Gene therapy for chronic lung diseases will require vectors capable of persistent transgene expression. This has been achieved for several non-viral vectors by judicious promoter selection and ...removal of CG dinucleotides from the plasmid DNA. To further study the cellular and molecular mechanisms involved, we investigated the potential use of two cell culture models to mimic the transgene silencing observed in vivo: the A549 lung cell line (ATCC) and human air-liqu id interface (ALI) primary respiratory epithelial ex vivo cultures (Epithelix, Sarl, Switzerland). Together these data demonstrate that non-viral transgene expression observed in mouse lungs following aerosol delivery is not recapitulated in A549 lung cells or in human ALI ex vivo cultures. Currently the reasons for this are unknown, but may be due to species and cell type differences.
The 19 conference papers in this collection deal with the relationship of various rhetorical theories and their practical applications to the rhetorical traditions that they are superseding. The ...papers deal with many topics, including the following: (1) a multidisciplinary approach to writing instruction; (2) the importance of writing as a human activity; (3) Michael Polanyi and the contexts of composing; (4) the role of rhetoric in the classical trivium and the school tradition, and the need for a new trivium; (5) four important models of discourse; (6) disharmonies in the new rhetoric; (7) invention and the composing process at the postsecondary level; (8) writing style; (9) sentence combining; (10) the invention aspects of the revision process; (11) audience awareness; (12) problems related to the rhetorical concept of "ethos"; (13) the complexities of writing evaluation; (14) the development of a curriculum, based on recent research and theory, for a college-level composition program; and (15) an inservice program for teachers that draws on the new rhetoric. An epilogue expands on the conference theme of reinventing the rhetorical tradition. (FL)
Gene therapy for cystic fibrosis lung disease will likely require repeated delivery of gene transfer agents (GTA) to the terminally differentiated cells of the airway epithelium. We are currently ...testing several non-viral GTAs for efficiency of gene transfer following aerosol delivery to the lungs of mice and sheep. The most sensitive method available to quantify gene expression from plasmid vectors is quantitative (TaqMan) reverse transcriptase (RT)-PCR, which has been widely used to quantify gene expression in pre-clinical and clinical samples. TaqMan PCR can potentially detect a single copy of the target sequence per reaction. We designed TaqMan assays to quantify both vector-specific mRNA and endogenous mRNA of a normalising gene. Specifically, assays were developed for plasmids containing the CMV promoter, or the human polyubiquitin C (UbC) promoter, shown to have improved duration of expression in the lung. In addition, assays have been developed to detect endogenous murine and ovine Cftr mRNA. Assays were designed to be specific for mRNA by placing one of the primer sequences across an exon splice site. In order to determine the absolute copy number of mRNA in a given sample, RNA standard curves were produced. DNA templates for the assays were constructed and included the PCR amplicon plus surrounding sequences totalling 160bp. RNA standards were generated incorporating RNase resistant ribonucleotides into RNA transcripts, using the Ambion Competitor Construction Kit. The mimics were quantified using the Molecular Probes RiboGreen assay and dilutions were produced containing 1 to 5 10 copies/μl. 1μl of each dilution was used in paired RT reactions with sequence specific primers and MultiScribe RT. Quantitative PCR was then carried out using an Applied Biosystems TaqMan 7700 Sequence Detector and standard curves generated for the four assays. When paired (multiplexed), the CMV and sheep Cftr assays were linear between 100 and 10 6 starting copies per reaction (R2 =0.984 and 0.981). The UbC and mouse Cftr assays were paired and the UbC assay was linear between 100 and 106 starting copies (R2 =0.987) while the mouse Cftr assay was linear between 1500 and 106 starting copies per reaction (R2 =0.890). Control samples lacking RT did not result in amplification, indicating that the standards were free of template DNA. These results show the high degree of sensitivity and linearity that can be obtained with TaqMan RT-PCR. These assays will now be used to quantify absolute levels of expression in our pre-clinical models to determine the optimal GTA formulation for future clinical studies.
