[show abstract][hide abstract] ABSTRACT: The response of S-phase cells labelled with
bromodeoxyuridine (BrdU) in sheep airways undergoing repair
in response to endobronchial brush biopsy was investigated
in this study. Separate sites within the airway tree of anaesthetised
sheep were biopsied at intervals prior to pulse labelling with
BrdU, which was administered one hour prior to euthanasia.
Both brushed and spatially disparate unbrushed (control) sites
were carefully mapped, dissected, and processed to facilitate
histological analysis of BrdU labelling. Our study indicated
that the number and location of BrdU-labelled cells varied
according to the age of the repairing injury. There was little
evidence of cell proliferation in either control airway tissues
or airway tissues examined six hours after injury. However,
by days 1 and 3, BrdU-labelled cells were increased
in number in the airway wall, both at the damaged site
and in the regions flanking either side of the injury. Thereafter,
cell proliferative activity largely declined by day 7 after injury,
when consistent evidence of remodelling in the airway wall could
be appreciated. This study successfully demonstrated the
effectiveness of in vivo pulse labelling in tracking cell
proliferation during repair which has a potential value in
exploring the therapeutic utility of stem cell approaches
in relevant lung disease models.
[show abstract][hide abstract] ABSTRACT: Chronic lung infection with Pseudomonas aeruginosa is a major contributor to morbidity, mortality and premature death in cystic fibrosis. A new paradigm for managing such infections is needed, as are relevant and translatable animal models to identify and test concepts. We sought to improve on limitations associated with existing models of infection in small animals through developing a lung segmental model of chronic Pseudomonas infection in sheep.
Using local lung instillation of P. aeruginosa suspended in agar beads we were able to demonstrate that such infection led to the development of a suppurative, necrotising and pyogranulomatous pneumonia centred on the instilled beads. No overt evidence of organ or systemic compromise was apparent in any animal during the course of infection. Infection persisted in the lungs of individual animals for as long as 66 days after initial instillation. Quantitative microbiology applied to bronchoalveolar lavage fluid derived from infected segments proved an insensitive index of the presence of significant infection in lung tissue (>10(4) cfu/g).
The agar bead model of chronic P. aeruginosa lung infection in sheep is a relevant platform to investigate both the pathobiology of such infections as well as novel approaches to their diagnosis and therapy. Particular ethical benefits relate to the model in terms of refining existing approaches by compromising a smaller proportion of the lung with infection and facilitating longitudinal assessment by bronchoscopy, and also potentially reducing animal numbers through facilitating within-animal comparisons of differential therapeutic approaches.
PLoS ONE 01/2013; 8(7):e67677. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Understanding the way in which the airway heals in response to injury is fundamental to dissecting the mechanisms underlying airway disease pathology. As only limited data is available in relation to the in vivo characterisation of the molecular features of repair in the airway we sought to characterise the dynamic changes in gene expression that are associated with the early response to physical injury in the airway wall.
We profiled gene expression changes in the airway wall using a large animal model of physical injury comprising bronchial brush biopsy in anaesthetised sheep. The experimental design featured sequential studies in the same animals over the course of a week and yielded data relating to the response at 6 hours, and 1, 3 and 7 days after injury. Notable features of the transcriptional response included the early and sustained preponderance of down-regulated genes associated with angiogenesis and immune cell activation, selection and differentiation. Later features of the response included the up-regulation of cell cycle genes at d1 and d3, and the latter pronounced up-regulation of extracellular matrix-related genes at d3 and d7.
It is possible to follow the airway wall response to physical injury in the same animal over the course of time. Transcriptional changes featured coordinate expression of functionally related genes in a reproducible manner both within and between animals. This characterisation will provide a foundation against which to assess the perturbations that accompany airway disease pathologies of comparative relevance.
