Factors affecting the efficiency of aerosolized salbutamol delivery via a metered dose inhaler and equine spacer device.

Despite frequent use of metered dose inhalers (MDIs) and spacers in equine practice, limited information exists on the efficiency of aerosol delivery using such devices. We determined the particle size distribution within an MDI-generated salbutamol aerosol delivered via an equine spacer using 'best practice' delivery technique and assessed the effect of variations in MDI use technique (shaking prior to each actuation, rapid repetitive actuations, and MDI angulation) on aerosol delivery efficiency. Under optimal conditions, only 53(±18) µg salbutamol per 100 µg actuation was delivered beyond the spacer. Although this aerosol had a high [89.6% (±2.4)] fine particle (<5 µm) fraction, and a low mass median aerodynamic diameter [2.52 (±0.29) µm], and particle size variability [geometric SD - 1.66 (±0.16) µm], within all particle size fractions, there was a high coefficient of variance (31-79%) of the percentage salbutamol delivered between experimental runs, thus impeding any effort to predict drug delivery to the patient during equine inhalation therapy. Despite observable trends and with the exception of minor statistically significant changes in the least abundant particle sizes, none of the deviations from a 'best practice' delivery technique significantly altered the relative salbutamol delivery beyond the spacer, a finding which has potential relevance with regard to maintaining user compliance.

A benchmark for evaluation of algorithms for identification of cellular correlates of clinical outcomes.

The Flow Cytometry: Critical Assessment of Population Identification Methods (FlowCAP) challenges were established to compare the performance of computational methods for identifying cell populations in multidimensional flow cytometry data. Here we report the results of FlowCAP-IV where algorithms from seven different research groups predicted the time to progression to AIDS among a cohort of 384 HIV+ subjects, using antigen-stimulated peripheral blood mononuclear cell (PBMC) samples analyzed with a 14-color staining panel. Two approaches (FlowReMi.1 and flowDensity-flowType-RchyOptimyx) provided statistically significant predictive value in the blinded test set. Manual validation of submitted results indicated that unbiased analysis of single cell phenotypes could reveal unexpected cell types that correlated with outcomes of interest in high dimensional flow cytometry datasets.

Toxicology study assessing efficacy and safety of repeated administration of lipid/DNA complexes to mouse lung.

For gene therapy to improve lung function in cystic fibrosis (CF) subjects, repeated administration of the gene transfer agent over the lifetime of patients is likely to be necessary. This requirement limits the utility of adenoviral and adeno-associated viral vectors (both previously evaluated in CF gene therapy trials) because of induced adaptive immune responses that render repeated dosing ineffective. For CF gene therapy trials, non-viral vectors are currently the only viable option. We previously showed that the cationic lipid formulation GL67A is the most efficient of several non-viral vectors analysed for airway gene transfer. Here, we assessed the efficacy and safety of administering 12 inhaled doses of GL67A complexed with pGM169, a CpG-free plasmid encoding human CFTR complementary DNA, into mice. We show that repeated administration of pGM169/GL67A to murine lungs is feasible, safe and achieves reproducible, dose-related and persistent gene expression (>140 days after each dose) using an aerosol generated by a clinically relevant nebuliser. This study supports progression into the first non-viral multidose lung trial in CF patients.

BrdU pulse labelling in vivo to characterise cell proliferation during regeneration and repair following injury to the airway wall in sheep.

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.

Lung disease in the cystic fibrosis mouse exposed to bacterial pathogens.

Lung disease is the major cause of death in cystic fibrosis (CF), but there is no evidence for overt lung involvement at birth. We show here that the same is true for the gene targeted cftrm1HGU mutant mouse. Furthermore, this CF mouse model demonstrates an impaired capacity to clear Staphylococcus aureus and Burkholderia (Pseudomonas) cepacia, two opportunistic lung pathogens closely associated with lung disease in CF subjects. The cftrm1HGU homozygotes display mucus retention and frank lung disease in response to repeated microbial exposure. Thus, lung disease in the cftrm1HGU mouse develops in response to bacterial infection, establishing a model to dissect the pathogenesis of CF pulmonary disease and providing a clinically relevant end point to assess the efficacy of pharmacologic or genetic interventions.