Non-viral mediated gene transfer is being investigated as an approach for the treatment of lung diseases such as Cystic Fibrosis (CF). However, an important limiting factor is the inflammatory ...responses generated by such vectors in patients and animal models. Gene delivery of plasmid DNA complexed with cationic lipid to the murine lung results in increased levels of pro-inflammatory cytokines, which includes tumour necrosis factor alpha (TNF-α) (Scheule et al., 1997, Human Gene Therapy, 8: 689-707). Clinical studies have also indicated an inflammatory response following dosing of plasmid DNA/lipid complexes to the lungs of patients with CF (Alton et al., 1999, Lancet, 353 947). It is believed that the inflammation occurs, at least in part, in response to the unmethylated bacterial CpG sequences delivered to the cytoplasm of the target cells which results in the activation of Nuclear Factor κβ (NF-κβ). NF-κβ is a key regulator of inflammatory and immune responses and is essential for the transcription of multiple pro-inflammatory molecules including TNF-α, interleukin 12 (IL-12) and interferons (IFN) and has become a target for anti-inflammatory treatment. Previous studies have employed an NF-κβ decoy strategy using synthetic oligodeoxynucleotides (ODNs) to block the binding of NF-κβ to the promoter region of its targeted genes following systemic delivery of non-viral vectors to the mouse lung. The addition of the oligodeoxynucleotides inhibited TNF-α in a dose-dependent manner (Tan et al., 2002, Molecular Therapy, 804-812). This study investigates the effect of NF-κβ decoy oligodeoxynucleotides on the inflammatory response following topical delivery of plasmid DNA complexed with Genzyme lipid 67 (GL67) to the mouse lung. Female BALB/c mice were dosed intranasally with 40μg of luciferase expressing plasmid pCIKLux and 40μg of synthetic single stranded phosphorothioate ODN, containing an NF-κβ consensus binding sequence (S+) complexed with cationic lipid GL67 in total volume of 100μl. A scrambled form of the ODN (S11), without a consenus NF-κβ binding site was used as a control. Mouse lungs were harvested 24hrs after dosing and luciferase activity and pro-inflammatory cytokine levels were measured. With topical delivery of the S+/pCIKLux/GL67 complex, there were 80% and 70% reductions in TNF-α (ANOVA with Fisher's PLSD, P=0.0001) and IFN-γ (P=0.004) respectively compared to dosing with pCIKLux/GL67. Furthermore, reporter gene expression was retained at maximum levels after co-delivery of the S+ ODN (ANOVA, P=0.820). This type of approach may allow reduced inflammation in the lung following delivery of GL67 complexes. Reduction in inflammation may also lead to improved duration of transgene expression by avoiding cytokine induced promoter attenuation. Further experiments are in progress to detect cytokine levels induced by intransanal delivery of other gene transfer formulations co-delivered with NF-κβ ODNs.
Preclinical models that can accurately predict outcomes in the clinic are much sought after in the field of cancer drug discovery and development. Existing models such as organoids and ...patient-derived xenografts have many advantages, but they suffer from the drawback of not contextually preserving human tumour architecture. This is a particular problem for the preclinical testing of immunotherapies, as these agents require an intact tumour human-specific microenvironment for them to be effective. In this review, we explore the potential of patient-derived explants (PDEs) for fulfilling this need. PDEs involve the ex vivo culture of fragments of freshly resected human tumours that retain the histological features of original tumours. PDE methodology for anti-cancer drug testing has been in existence for many years, but the platform has not been widely adopted in translational research facilities, despite strong evidence for its clinical predictivity. By modifying PDE endpoint analysis to include the spatial profiling of key biomarkers by using multispectral imaging, we argue that PDEs offer many advantages, including the ability to correlate drug responses with tumour pathology, tumour heterogeneity and changes in the tumour microenvironment. As such, PDEs are a powerful model of choice for cancer drug and biomarker discovery programmes.
