Enhanced avidity from a multivalent fluorescent antimicrobial peptide enables pathogen detection in a human lung model.

Rapid in situ detection of pathogens coupled with high resolution imaging in the distal human lung has the potential to provide new insights and diagnostic utility in patients in whom pneumonia is suspected. We have previously described an antimicrobial peptide (AMP) Ubiquicidin (fragment UBI) labelled with an environmentally sensitive fluorophore that optically detected bacteria in vitro but not ex vivo. Here, we describe further chemical development of this compound and demonstrate that altering the secondary structure of the AMP to generate a tri-branched dendrimeric scaffold provides enhanced signal in vitro and ex vivo and consequently allows the rapid detection of pathogens in situ in an explanted human lung.

In situ identification of Gram-negative bacteria in human lungs using a topical fluorescent peptide targeting lipid A.

Respiratory infections in mechanically ventilated patients caused by Gram-negative bacteria are a major cause of morbidity. Rapid and unequivocal determination of the presence, localization, and abundance of bacteria is critical for positive resolution of the infections and could be used for patient stratification and for monitoring treatment efficacy. Here, we developed an in situ approach to visualize Gram-negative bacterial species and cellular infiltrates in distal human lungs in real time.

Nebulisation of synthetic lamellar lipids mitigates radiation-induced lung injury in a large animal model.

Methods to protect against radiation-induced lung injury (RILI) will facilitate the development of more effective radio-therapeutic protocols for lung cancer and may provide the means to protect the wider population in the event of a deliberate or accidental nuclear or radiological event. We hypothesised that supplementing lipid membranes through nebulization of synthetic lamellar lipids would mitigate RILI. Following pre-treatment with either nebulised lamellar lipids or saline, anaesthetised sheep were prescribed fractionated radiotherapy (30 Gray (Gy) total dose in five 6 Gy fractions at 3-4 days intervals) to a defined unilateral lung volume.

Comparing microbiotas in the upper aerodigestive and lower respiratory tracts of lambs.

Background: Recently, the importance of the lung microbiota during health and disease has been examined in humans and in small animal models. Whilst sheep have been proposed as an appropriate large animal model for studying the pathophysiology of a number of important human respiratory diseases, it is clearly important to continually define the limits of agreement between these systems as new concepts emerge. In humans, it has recently been established that the lung microbiota is seeded by microbes from the oral cavity.

Microbiota in Exhaled Breath Condensate and the Lung.

The lung microbiota is commonly sampled using relatively invasive bronchoscopic procedures. Exhaled breath condensate (EBC) collection potentially offers a less invasive alternative for lung microbiota sampling. We compared lung microbiota samples retrieved by protected specimen brushings (PSB) and exhaled breath condensate collection.

Variability of the Sheep Lung Microbiota.

Unlabelled: Sequencing technologies have recently facilitated the characterization of bacterial communities present in lungs during health and disease. However, there is currently a dearth of information concerning the variability of such data in health both between and within subjects. This study seeks to examine such variability using healthy adult sheep as our model system.

Lung Microbiota Changes Associated with Chronic Pseudomonas aeruginosa Lung Infection and the Impact of Intravenous Colistimethate Sodium.

Background: Exacerbations associated with chronic lung infection with Pseudomonas aeruginosa are a major contributor to morbidity, mortality and premature death in cystic fibrosis. Such exacerbations are treated with antibiotics, which generally lead to an improvement in lung function and reduced sputum P. aeruginosa density.

Repeated nebulisation of non-viral CFTR gene therapy in patients with cystic fibrosis: a randomised, double-blind, placebo-controlled, phase 2b trial.

Background: Lung delivery of plasmid DNA encoding the CFTR gene complexed with a cationic liposome is a potential treatment option for patients with cystic fibrosis. We aimed to assess the efficacy of non-viral CFTR gene therapy in patients with cystic fibrosis.

