High-resolution mucociliary transport measurement in live excised large animal trachea using synchrotron X-ray imaging

Background: The Australian Synchrotron Imaging and Medical Beamline (IMBL) was designed as the world's widest synchrotron X-ray beam, enabling both clinical imaging and therapeutic applications for humans as well as the imaging of large animal models. Our group is developing methods for imaging the airways of newly developed CF animal models that display human-like lung disease, such as the CF pig, and we expect that the IMBL can be utilised to image airways in animals of this size. Methods: This study utilised samples of excised tracheal tissue to assess the feasibility, logistics and protocols required for airway imaging in large animal models such as pigs and sheep at the IMBL. We designed an image processing algorithm to automatically track and quantify the tracheal mucociliary transport (MCT) behaviour of 103 μm diameter high refractive index (HRI) glass bead marker particles deposited onto the surface of freshly-excised normal sheep and pig tracheae, and assessed the effects of airway rehydrating aerosols. Results: We successfully accessed and used scavenged tracheal tissue, identified the minimum bead size that is visible using our chosen imaging setup, verified that MCT could be visualised, and that our automated tracking algorithm could quantify particle motion. The imaging sequences show particles propelled by cilia, against gravity, up the airway surface, within a well-defined range of clearance speeds and with examples of 'clumping' behaviour that is consistent with the in vivo capture and mucus-driven transport of particles. Conclusion: This study demonstrated that the wide beam at the IMBL is suitable for imaging MCT in ex vivo tissue samples. We are now transitioning to in vivo imaging of MCT in live pigs, utilising higher X-ray energies and shorter exposures to minimise motion blur.Martin Donnelley, Kaye S. Morgan, Maged Awadalla, Nigel R. Farrow, Chris Hall and David W. Parson

An approach for the calculation of one-loop effective actions, vacuum energies, and spectral counting functions

In this paper, we provide an approach for the calculation of one-loop effective actions, vacuum energies, and spectral counting functions and discuss the application of this approach in some physical problems. Concretely, we construct the equations for these three quantities; this allows us to achieve them by directly solving equations. In order to construct the equations, we introduce shifted local one-loop effective actions, shifted local vacuum energies, and local spectral counting functions. We solve the equations of one-loop effective actions, vacuum energies, and spectral counting functions for free massive scalar fields in $\mathbb{R}^{n}$, scalar fields in three-dimensional hyperbolic space $H_{3}$ (the Euclidean Anti-de Sitter space $AdS_{3}$), in $H_{3}/Z$ (the geometry of the Euclidean BTZ black hole), and in $S^{1}$, and the Higgs model in a $(1+1)$-dimensional finite interval. Moreover, in the above cases, we also calculate the spectra from the counting functions. Besides exact solutions, we give a general discussion on approximate solutions and construct the general series expansion for one-loop effective actions, vacuum energies, and spectral counting functions. In doing this, we encounter divergences. In order to remove the divergences, renormalization procedures are used. In this approach, these three physical quantities are regarded as spectral functions in the spectral problem.Comment: 37 pages, no figure. This is an enlarged and improved version of the paper published in JHE

Non-invasive airway health assessment: Synchrotron imaging reveals effects of rehydrating treatments on mucociliary transit in-vivo

To determine the efficacy of potential cystic fibrosis (CF) therapies we have developed a novel mucociliary transit (MCT) measurement that uses synchrotron phase contrast X-ray imaging (PCXI) to non-invasively measure the transit rate of individual micron-sized particles deposited into the airways of live mice. The aim of this study was to image changes in MCT produced by a rehydrating treatment based on hypertonic saline (HS), a current CF clinical treatment. Live mice received HS containing a long acting epithelial sodium channel blocker (P308); isotonic saline; or no treatment, using a nebuliser integrated within a small-animal ventilator circuit. Marker particle motion was tracked for 20 minutes using PCXI. There were statistically significant increases in MCT in the isotonic and HS-P308 groups. The ability to quantify in vivo changes in MCT may have utility in pre-clinical research studies designed to bring new genetic and pharmaceutical treatments for respiratory diseases into clinical trials.Martin Donnelley, Kaye S. Morgan, Karen K. W. Siu, Nigel R. Farrow, Charlene S. Stahr, Richard C. Boucher, Andreas Fouras & David W. Parson

The Impact of HAART on the Respiratory Complications of HIV Infection: Longitudinal Trends in the MACS and WIHS Cohorts