Ligand‐enabled aza‐Heck cyclizations and cascades of N‐(pentafluorobenzoyloxy)carbamates are described. These studies encompass the first examples of efficient non‐biased 6‐exo aza‐Heck cyclizations. ...The methodology provides direct and flexible access to carbamate protected pyrrolidines and piperidines.
Direct and flexible: Ligand‐enabled aza‐Heck cyclizations and cascades of N‐(pentafluorobenzoyloxy)carbamates are described. These studies encompass the first examples of efficient non‐biased 6‐exo aza‐Heck cyclizations. The methodology provides direct and flexible access to carbamate protected pyrrolidines and piperidines.
Lung pathology in cystic fibrosis is linked to dehydration of the airways epithelial surface which in part results from inappropriately raised sodium reabsorption through the epithelial sodium ...channel (ENaC). To identify a small-interfering RNA (siRNA) which selectively inhibits ENaC expression, chemically modified 21-mer siRNAs targeting human ENaCα were designed and screened. GSK2225745, was identified as a potent inhibitor of ENaCα mRNA (EC 50 (half maximal effective concentration) = 0.4 nmol/l, maximum knockdown = 85%) and protein levels in A549 cells. Engagement of the RNA interference (RNAi) pathway was confirmed using 5variant prime RACE. Further profiling was carried out in therapeutically relevant human primary cells. In bronchial epithelial cells, GSK2225745 elicited potent suppression of ENaCα mRNA (EC 50 = 1.6 nmol/l, maximum knockdown = 82%). In human nasal epithelial cells, GSK2225745 also produced potent and long-lasting (≥72 hours) suppression of ENaCα mRNA levels which was associated with significant inhibition of ENaC function (69% inhibition of amiloride-sensitive current in cells treated with GSK2225745 at 10 nmol/l). GSK2225745 showed no evidence for potential to stimulate toll-like receptor (TLR)3, 7 or 8. In vivo, topical delivery of GSK2225745 in a lipid nanoparticle formulation to the airways of mice resulted in significant inhibition of the expression of ENaCα in the lungs. In conclusion, GSK2225745 is a potent inhibitor of ENaCα expression and warrants further evaluation as a potential novel inhaled therapeutic for cystic fibrosis.Molecular Therapy -- Nucleic Acids (2013) 2, e65; doi:10.1038/mtna.2012.57; published online 15 January 2013
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.
Lay Summary
Invasive rose-ringed parakeets caused behavioral changes in native garden birds that reduced their feeding rates. Understanding how invasive species impact native species can be complex, ...especially in urban environments where many other factors are also at play. We therefore used an experiment to disentangle these factors and demonstrate that parakeets are more disruptive than a dominant native competitor.
Resource competition is one potential behavioral mechanism by which invasive species can impact native species, but detecting this competition can be difficult due to the interactions that variable environmental conditions can have on species behavior. This is particularly the case in urban habitats where the disturbed environment can alter natural behavior from that in undisturbed habitats. The rose-ringed parakeet (Psittacula krameri), is an increasingly common invasive species, predominantly associated with large urban centers. Using an experimental approach, we tested the behavioral responses of native garden birds in response to the presence of a rose-ringed parakeet versus the presence of a similarly sized and dominant native bird, the great spotted woodpecker (Dendrocopos major). Parakeet presence significantly reduced feeding rates and increased vigilance among native birds compared with our control treatments. Of visits made by native birds in the presence of a parakeet, feeding was more likely to occur in sites within the parakeet range compared with sites outside, suggesting some habituation of native birds has occurred following prior exposure to parakeets but overall foraging behavior is still disrupted. The results of our study suggest that nonnative species can have complex and subtle impacts on native fauna and show that a nonnative competitor can impact native species simply through their presence near resources.
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