Validation of recombinant Sendai virus in a non-natural host model.

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.

Pre-clinical evaluation of three non-viral gene transfer agents for cystic fibrosis after aerosol delivery to the ovine lung.

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.

Analysis of airway epithelial regeneration and repair following endobronchial brush biopsy in sheep.

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.

Testing for group structure in high-dimensional data.

With the use of finite mixture models for the clustering of a data set, the crucial question of how many clusters there are in the data can be addressed by testing for the smallest number of components in the mixture model compatible with the data. We investigate the performance of a resampling approach to this latter problem in the context of high-dimensional data, where the number of variables p is extremely large relative to the number of observations n. In order to be able to fit normal mixture models to such data, some form of dimension reduction has to be performed. This raises the question of whether a practically significant bias results if the bootstrapping is undertaken solely on the basis of the reduced dimensional form of the data, rather than using the full data from which to draw the bootstrap sample replications.

A microbiome analysis of equine peripheral dental caries using next generation sequencing.

BACKGROUND: Although, peripheral caries (PC) affects almost half of UK horses, no comprehensive microbiological study has been performed on this disorder. As a high proportion of oral bacteria cannot be conventionally cultured, molecular microbiological techniques such as Next Generation Sequencing are required to examine the complex oral bacteria community. OBJECTIVES: To identify the microbiota involved in equine PC, including comparing microbiota at the more commonly and severely affected three caudal cheek teeth with the less commonly affected three rostral cheek teeth. STUDY DESIGN AND METHODS: Equine dental plaque samples were collected from the palatal aspects of cheek teeth of 63 horses. DNA was isolated and amplified using PCR, targeting the V4 region of the 16S rRNA gene and Next Generation Sequencing of these gene amplicons was performed. The acquired data were processed and analysed using Mothur and R. RESULTS: Streptococcus species was the genus most commonly associated with equine PC, whereas Gemella species was the genus most associated with the control group. In a further analysis where the rostral and caudal cheek teeth were compared with each other and with the control group. Veillonella species was the most commonly associated genus with PC of the rostral cheek teeth, Streptococcus species was the most associated genus with the caudal cheek teeth, and Corynebacterium with the control group. MAIN LIMITATIONS: Some bacteria can have multiple heterogeneous copies of the 16S rRNA gene, which can affect the estimation of their relative abundance. CONCLUSIONS: Similar to caries studies in other species, acidogenic and aciduric microorganisms including Streptococcus species were found to be associated with equine peripheral caries.

A mixture model with random-effects components for clustering correlated gene-expression profiles.

MOTIVATION: The clustering of gene profiles across some experimental conditions of interest contributes significantly to the elucidation of unknown gene function, the validation of gene discoveries and the interpretation of biological processes. However, this clustering problem is not straightforward as the profiles of the genes are not all independently distributed and the expression levels may have been obtained from an experimental design involving replicated arrays. Ignoring the dependence between the gene profiles and the structure of the replicated data can result in important sources of variability in the experiments being overlooked in the analysis, with the consequent possibility of misleading inferences being made. We propose a random-effects model that provides a unified approach to the clustering of genes with correlated expression levels measured in a wide variety of experimental situations. Our model is an extension of the normal mixture model to account for the correlations between the gene profiles and to enable covariate information to be incorporated into the clustering process. Hence the model is applicable to longitudinal studies with or without replication, for example, time-course experiments by using time as a covariate, and to cross-sectional experiments by using categorical covariates to represent the different experimental classes. RESULTS: We show that our random-effects model can be fitted by maximum likelihood via the EM algorithm for which the E(expectation)and M(maximization) steps can be implemented in closed form. Hence our model can be fitted deterministically without the need for time-consuming Monte Carlo approximations. The effectiveness of our model-based procedure for the clustering of correlated gene profiles is demonstrated on three real datasets, representing typical microarray experimental designs, covering time-course, repeated-measurement and cross-sectional data. In these examples, relevant clusters of the genes are obtained, which are supported by existing gene-function annotation. A synthetic dataset is considered too. AVAILABILITY: A Fortran program blue called EMMIX-WIRE (EM-based MIXture analysis WIth Random Effects) is available on request from the corresponding author.