Methods: We did this randomised, double-blind, placebo-controlled, phase 2b trial in two cystic fibrosis centres with patients recruited from 18 sites in the UK.

Sheep lung segmental delivery strategy demonstrates adenovirus priming of local lung responses to bacterial LPS and the role of elafin as a response modulator.

Viral lung infections increase susceptibility to subsequent bacterial infection. We questioned whether local lung administration of recombinant adenoviral vectors in the sheep would alter the susceptibility of the lung to subsequent challenge with bacterial lipopolysaccharide (LPS). We further questioned whether local lung expression of elafin, a locally produced alarm anti-LPS/anti-bacterial molecule, would modulate the challenge response.

The safety profile of a cationic lipid-mediated cystic fibrosis gene transfer agent following repeated monthly aerosol administration to sheep.

Clinically effective gene therapy for Cystic Fibrosis has been a goal for over 20 years. A plasmid vector (pGM169) that generates persistent expression and reduced host inflammatory responses in mice has raised prospects for translation to the clinic. The UK CF Gene Therapy Consortium is currently evaluating long-term repeated delivery of pGM169 complexed with the cationic lipid GL67A in a large Multidose Trial.

Gene expression changes associated with the airway wall response to injury.

Background: 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.

Methodology/principal Findings: We profiled gene expression changes in the airway wall using a large animal model of physical injury comprising bronchial brush biopsy in anaesthetised sheep.

Secreted Gaussia luciferase as a sensitive reporter gene for in vivo and ex vivo studies of airway gene transfer.

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.

An immunocytochemical assay to detect human CFTR expression following gene transfer.

Background: To assess gene therapy treatment for cystic fibrosis (CF) in clinical trials it is essential to develop robust assays that can accurately detect transgene expression in human airway epithelial cells. Our aim was to develop a reproducible immunocytochemical assay for human CFTR protein which can measure both endogenous CFTR levels and augmented CFTR expression after gene delivery.

Methods: We characterised an antibody (G449) which satisfied the criteria for use in clinical trials.

Enhanced lung gene expression after aerosol delivery of concentrated pDNA/PEI complexes.

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.

Animal-handling teaching at the Royal (Dick) School of Veterinary Studies, University of Edinburgh.

This article describes the teaching of animal handling at the Royal (Dick) School of Veterinary Studies, University of Edinburgh, as part of an animal husbandry course during the first two years of the veterinary curriculum. Basic methods of handling and restraint appropriate for the wide range of animal species that might be encountered in veterinary practice are demonstrated in practical handling classes. Students are given opportunities to practice the techniques under supervision.

Local lung responses following endobronchial elastase and lipopolysaccharide instillation in sheep.

Chronic lipopolysaccharide (LPS) exposure may contribute to the pathogenesis of a number of lung diseases including COPD and emphysema. We sought to develop a large-animal model of emphysema using repeated LPS administration into sheep lung segments. An experimental protocol was designed to facilitate comparisons with elastase-treated and control segments within the same lung of individual sheep.

Role of alveolar macrophages in respiratory transmission of visna/maedi virus.

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.

Electroporation enhances reporter gene expression following delivery of naked plasmid DNA to the lung.

Background: 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.

Optimizing aerosol gene delivery and expression in the ovine lung.

We have developed the sheep as a large animal model for optimizing cystic fibrosis gene therapy protocols. We administered aerosolized gene transfer agents (GTAs) to the ovine lung in order to test the delivery, efficacy, and safety of GTAs using a clinically relevant nebulizer. A preliminary study demonstrated GTA distribution and reporter gene expression throughout the lung after aerosol administration of plasmid DNA (pDNA):GL67 and pDNA:PEI complexes.

Integrin-alphavbeta6, a putative receptor for foot-and-mouth disease virus, is constitutively expressed in ruminant airways.

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.

Human-specific cystic fibrosis transmembrane conductance regulator antibodies detect in vivo gene transfer to ovine airways.

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.