PLoS ONE 01/2013; 8(4):e58930. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Understanding the fundamental processes involved in repairing the airway wall following injury is fundamental to understanding the way in which these processes are perturbed during disease pathology. Indeed complex diseases such as asthma and chronic obstructive pulmonary disease (COPD) have at their core evidence of airway wall remodeling processes that play a crucial functional role in these diseases. The authors sought to understand the dynamic cellular events that occur during bronchial airway epithelial repair in sheep. The injury was induced by endobronchial brush biopsy (BBr), a process that causes epithelial débridement and induces a consequential repair process. In addition, the current experimental protocol allowed for the time-dependent changes in airway wall morphology to be studied both within and between animals. The initial débridement was followed by evidence of dedifferentiation in the intact epithelium at the wound margins, followed by proliferation of cells both within the epithelium and in the deeper wall structures, notably in association with the submucosal glands and smooth muscle bundles. Seven days after injury, although the airway wall was thickened at the site of damage, the epithelial layer was intact, with evidence of redifferentiation. These studies, in demonstrating broad agreement with previous studies in small animals, indicate the wider relevance of this system as a comparative model and should provide a solid basis upon which to further characterize the critical cellular and molecular interactions that underlie both effective restitution and pathological repair.
Experimental Lung Research 09/2011; 37(9):519-35. · 1.47 Impact Factor
[show abstract][hide abstract] ABSTRACT: The cationic lipid GL67A is one of the more efficient non-viral gene transfer agents (GTAs) for the lungs, and is currently being evaluated in an extensive clinical trial programme for cystic fibrosis gene therapy. Despite conferring significant expression of vector-specific mRNA following transfection of differentiated human airway cells cultured on air liquid interfaces (ALI) cultures and nebulisation into sheep lung in vivo we were unable to detect robust levels of the standard reporter gene Firefly luciferase (FLuc). Recently a novel secreted luciferase isolated from Gaussia princeps (GLuc) has been described. Here, we show that (1) GLuc is a more sensitive reporter gene and offers significant advantages over the traditionally used FLuc in pre-clinical models for lung gene transfer that are difficult to transfect, (2) GL67A-mediated gene transfection leads to significant production of recombinant protein in these models, (3) promoter activity in ALI cultures mimics published in vivo data and these cultures may, therefore, be suitable to characterise promoter activity in a human ex vivo airway model and (4) detection of GLuc in large animal broncho-alveolar lavage fluid and serum facilitates assessment of duration of gene expression after gene transfer to the lungs. In summary, we have shown here that GLuc is a sensitive reporter gene and is particularly useful for monitoring gene transfer in difficult to transfect models of the airway and lung. This has allowed us to validate that GL67A, which is currently in clinical use, can generate significant amounts of recombinant protein in fully differentiated human air liquid interface cultures and the ovine lung in vivo.
[show abstract][hide abstract] ABSTRACT: We use both large and small animal models in our pre-clinical evaluation of gene transfer agents (GTAs) for cystic fibrosis (CF) gene therapy. Here, we report the use of a large animal model to assess three non-viral GTAs: 25 kDa-branched polyethyleneimine (PEI), the cationic liposome (GL67A) and compacted DNA nanoparticle formulated with polyethylene glycol-substituted lysine 30-mer. GTAs complexed with plasmids expressing human cystic fibrosis transmembrane conductance regulator (CFTR) complementary DNA were administered to the sheep lung (n = 8 per group) by aerosol. All GTAs gave evidence of gene transfer and expression 1 day after treatment. Vector-derived mRNA was expressed in lung tissues, including epithelial cell-enriched bronchial brushing samples, with median group values reaching 1-10% of endogenous CFTR mRNA levels. GL67A gave the highest levels of expression. Human CFTR protein was detected in small airway epithelial cells in some animals treated with GL67A (two out of eight) and PEI (one out of eight). Bronchoalveolar lavage neutrophilia, lung histology and elevated serum haptoglobin levels indicated that gene delivery was associated with mild local and systemic inflammation. Our conclusion was that GL67A was the best non-viral GTA currently available for aerosol delivery to the sheep lung, led to the selection of GL67A as our lead GTA for clinical trials in CF patients. Gene Therapy (2011) 18, 996-1005; doi:10.1038/gt.2011.55; published online 21 April 2011
[show abstract][hide abstract] ABSTRACT: We have previously shown that recombinant Sendai virus (SeV) vector, derived from murine parainfluenza virus, is one of the most efficient vectors for airway gene transfer. We have also shown that SeV-mediated transfection on second administration, although reduced by 60% when compared with levels achieved after a single dose, is still high because of the efficient transfection achieved by SeV vector in murine airways. Here, we show that these levels further decrease on subsequent doses. In addition, we validated SeV vector repeat administration in a non-natural host model, the sheep. As part of these studies we first assessed viral stability in a Pari LC Plus nebuliser, a polyethylene catheter (PEC) and the Trudell AeroProbe. We also compared the distribution of gene expression after PEC and Trudell AeroProbe administration and quantified virus shedding after sheep transduction. In addition, we show that bronchial brushings and biopsies, collected in anaesthetized sheep, can be used to assess SeV-mediated gene expression over time. Similar to mice, gene expression in sheep was transient and had returned to baseline values by day 14. In conclusion, the SeV vector should be strongly considered for lung-related applications requiring a single administration of the vector even though it might not be suitable for diseases requiring repeat administration.