Clustering.

Clustering techniques are used to arrange genes in some natural way, that is, to organize genes into groups or clusters with similar behavior across relevant tissue samples (or cell lines). These techniques can also be applied to tissues rather than genes. Methods such as hierarchical agglomerative clustering, k-means clustering, the self-organizing map, and model-based methods have been used. Here we focus on mixtures of normals to provide a model-based clustering of tissue samples (gene signatures) and of gene profiles, including time-course gene expression data.

An epidemiological survey on the prevalence of equine peripheral dental caries in the United Kingdom and possible risk factors for its development.

BACKGROUND: Equine peripheral caries (PC) is an increasingly recognised disorder that causes premature wear of teeth and dental fractures and thus has major welfare implications. Little information is available on its prevalence or severity in UK horses and there are no proven associations with any risk factors. OBJECTIVES: To document the prevalence of PC over a wide area of the UK, assess its intraoral distribution and severity in affected horses and examine for potential risk factors for its development. STUDY DESIGN: Cross sectional study. METHODS: Experienced personnel were recruited for a UK wide dental survey on their patients during dental examinations. Established guidelines were used for grading PC. Frequency of PC occurrence was compared between teeth and dental arcades using McNemar's tests. Potential risk factors for PC were screened using univariable logistic regression prior to building a multivariable model. RESULTS: A total of 706 horses were examined by 25 participants, showing a 51.7% prevalence of PC (365/706). Some regional differences in prevalence were found. The PC primarily affected the cheek teeth with the 12 caudal being significantly more commonly affected than the 12 rostral cheek teeth. Most of the hypothesised risk factors including age, breed, sex, time at pasture and feeding of silage (haylage) were unproven. A limited association with moderate levels of concentrate feeding was observed. The presence of concurrent dental abnormalities were significantly associated with the likelihood of having PC. MAIN LIMITATIONS: Not all regions in UK were included and there may be inconsistencies between examiners. CONCLUSIONS: Peripheral caries is common in British horses, primarily affecting the caudal cheek teeth. There was limited evidence of an association between feeding and PC. The association between PC and concurrent dental disorders indicates that these should be addressed in affected horses.

Preclinical analysis of the analinoquinazoline AG1478, a specific small molecule inhibitor of EGF receptor tyrosine kinase.

The tyrphostin 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478) is a potent and specific inhibitor of EGFR tyrosine kinase whose favourable preclinical profile supports progression towards clinical trials. Microphysiometric evaluation revealed a short (<24 min) effective inhibition of cellular receptor response to EGF challenge in BaF/ERX cells indicating a need to maintain sustained levels of inhibitor. Initial pharmacokinetic evaluation in mice of novel AG1478 formulations in a beta-cyclodextrin (Captisol) showed monoexponential elimination from plasma (half-life 30 min) following subcutaneous administration. A two-fold dose escalation gave a 2.4-fold increase in the total AUC. Bolus i.v. and 6 h continuous infusion were investigated in rats to mimic a more clinically relevant administration regimen. Drug elimination following bolus i.v. administration was biphasic (terminal elimination half-life 30-48 min). The linear relationship between dose and AUC(0-->infinity) (r2=0.979) enabled the prediction of infusion rates and doses for sustained delivery using continuous 6 h infusions, where steady state was reached in 120 min. Plasma levels of AG1478>10 microM were achieved over the duration of the infusion. At the lowest dose, plasma drug levels after the cessation of infusion declined with a half-life of approximately 43 min. EGFR activity, measured both by autophosphorylation and downstream signalling, was inhibited in a dose-dependent manner by injection of AG1478 in mice bearing xenografts of the human glioblastoma cell line U87MG.delta2-7, which expresses a constitutively active variant of the EGF receptor. Taken together, these experiments provide essential data to assess the anti-tumour efficacy of AG1478 and will assist in the rational design of dose regimens for clinical studies.

Meta-analysis of the diagnostic accuracy of laparoscopic ultrasonography and intraoperative cholangiography in detection of common bile duct stones.