Objective: To review the incidence of respiratory conditions and their effect on mortality in HIV-infected and uninfected individuals prior to and during the era of highly active antiretroviral therapy (HAART). Design: Two large observational cohorts of HIV-infected and HIV-uninfected men (Multicenter AIDS Cohort Study [MACS]) and women (Women's Interagency HIV Study [WIHS]), followed since 1984 and 1994, respectively. Methods: Adjusted odds or hazards ratios for incident respiratory infections or non-infectious respiratory diagnoses, respectively, in HIV-infected compared to HIV-uninfected individuals in both the pre-HAART (MACS only) and HAART eras; and adjusted Cox proportional hazard ratios for mortality in HIV-infected persons with lung disease during the HAART era. Results: Compared to HIV-uninfected participants, HIV-infected individuals had more incident respiratory infections both pre-HAART (MACS, odds ratio [adjusted-OR], 2.4; 95% confidence interval [CI], 2.2-2.7; p<0.001) and after HAART availability (MACS, adjusted-OR, 1.5; 95%CI 1.3-1.7; p<0.001; WIHS adjusted-OR, 2.2; 95%CI 1.8-2.7; p<0.001). Chronic obstructive pulmonary disease was more common in MACS HIV-infected vs. HIV-uninfected participants pre-HAART (hazard ratio [adjusted-HR] 2.9; 95%CI, 1.02-8.4; p = 0.046). After HAART availability, non-infectious lung diseases were not significantly more common in HIV-infected participants in either MACS or WIHS participants. HIV-infected participants in the HAART era with respiratory infections had an increased risk of death compared to those without infections (MACS adjusted-HR, 1.5; 95%CI, 1.3-1.7; p<0.001; WIHS adjusted-HR, 1.9; 95%CI, 1.5-2.4; p<0.001). Conclusion: HIV infection remained a significant risk for infectious respiratory diseases after the introduction of HAART, and infectious respiratory diseases were associated with an increased risk of mortality. © 2013 Gingo et al

Continuous monitoring of the bronchial epithelial lining fluid by microdialysis

<p>Abstract</p> <p>Background</p> <p>Contents of the epithelial lining fluid (ELF) of the bronchi are of central interest in lung diseases, acute lung injury and pharmacology. The most commonly used technique broncheoalveolar lavage is invasive and may cause lung injury. Microdialysis (MD) is a method for continuous sampling of extracellular molecules in the immediate surroundings of the catheter. Urea is used as an endogenous marker of dilution in samples collected from the ELF. The aim of this study was to evaluate bronchial MD as a continuous monitor of the ELF.</p> <p>Methods</p> <p>Microdialysis catheters were introduced into the right main stem bronchus and into the right subclavian artery of five anesthetized and normoventilated pigs. The flowrate was 2 μl/min and the sampling interval was 60 minutes. Lactate and fluorescein-isothiocyanate-dextran 4 kDa (FD-4) infusions were performed to obtain two levels of steady-state concentrations in blood. Accuracy was defined as [bronchial-MD] divided by [arterial-MD] in percent. Data presented as mean ± 95 percent confidence interval.</p> <p>Results</p> <p>The accuracy of bronchial MD was calculated with and without correction by the arteriobronchial urea gradient. The arteriobronchial lactate gradient was 1.2 ± 0.1 and FD-4 gradient was 4.0 ± 1.2. Accuracy of bronchial MD with a continuous lactate infusion was mean 25.5% (range 5.7–59.6%) with a coefficient of variation (CV) of 62.6%. With correction by the arteriobronchial urea gradient accuracy was mean 79.0% (57.3–108.1%) with a CV of 17.0%.</p> <p>Conclusion</p> <p>Urea as a marker of catheter functioning enhances bronchial MD and makes it useful for monitoring substantial changes in the composition of the ELF.</p

Antibacterial resistance and their genetic location in MRSA isolated in Kuwait hospitals, 1994-2004

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) continues to be a major cause of serious infections in hospitals and in the community worldwide. In this study, MRSA isolated from patients in Kuwait hospitals were analyzed for resistance trends and the genetic location of their resistance determinants. METHODS: Between April 1994 and December 2004, 5644 MRSA isolates obtained from different clinical samples were studied for resistance to antibacterial agents according to guidelines from the National Committee for Clinical Laboratory Standards and the British Society for Antimicrobial Chemotherapy. The genetic location of their resistance determinants was determined by curing and transfer experiments. RESULTS: They were resistant to aminoglycosides, erythromycin, tetracycline, trimethoprim, fusidic acid, ciprofloxacin, chloramphenicol, rifampicin, mupirocin, cadmium acetate, mercuric chloride, propamidine isethionate and ethidium bromide but susceptible to vancomycin, teicoplanin and linezolid. The proportion of the isolates resistant to erythromycin, ciprofloxacin and fusidic acid increased during the study period. In contrast, the proportion of isolates resistant to gentamicin, tetracycline, chloramphenicol and trimethoprim declined. High-level mupirocin resistance increased rapidly from 1996 to 1999 and then declined. They contained plasmids of 1.9, 2.8, 3.0, 4.4, 27 and 38 kilobases. Genetic studies revealed that they carried plasmid-borne resistance to high-level mupirocin resistance (38 kb), chloramphenicol (2.8 – 4.4 kb), erythromycin (2.8–3.0 kb) and cadmium acetate, mercuric chloride, propamidine isethionate and ethidium bromide (27 kb) and chromosomal location for methicillin, the aminoglycosides, tetracycline, fusidic acid, ciprofloxacin and trimethoprim resistance. Thus, the 27 kb plasmids had resistance phenotypes similar to plasmids reported in MRSA isolates in South East Asia. CONCLUSION: The prevalence of resistance to erythromycin, ciprofloxacin, high-level mupirocin and fusidic acid increased whereas the proportion of isolates resistant to gentamicin, tetracycline, chloramphenicol and trimethoprim declined during the study period. They contained 27-kb plasmids encoding resistance to cadmium acetate, mercuric chloride, propamidine isethionate and ethidium bromide similar to plasmids isolated in MRSA from South East Asia. Molecular typing of these isolates will clarify their relationship to MRSA from South East Asia

