found in asthma and allows accurate and reproducible measurement of sub-epithelial extra-cellular matrix deposition. As far as we are aware, this is the first demonstration of increased sub-epithelial proteoglycan deposition in an animal model of airway remodelling. This model will be useful for measurement of other matrix components, as well as for assessment of the molecular mechanisms contributing to, and agents to modulate airway remodelling.


Subject(s)
Asthma/metabolism , Collagen/metabolism , Disease Models, Animal , Lung/metabolism , Proteoglycans/metabolism , Acute Disease , Animals , Asthma/chemically induced , Extracellular Matrix/metabolism , Mice , Mice, Inbred C57BL , Ovalbumin , Respiratory Mucosa/metabolism , Tissue Distribution
8.
Am J Pathol ; 165(5): 1663-76, 2004 Nov.
Article in English | MEDLINE | ID: mdl-15509536

ABSTRACT

Levels of prostaglandin E(2) (PGE(2)), a potent inhibitor of fibroblast function, are decreased in the lungs of patients with pulmonary fibrosis, which has been shown to be because of limited expression of cyclooxygenase-2 (COX-2). To further investigate the relative importance of COX-2 and PGE(2) in the development of fibrosis we have used a selective COX-2 inhibitor and COX-2-deficient ((-/-) and (+/-)) mice in studies of bleomycin-induced lung fibrosis. We demonstrate in wild-type mice that bleomycin-induced lung PGE(2) production is predominantly COX-2 mediated. Furthermore, COX-2(+/-) mice show limited induction of PGE(2) and an enhanced fibrotic response with increased lung collagen content compared with wild-type mice after bleomycin injury (P < 0.001). In contrast, COX-2(-/-) mice show increased levels of lung PGE(2), compared with wild-type mice after injury (P < 0.05), because of compensatory up-regulation of COX-1, which appears to be associated with macrophage/monocytes but not fibroblasts derived from these mice. COX-2(-/-) mice show an enhanced and persistent inflammatory response to bleomycin, however the fibrotic response to injury was unaltered compared with wild-type animals. These data provide further direct evidence for the importance of up-regulating COX-2 and PGE(2) expression in protecting against the development of fibrosis after lung injury.


Subject(s)
Dinoprostone/biosynthesis , Isoenzymes/physiology , Lung Injury , Prostaglandin-Endoperoxide Synthases/physiology , Animals , Bleomycin/pharmacology , Blotting, Western , Bronchoalveolar Lavage , Collagen/metabolism , Cyclooxygenase 2 , Dinoprostone/metabolism , Female , Fibroblasts/metabolism , Fibrosis/pathology , Heterozygote , Immunohistochemistry , Isoenzymes/genetics , Leukotrienes/metabolism , Lung/pathology , Macrophages/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Monocytes/metabolism , Prostaglandin-Endoperoxide Synthases/genetics , Time Factors , Up-Regulation
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