[show abstract][hide abstract] ABSTRACT: A major limitation of many self-assembling nonviral gene transfer formulations is that they are commonly prepared at relatively low component concentrations. While this typically has little impact on their use in cell culture, it can severely limit the progress of in vivo studies. In order to overcome this, we have developed a simple, scalable, pharmaceutically acceptable concentration method that has allowed us to increase the concentration of a commonly used pDNA/PEI formulation from 0.2 to >8 mg/ml plasmid DNA (pDNA). Crucially, the concentration method was found to have only minimal impact on the electrostatic properties or size of the pDNA/PEI particles. When delivered as an aerosol to the mouse lung, the concentrated pDNA/PEI formulations resulted in a 15-fold increase in lung reporter gene expression, with minimal impact in terms of inflammation or toxicity. Importantly, this performance advantage was replicated after aerosol administration to sheep lungs, with reporter gene expression being similarly approximately 15-fold higher than with the conventional pDNA/PEI formulation, and lung inflammation falling to background levels. These findings demonstrate that concentrated pDNA/PEI formulations offer increased aerosol gene transfer with decreased inflammatory sequelae, and represent a promising advance in the field of nonviral lung gene transfer. It seems likely that similar benefits might be achievable with alternative delivery routes and with other nonviral formulations.
[show abstract][hide abstract] ABSTRACT: A major route of transmission of Visna/maedi virus (VMV), an ovine lentivirus, is thought to be via the respiratory tract, by inhalation of either cell-free or cell-associated virus. In previous studies, we have shown that infection via the lower respiratory tract is much more efficient than via upper respiratory tissues (T. N. McNeilly, P. Tennant, L. Lujan, M. Perez, and G. D. Harkiss, J. Gen. Virol. 88:670-679, 2007). Alveolar macrophages (AMs) are prime candidates for the initial uptake of virus in the lower lung, given their in vivo tropism for VMV, abundant numbers, location within the airways, and role in VMV-induced inflammation. Furthermore, AMs are the most likely cell type involved in the transmission of cell-associated virus. In this study, we use an experimental in vivo infection model that allowed the infection of specific segments of the ovine lung. We demonstrate that resident AMs are capable of VMV uptake in vivo and that this infection is associated with a specific up-regulation of AM granulocyte-macrophage colony-stimulating factor mRNA expression (P < 0.05) and an increase in bronchoalveolar lymphocyte numbers (P < 0.05), but not a generalized inflammatory response 7 days postinfection. We also demonstrate that both autologous and heterologous VMV-infected AMs are capable of transmitting virus after lower, but not upper, respiratory tract instillation and that this transfer of virus appears not to involve the direct migration of virus-infected AMs from the airspace. These results suggest that virus is transferred from AMs into the body via an intermediate route. The results also suggest that the inhalation of infected AMs represents an additional mechanism of virus transmission.
Journal of Virology 03/2008; 82(3):1526-36. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Existing methods of non-viral airway gene transfer suffer from low levels of efficiency. Electroporation has been used to enhance gene transfer in a range of tissues. Here we assess the usefulness of electroporation for enhancing gene transfer in the lungs of mice and sheep.