Lipoxin A4 Stimulates Calcium-Activated Chloride Currents and Increases Airway Surface Liquid Height in Normal and Cystic Fibrosis Airway Epithelia

Cystic Fibrosis (CF) is a genetic disease characterised by a deficit in epithelial Cl− secretion which in the lung leads to airway dehydration and a reduced Airway Surface Liquid (ASL) height. The endogenous lipoxin LXA4 is a member of the newly identified eicosanoids playing a key role in ending the inflammatory process. Levels of LXA4 are reported to be decreased in the airways of patients with CF. We have previously shown that in normal human bronchial epithelial cells, LXA4 produced a rapid and transient increase in intracellular Ca2+. We have investigated, the effect of LXA4 on Cl− secretion and the functional consequences on ASL generation in bronchial epithelial cells obtained from CF and non-CF patient biopsies and in bronchial epithelial cell lines. We found that LXA4 stimulated a rapid intracellular Ca2+ increase in all of the different CF bronchial epithelial cells tested. In non-CF and CF bronchial epithelia, LXA4 stimulated whole-cell Cl− currents which were inhibited by NPPB (calcium-activated Cl− channel inhibitor), BAPTA-AM (chelator of intracellular Ca2+) but not by CFTRinh-172 (CFTR inhibitor). We found, using confocal imaging, that LXA4 increased the ASL height in non-CF and in CF airway bronchial epithelia. The LXA4 effect on ASL height was sensitive to bumetanide, an inhibitor of transepithelial Cl− secretion. The LXA4 stimulation of intracellular Ca2+, whole-cell Cl− currents, conductances and ASL height were inhibited by Boc-2, a specific antagonist of the ALX/FPR2 receptor. Our results provide, for the first time, evidence for a novel role of LXA4 in the stimulation of intracellular Ca2+ signalling leading to Ca2+-activated Cl− secretion and enhanced ASL height in non-CF and CF bronchial epithelia

Synchrotron phase-contrast X-ray imaging reveals fluid dosing dynamics for gene transfer into mouse airways

Although airway gene transfer research in mouse models relies on bolus fluid dosing into the nose or trachea, the dynamics and immediate fate of delivered gene transfer agents are poorly understood. In particular, this is because there are no in vivo methods able to accurately visualize the movement of fluid in small airways of intact animals. Using synchrotron phase-contrast X-ray imaging, we show that the fate of surrogate fluid doses delivered into live mouse airways can now be accurately and non-invasively monitored with high spatial and temporal resolution. This new imaging approach can help explain the non-homogenous distributions of gene expression observed in nasal airway gene transfer studies, suggests that substantial dose losses may occur at deliver into mouse trachea via immediate retrograde fluid motion and shows the influence of the speed of bolus delivery on the relative targeting of conducting and deeper lung airways. These findings provide insight into some of the factors that can influence gene expression in vivo, and this method provides a new approach to documenting and analyzing dose delivery in small-animal models.M Donnelley, KKW Siu, RA Jamison and DW Parson

Ileal mucosal bile acid absorption is increased in Cftr knockout mice

BACKGROUND: Excessive loss of bile acids in stool has been reported in patients with cystic fibrosis. Some data suggest that a defect in mucosal bile acid transport may be the mechanism of bile acid malabsorption in these individuals. However, the molecular basis of this defect is unknown. This study examines the expression of the ileal bile acid transporter protein (IBAT) and rates of diffusional (sodium independent) and active (sodium dependent) uptake of the radiolabeled bile acid taurocholate in mice with targeted disruption of the cftr gene. METHODS: Wild-type, heterozygous cftr (+/-) and homozygous cftr (-/-) mice were studied. Five one-cm segments of terminal ileum were excised, everted and mounted onto thin stainless steel rods and incubated in buffer containing tracer (3)H-taurocholate. Simultaneously, adjacent segments of terminal ileum were taken and processed for immunohistochemistry and Western blots using an antibody against the IBAT protein. RESULTS: In all ileal segments, taurocholate uptake rates were fourfold higher in cftr (-/-) and two-fold higher in cftr (+/-) mice compared to wild-type mice. Passive uptake was not significantly higher in cftr (-/-) mice than in controls. IBAT protein was comparably increased. Immuno-staining revealed that the greatest increases occurred in the crypts of cftr (-/-) animals. CONCLUSIONS: In the ileum, IBAT protein densities and taurocholate uptake rates are elevated in cftr (-/-) mice > cftr (+/-) > wild-type mice. These findings indicate that bile acid malabsorption in cystic fibrosis is not caused by a decrease in IBAT activity at the brush border. Alternative mechanisms are proposed, such as impaired bile acid uptake caused by the thick mucus barrier in the distal small bowel, coupled with a direct negative regulatory role for cftr in IBAT function