Previous Influenza Infection Exacerbates Allergen Specific Response and Impairs Airway Barrier Integrity in Pre-Sensitized Mice.

In this study we assessed the effects of antigen exposure in mice pre-sensitized with allergen following viral infection on changes in lung function, cellular responses and tight junction expression. Female BALB/c mice were sensitized to ovalbumin and infected with influenza A before receiving a second ovalbumin sensitization and challenge with saline, ovalbumin (OVA) or house dust mite (HDM). Fifteen days post-infection, bronchoalveolar inflammation, serum antibodies, responsiveness to methacholine and barrier integrity were assessed. There was no effect of infection alone on bronchoalveolar lavage cellular inflammation 15 days post-infection; however, OVA or HDM challenge resulted in increased bronchoalveolar inflammation dominated by eosinophils/neutrophils or neutrophils, respectively. Previously infected mice had higher serum OVA-specific IgE compared with uninfected mice. Mice previously infected, sensitized and challenged with OVA were most responsive to methacholine with respect to airway resistance, while HDM challenge caused significant increases in both tissue damping and tissue elastance regardless of previous infection status. Previous influenza infection was associated with decreased claudin-1 expression in all groups and decreased occludin expression in OVA or HDM-challenged mice. This study demonstrates the importance of the respiratory epithelium in pre-sensitized individuals, where influenza-infection-induced barrier disruption resulted in increased systemic OVA sensitization and downstream effects on lung function.

Complementary Approaches to Imaging Subcellular Lipid Architectures in Live Bacteria Using Phosphorescent Iridium Complexes and Raman Spectroscopy.

A family of three neutral iridium(III) tetrazolato complexes are investigated as bacterial imaging agents. The complexes offer a facile tuning of the emission colour from green (520 nm) to red (600 nm) in aqueous media, while keeping the excitation wavelength unchanged. The three complexes do not inhibit the bacterial growth of Bacillus Cereus, used as a model in this study, and exhibit extremely fast cellular uptake. After a minute incubation time, the nontoxic complexes show subcellular localisation in spherical structures identified as lipid vacuoles. Confocal Raman imaging has been exploited for the first time on live bacteria, to provide direct and label-free mapping of the lipid-enriched organelles within B. cereus, complementing the use of luminescent probes. Examination of the Raman spectra not only confirmed the presence of lipophilic inclusions in B. cereus but offered additional information about their chemical composition, suggesting that the lipid vacuoles may contain polyhydroxybutyrate (PHB).

Visualisation of Multiple Tight Junctional Complexes in Human Airway Epithelial Cells.

BACKGROUND: Apically located tight junctions in airway epithelium perform a fundamental role in controlling macromolecule migration through paracellular spaces. Alterations in their expression may lead to disruptions in barrier integrity, which subsequently facilitates entry of potential bacterial and other pathogens into the host. Furthermore, there is emerging evidence that the barrier integrity of the airway in certain airway inflammatory diseases may be altered. However, there is little consensus on the way this is assessed and measured and the type of cells used to achieve this. METHODS: Here, we assessed four fixation methods including; (i) 4% (v/v) paraformaldehyde; (ii) 100% methanol; (iii) acetone or; (iv) 1:1 methanol: acetone. Pre-extraction with Triton X-100 was also performed and assessed on cells prior to fixation with either methanol or paraformaldehyde. Cells were also permeabilized with 0.1% (v/v) Saponin in 1× TBS following fixation and subsequently stained for tight junction proteins. Confocal microscopy was then used to visualise, compare and evaluate staining intensity of the tight junctional complexes in order to determine a standardised workflow of reproducible staining. RESULTS: Positive staining was observed following methanol fixation for claudin-1 and ZO-1 tight junction proteins but no staining was detected for occludin in 16HBE14o- cells. Combinatorial fixation with methanol and acetone also produced consistent positive staining for both occludin and ZO-1 tight junction proteins in these cells. When assessed using primary cells cultured at air-liquid interface, similar positive staining for claudin-1 and ZO-1 was observed following methanol fixation, while similar positive staining for occludin and ZO-1 was observed following the same combinatorial fixation with methanol and acetone. CONCLUSIONS: The present study demonstrates the importance of a personalised approach to optimise staining for the visualisation of different tight junction proteins. Of significance, the workflow, once optimised, can readily be translated into primary airway epithelial cell air-liquid interface cultures where it can be used to assess barrier integrity in chronic lung diseases.

Effect of human rhinovirus infection on airway epithelium tight junction protein disassembly and transepithelial permeability.

RATIONALE: No studies have assessed the effects of human rhinovirus (HRV) infection on epithelial tight junctions (TJs) and resultant barrier function. AIM OF THE STUDY: To correlate viral infection with TJ disassembly, epithelial barrier integrity, and function. MATERIALS AND METHODS: Human airway epithelial cells were infected with HRV minor serotype 1B (HRV-1B) at various 50% tissue culture infectivity doses (TCID50) over 72 hours. HRV replication was assessed by quantitative-polymerase chain reaction (qPCR) while cell viability and apoptosis were assessed by proliferation and apoptotic assays, respectively. Protein expression of claudin-1, occludin, and zonula occludens protein-1 (ZO-1) was assessed using In-Cell™ Western assays. Transepithelial permeability assays were performed to assess effects on barrier functionality. RT2 Profiler focused qPCR arrays and pathway analysis evaluating associations between human TJ and antiviral response were performed to identify potential interactions and pathways between genes of interests. RESULTS: HRV-1B infection affected viability that was both time and TCID50 dependent. Significant increases in apoptosis and viral replication post-infection correlated with viral titer. Viral infection significantly decreased claudin-1 protein expression at the lower TCID50, while a significant decrease in all three TJ protein expressions occurred at higher TCID50. Decrease in protein expression was concomitant with significant increases in epithelial permeability of fluorescein isothiocynate labeled-dextran 4 and 20 kDa. Analysis of focused qPCR arrays demonstrated a significant decrease in ZO-1 gene expression. Furthermore, network analysis between human TJ and antiviral response genes revealed possible interactions and regulation of TJ genes via interleukin (IL)-15 in response to HRV-1B infection. CONCLUSION: HRV-1B infection directly alters human airway epithelial TJ expression leading to increased epithelial permeability potentially via an antiviral response of IL-15.