Naked plasmid DNA (pDNA) expressing either luciferase or green fluorescent protein (GFP) was delivered to mouse lungs by instillation. Following surgical visualisation, the lungs were directly electroporated and the level and duration of luciferase activity was assessed and cell types that were positive for GFP were identified in lung cryosections. Naked pDNA was nebulised to the sheep lung and electrodes attached to the tip of a bronchoscope were used to electroporate airway segment bifurcations, Luciferase activity was assessed in electroporated and control non-electroporated regions, after 24 h.
Following delivery of naked pDNA to the mouse lung, electroporation resulted in up to 400-fold higher luciferase activity than naked pDNA alone when luciferase was under the control of a cytomegalovirus (CMV) promoter. Following delivery of a plasmid containing the human polyubiquitin C (UbC) promoter, electroporation resulted in elevated luciferase activity for at least 28 days. Visualisation of GFP indicated that electroporation resulted in increased GFP detection compared with non-electroporated controls. In the sheep lung electroporation of defined sites in the airways resulted in luciferase activity 100-fold greater than naked pDNA alone.
These results indicate that electroporation can be used to enhance gene transfer in the lungs of mice and sheep without compromising the duration of expression.
The Journal of Gene Medicine 06/2007; 9(5):369-80. · 2.16 Impact Factor
[show abstract][hide abstract] ABSTRACT: A panel of 11 human cystic fibrosis transmembrane conductance regulator (hCFTR) antibodies were tested in ovine nasal, tracheal, and bronchial epithelial brushings. Two of these, G449 (polyclonal) and MATG1104 (monoclonal), recognized hCFTR but did not cross react with endogenous sheep CFTR. This specificity allows immunologic detection of hCFTR expressed in gene transfer studies in sheep against the background of endogenous ovine CFTR, thus enhancing the value of the sheep as a model animal in which to study CFTR gene transfer. Studies on mixed populations of human and sheep nasal epithelial cells showed that detection of hCFTR by these two antibodies was possible even at the lowest proportion of human cells (1:100). The hCFTR gene was delivered in vivo by local instillation using polyethylenimine-mediated gene transfer to the ventral surface of the ovine trachea and hCFTR mRNA and protein levels scored in a blinded fashion. Despite abundant hCFTR mRNA expression, the number of cells expressing hCFTR protein detectable by G449 was low (approximately 0.006-0.05%). Immunohistochemistry for hCFTR in animals treated by whole-lung aerosol demonstrated positive cells in sections of tracheal epithelium and in distal conducting airways. The strategic use of hCFTR-specific antibodies supports the utility of the normal sheep as a model for hCFTR gene transfer studies.
American Journal of Respiratory Cell and Molecular Biology 08/2006; 35(1):72-83. · 4.15 Impact Factor
[show abstract][hide abstract] ABSTRACT: Evolved functions of integrin-alpha(v)beta(6) include roles in epithelial cell-extracellular matrix protein interactions and in the binding and activation of latent TGF-beta(1). Integrin-alpha(v)beta(6) is also exploited as a receptor by foot-and-mouth disease virus (FMDV) and may play a significant role in its transmission and pathogenesis. The ovine beta(6) integrin subunit was cloned and sequenced (EMBL accession no. AJ439062). Screening of normal ovine tissues by RT-PCR and immunocytochemistry confirmed that integrin-alphavbeta6 is restricted to sheep epithelial cells. Integrin-alphavbeta6 expression was detected in epithelia of the airways, oral cavity, gastrointestinal tract, kidney, sweat glands, hair follicle sheaths, and the epidermis of pedal coronary band (PB) but not of normal skin. Consistent with FMDV tropism, integrin-alphavbeta6 was detected within the basal layers of the stratified squamous epithelium of the oral mucosa and PB. In addition, integrin-alphavbeta6 appears to be constitutively expressed in the normal airways of both cattle and sheep. The latter finding suggests that ruminant airway epithelium presents a highly accessible target for initiation of infection with FMDV by inhalation.