Introduction During laparoscopic cholecystectomy, intraoperative cholangiography (IOC) is currently regarded as the gold standard in the detection of choledocholithiasis. Laparoscopic ultrasonography (LUS) is an attractive alternative with several potential advantages. Methods A systematic review was undertaken of the published literature comparing LUS with IOC in the assessment of common bile duct (CBD) stones. Results Twenty-one comparative studies were analysed. There were 4,566 patients in the IOC group and 5,044 in the LUS group. The combined sensitivity and specificity of IOC in the detection of CBD stones were 0.87 (95% confidence interval [CI]: 0.83-0.89) and 0.98 (95% CI: 0.98-0.98) respectively with a pooled area under the curve (AUC) of 0.985 and a diagnostic odds ratio (OR) of 260.65 (95% CI: 160.44-423.45). This compares with a sensitivity and specificity for LUS of 0.90 (95% CI: 0.87-0.92) and 0.99 (95% CI: 0.99-0.99) respectively with a pooled AUC of 0.982 and a diagnostic OR of 765.15 (95% CI: 450.78-1,298.76). LUS appeared to be more successful in terms of coming to a clinical decision regarding CBD stones than IOC (random effects, risk ratio: 0.95, 95% CI: 0.93-0.98, df=20, z=-3.7, p<0.005). Furthermore, LUS took less time (random effects, standardised mean difference: 0.95, 95% CI: 0.93-0.98, df=20, z=-3.7, p<0.005). Conclusions LUS is comparable with IOC in the detection of CBD stones. The main advantages of LUS are that it does not involve ionising radiation, is quicker to perform, has a lower failure rate and can be repeated during the procedure as required.

pSURF-2, a modified BAC vector for selective YAC cloning and functional analysis.

A modified bacterial artificial chromosome (BAC) vector, pSURF-2, adapted for the selective subcloning of yeast artificial chromosome (YAC) sequences was constructed. DH10B-U, a pyrF derivative of the highly transformable E. coli strain DH10B was also constructed and used for the detection of Ura+ recombinants carrying DNA linked to YAC right arms. The vector's properties were illustrated in two main ways. (i) An intact 25-kb YAC containing a mouse tyrosinase minigene was cloned into pSURF-2. Appropriately spliced tyrosinase RNA was detected by reverse transcription (RT)-PCR in extracts of cells transiently lipofected with the cloned YAC. (ii) Cells expressing human cystic fibrosis transmembrane conductance regulator (CFTR) from an integrated pSURF-2 recombinant containing a cDNA expression cassette were selected using the hygromycin-resistance (HyTK) marker of the vector and characterized by RT-PCR and immunoprecipitation. The unique I-SceI site and HyTK marker of pSURF-2 are designed to facilitate subsequent functional studies of cloned DNA.

An analysis of valve re-replacement after aortic valve replacement with biologic devices.

Biologic valve re-replacement was examined in a series of 1343 patients who underwent aortic valve replacement at The Prince Charles Hospital, Brisbane, with a cryopreserved or 4 degrees C stored allograft valve or a xenograft valve. A parametric model approach was used to simultaneously model the competing risks of death without re-replacement and re-replacement before death. One hundred eleven patients underwent a first re-replacement for a variety of reasons (69 patients with xenograft valves, 28 patients with 4 degrees C stored allograft valves, and 14 patients with cryopreserved allograft valves). By multivariable analysis younger age at operation was associated with xenograft, 4 degrees C stored allograft, and cryopreserved allograft valve re-replacement. However, this effect was examined in the context of longer survival of younger patients, which increases their exposure to the risk of re-replacement as compared with that in older patients whose decreased survival reduced their probability of requiring valve re-replacement. In patients older than 60 years at the time of aortic valve replacement, the probability of re-replacement (for any reason) before death was similar for xenografts and cryopreserved allograft valves but higher for 4 degrees C stored valves. However, in patients younger than 60 years, the probability of re-replacement at any time during the remainder of the life of the patient was lower with the cryopreserved allograft valve compared with the xenograft valve and 4 degrees C stored allografts.