Journal of Histochemistry and Cytochemistry 08/2006; 54(7):807-16. · 2.26 Impact Factor
[show abstract][hide abstract] ABSTRACT: Recombinant Sendai-virus (SeV) vector transduces airway epithelium efficiently via the apical membrane and leads to high, but transient gene expression. The treatment of chronic diseases may require repeated administration of the virus. We have previously shown that three repeat administrations of recombinant Sendai virus (DF/SeV) did not lead to measurable luciferase (lux) expression after the third administration. However, as part of these experiments we noted that even after one administration, lux expression was lower than expected for a given virus dose. Sequence analysis revealed that the lux gene contained sequences that may be recognised by SeV as “stuttering” sequences and may interfere with gene expression.In an attempt to improve SeV-mediated lux expression we introduced several silent mutations to modify these putative stuttering sequences. SeV-mediated lux expression in the lung was increased 2.5-fold (n=10, p
[show abstract][hide abstract] ABSTRACT: Molecular Therapy (2005) 11, S327|[ndash]|S327; doi: 10.1016/j.ymthe.2005.07.385
842. Repeated Administration of Sendai Virus into Lung
Uta Griesenbach1,4, Gerry McLachlan2,4, Luci Somerton1,4, David Collie2,4, Makoto Inoue3,|[ast]|, Takashi Hironaka3,|[ast]|, Toshiyuki Owaki3,|[ast]|, Duncan M. Geddes1,4, Mamoru Hasegawa3,|[ast]| and Eric W. F. W. Alton1,4,|[dagger]|1Department of Gene Therapy, Imperial College, London, United Kingdom2College of Medicine and Veterinary Medicine, University of Edinburgh, Edinburgh, United Kingdom3DNAVEC Corporation, Tsukuba, Ibaraki, Japan4UK Cystic Fibrosis Gene Therapy Consortium, United Kingdom|[ast]|Some authors are employed by DNAVEC Corporation|[dagger]|EWFWA is a consultant for DNAVEC Corporation.
[show abstract][hide abstract] ABSTRACT: Current non-viral vectors for lung gene transfer are limited by inefficient uptake, and low levels of expression in cells throughout the lung. The use of electroporation to enhance reporter gene expression from naked plasmid DNA (pDNA) in the mouse lung has been assessed. A direct electroporation model has been established, in which following delivery of pDNA and electroporation of the left lung, luciferase (Lux) reporter gene expression has been enhanced by up to 500 fold compared to the non-electroporated right lung depending on the combination of plasmid, field strength (V/cm) and pulse length employed. Furthermore, following delivery of a GFP expressing plasmid, up to 5% of the left lung cells were positive for GFP after electroporation. Now, the duration of Lux activity following electroporation of the mouse lung has been assessed. Female BALB/c mice were anaesthetised with isoflurane and received 100μg pUbLux (human ubiquitin C promoter, Lux transgene) in 150μl water. The left lungs of mice were exposed by performing a lateral thoracotomy. Electroporation of the left lung (8 pulses of 800V/cm, 2ms with a 1sec interval) was carried out using the BTX ECM®830 Generator with 2-Needle electrodes with 5mm gap with the right lungs remaining non-electroporated. Lux activity was measured at 2, 14 and 28 days post dosing. At day 2 post dosing, mean Lux activity in the left lungs of mice was 40 fold higher than the right lung (Mann-Whitney U: P = 0.016). The Lux activity in the left lungs at day 28 post dosing was no different to the level at day 2 (P=0.512) and still significantly higher than the activity observed in the right lung (P = 0.050). Therefore this study has established that direct electroporation of the mouse lung can result in high levels of Lux activity from naked pDNA and that this level persists for at least 28 days post dosing. In similar studies with the plasmid pCIKLux (CMV promoter, Lux transgene), electroporation resulted in far greater levels of expression at day 2 post dosing, but by day 14, the Lux activity had fallen to background. The safety and efficacy of lung electroporation has therefore been well established in the mouse lung. Further development of lung electroporation is now planned in the sheep lung, as a larger animal model will be more relevant for clinical development of this approach. A series of wire electrodes have been constructed from Teflon insulated stainless steel up to 80cm in length with a diameter of just 0.3mm. In an ex vivo mouse lung model, electroporation with these wires following pCIKLux intranasal delivery (100μg pDNA/150μl) resulted in equivalent 200 fold increases in Lux activity to commercially available needle electrodes. These wire electrodes will be placed within the sheep airways with a bronchoscope allowing for a range of electroporation studies to be carried out. These studies will determine if the electric field can be efficiently and safely applied over a large area to allow electroporation to be used for improving gene transfer throughout the whole lung.
[show abstract][hide abstract] ABSTRACT: We have described gene delivery in sheep as a large animal model for airway gene transfer. This model allows comparison of different GTAs in terms of efficacy and safety using doses, delivery methods and assays, relevant to human studies. Proof-of-principle for non-viral gene transfer has been established however, levels of transgene expression are too low on a per cell basis to characterise which cells are targeted. Our preferred method for delivery is by aerosol however, not all GTAs are stable following aerosolisation in conventional jet or ultrasonic nebulisers and the cost of producing GTAs in sufficient quantities for whole lung aerosol studies can be prohibitive. These issues were behind our initial studies with delivery by instillation to individual segments. Unfortunately this approach is associated with increased toxicity as a result of pooling of material in the terminal bronchiole/alveolar duct regions. The Trudell AeroProbe* catheter develops an aerosol at the tip and can be passed through a bronchoscope. This allows localised delivery of GTAs, reducing the amount of material required. A further advantage of the AeroProbe* is that it is a single-pass aerosol device and subjects the GTA to less shear stress. An important question to be addressed when comparing delivery with different devices, is how their respective distribution patterns compare. Here we used a recombinant Sendai virus (DF/SeV-LacZ) vector, shown to be highly efficient at transducing airway epithelial cells in a variety of species, to investigate distribution patterns of delivery methods. SeV was delivered either by instillation to single lung segments via a polyethylene catheter (PEC), by AeroProbe* directed at segments or by AeroProbe* directed at the whole lung. In terms of stability, 100% and 49 +/− 3% of infectious virus particles survived the PEC and AeroProbe* respectively (n=3/group). In vivo studies show the variation in distribution of the different delivery methods. Instillation of 5ml SeV (3e9 CIU) to the same segment of different animals (n=3) results in varied distribution. Data from these animals confirm the absence of spillover as determined by histological assessment of adjacent segments. Segmentally-directed delivery of 5ml SeV (1e10 CIU) via AeroProbe* gives a different distribution. We observed areas of expression at airway bifurcations from impaction of aerosol particles at these sites and expression around sub-mucosal gland openings and within the glands themselves. Spillover to adjacent segments was observed in this case. Delivery of 24ml SeV (4e10 CIU) to the whole lung via the AeroProbe* gave a different type of expression pattern again. SeV was distributed throughout the lung. An even pattern of distribution all the way to the peripheral alveolar regions can be observed in several segments although the extent varies. These distribution patterns will have implications for sampling. It is clear from these studies that the properties of SeV make it an ideal vector to study the distribution pattern of different devices in our ovine model.
[show abstract][hide abstract] ABSTRACT: We sought to determine the normal within-sheep variability of bronchoalveolar cellular parameters and to infer the numbers of sheep required to detect a defined significant change within the same. We further sought to examine the effect of repeated bronchoalveolar lavage (BAL) on those parameters. Neither the total cell number nor the absolute numbers of any given cell type changed significantly following repeated BAL. Additionally, there was no significant change in percentage differential cell populations over time in randomly sampled lobes. However for lobes sampled repeatedly, a significant change in the percentage of lymphocytes was detected. Although the percentages of neutrophils in repeatedly and randomly sampled lobes were significantly correlated, the percentage found in the former tended to be greater and more variable than in the latter. As a consequence, larger group sizes are required to detect given changes in neutrophil percentages in repeatedly sampled lobes over time when compared with detecting equivalent changes in randomly selected lobes.
Research in Veterinary Science 11/1999; 67(2):137-40. · 1.77 Impact Factor