An analysis of risk factors for death and mode-specific death after aortic valve replacement with allograft, xenograft, and mechanical valves.

From September 1967 to January 1990, a total of 2100 patients underwent 2366 aortic valve replacements with a variety of allograft, xenograft, and mechanical valves. Concomitant procedures were performed in 764 patients. Actuarial survival at 12 years was 59.6% (70% confidence limits 57.8% to 61.4%). Hazard function for death was highest immediately after operation, falling to merge with a slowly rising phase of risk at approximately 3 months. Actuarial freedom from sudden death at 12 years was 88.0% (70% confidence limits 86.7% to 89.3%). The shape of the hazard function for sudden death was similar to that for death. Actuarial freedom from death with cardiac failure at 12 years was 87.9% (70% confidence limits 86.5% to 89.2%). The shape of the hazard function for death with cardiac failure was also similar to that for death. Risk factor analysis revealed the important deleterious impact on long-term survival resulting from impaired left ventricular structure and function because of aortic valve disease. No current-era valve used in this study (allograft, xenograft, or mechanical) was a risk factor for death. Both aortic wall disease and endocarditis necessitating aortic valve replacement substantially decreased long-term patient survival. Aortic valve replacement is advisable much earlier in the natural history of aortic valve disease before secondary left ventricular damage occurs.

Aortic valve infection. Risk factors for death and recurrent endocarditis after aortic valve replacement.

Patients (n = 195) undergoing aortic valve replacement (n = 209) for native or prosthetic valve endocarditis were studied to determine risk factors for death and recurrent endocarditis and also to determine the valve type least likely to be associated with recurrent endocarditis. Ten-year survival was 60%, the highest risk of dying occurring within the first 3 postoperative months. Risk factors for death in this early phase included increased urea concentration, higher New York Heart Association functional class, prosthetic valve endocarditis, infection status (lower in patients with healed endocarditis), longer duration of cardiopulmonary bypass, and nonuse of an allograft valve. In the late phase (beyond 3 months), risk factors included age at operation and Staphylococcus aureus infection (only in New York Heart Association functional class V). Ten years after aortic valve replacement, 79% of valves were free of recurrent endocarditis. The highest risk of recurrence was in the first 4 months. Longer duration of cardiopulmonary bypass was a weak risk factor for recurrent endocarditis in the early phase, and in the late phase risk factors were S. aureus infection (only in New York Heart Association functional classes III, IV, and V) and the use of now discontinued biologic valves. Allograft aortic valve replacement was shown to be associated with a low and constant risk of recurrent endocarditis, whereas other valve types were associated with a high early risk. The allograft valve should be the preferred replacement device for aortic root infection.

Relationship of platelet aggregation to bleeding after cardiopulmonary bypass.

Excessive bleeding after cardiopulmonary bypass operations is a persistent problem. This study assessed the influence of platelet function on blood loss for 134 patients undergoing cardiopulmonary bypass. Platelet function was measured by platelet aggregation in platelet-rich plasma and whole blood using collagen as the agonist. Adenosine triphosphate release was assessed concurrently. Measurements were made 1 day before operation and 1 hour after the cessation of cardiopulmonary bypass. Three important findings were made. First, statistically significant correlations were shown between preoperative and postoperative platelet aggregation and blood drainage for the first 3 hours postoperatively. Second, correlations were greatest when preoperative measurement was performed on whole blood and postoperative measurement was performed on platelet-rich plasma. Third, patients with reduced postoperative platelet aggregation in platelet-rich plasma had significantly greater transfusion requirements in the first 24 hours postoperatively. In defining the 16 patients who bled excessively among the 134 patients studied, the preoperative aggregation in whole blood had a sensitivity of 62%, specificity of 75%, positive predictive value of 26%, and negative predictive value of 94%. The postoperative aggregation in platelet-rich plasma had a sensitivity of 86%, specificity of 69%, positive predictive value of 28%, and negative predictive value of 97%. These results indicate that preoperative and postoperative measurement of platelet aggregation may provide a rationale for the prophylaxis or treatment of patients to reduce blood loss after cardiopulmonary bypass.