Flow cytometry as a rapid and reliable method to quantify sperm viability in the honeybee Apis mellifera.

An important measure of male quality is sperm viability; i.e., the percentage of live sperm within an ejaculate, as this provides an accurate measure of the number of sperm potentially available for egg fertilization. Sperm viability is often determined by fluorescence microscopy using dyes that differentially stain viable and nonviable sperm, but the technique has a number of limitations. Here, a flow cytometry (FCM) method was developed, which allows the rapid determination of honeybee sperm viability, facilitating high throughput analyses. Using samples with known sperm viabilities, it was found that data obtained from FCM were more accurate and less variable compared with data obtained for the same samples using fluorescence microscopy. It was also found that a previously reported additional population of honeybee sperm found in datasets using FCM is caused by freeze-thawing samples. In conclusion, the method described here allows to quantify sperm viability of honeybees quickly and with high accuracy. This will be of great value for future scientific research and could also be of value to guide future bee breeding programs, given the agricultural importance of honeybees as pollinators.

Vascular normalization in Rgs5-deficient tumours promotes immune destruction.

The vasculature of solid tumours is morphologically aberrant and characterized by dilated and fragile vessels, intensive vessel sprouting and loss of hierarchical architecture. Constant vessel remodelling leads to spontaneous haemorrhages and increased interstitial fluid pressure in the tumour environment. Tumour-related angiogenesis supports tumour growth and is also a major obstacle for successful immune therapy as it prevents migration of immune effector cells into established tumour parenchyma. The molecular mechanisms for these angiogenic alterations are largely unknown. Here we identify regulator of G-protein signalling 5 (Rgs5) as a master gene responsible for the abnormal tumour vascular morphology in mice. Loss of Rgs5 results in pericyte maturation, vascular normalization and consequent marked reductions in tumour hypoxia and vessel leakiness. These vascular and intratumoral changes enhance influx of immune effector cells into tumour parenchyma and markedly prolong survival of tumour-bearing mice. This is the first demonstration, to our knowledge, of reduced tumour angiogenesis and improved immune therapeutic outcome on loss of a vascular gene function and establishes a previously unrecognized role of G-protein signalling in tumour angiogenesis.

Receptor activator of NF-κB ligand promotes proliferation of a putative mammary stem cell unique to the lactating epithelium.

In mice, CD49f(hi) mammary stem cells (MaSCs) asymmetrically divide to generate CD49f(+) committed progenitor cells that differentiate into CD49f(-) phenotypes of the milk-secreting tissue at the onset of pregnancy. We show CD49f(+) primary mammary epithelial cells (PMECs) isolated from lactating tissue uniquely respond to pregnancy-associated hormones (PAH) compared with CD49f(+) cells from nonlactating tissue. Differentiation of CD49f(+) PMEC in extracellular matrix produces CD49f(-) luminal cells to form differentiated alveoli. The PAH prolactin and placental lactogen specifically stimulate division of CD49f(-) luminal cells, while receptor activator of nuclear factor (NF)-κB ligand (RANKL) specifically stimulates division of basal CD49f(+) cells. In nondifferentiating conditions, we observed a greater proportion of multipotent self-renewing cells, and RANKL treatment activated the RANK pathway in these cultures. Furthermore, we observed the deposition of calcium nodules in a proportion of these cells. These data imply that a MaSC unique to the lactating breast exists in humans, which generates progeny with discrete lineages and distinct response to PAH.

Image analysis of liver collagen using sirius red is more accurate and correlates better with serum fibrosis markers than trichrome.

BACKGROUND: Collagen proportional area (CPA) determined by quantitative digital image analysis better quantifies liver fibrosis than histological stage; however, its clinical use has been limited by non-standardized methods. AIM: This study aimed to compare CPA obtained using different staining methods, magnifications and biopsy sizes. METHODS: Two hundred and forty-nine patients with chronic hepatitis C who had a liver biopsy and serum fibrosis markers performed were included. CPA was measured either using a sirius red (CPAs) or a trichrome (CPAt) stain. RESULTS: CPAs measured at 20× and 40× magnifications generated similar outcomes with interclass correlation (ICC) coefficient of 0.98. Compared with trichrome, sirius red staining had much less variation with an ICC coefficient of 0.99 for slides stained in the same batch and 0.92 in different batches. Mean CPAs was higher than mean CPAt by 3.53%, P < 0.001. Morphological analysis found that sirius red detected delicate fibrous septa and spurs better than trichrome. Both CPAs and CPAt correlated well with Metavir stage, whereas CPAs had better ability to detect cirrhosis with the area under ROC curve of 0.95. Overall CPA had superior correlation with serum markers of fibrosis in Metavir F2-F4 than that in F0-F1 and CPAs correlated better with serum fibrosis markers than CPAt in Metavir F0-F1. Multivairate analysis found that HA, α2-macroglobulin, platelet count and albumin were independently correlated with CPAs and only HA was independently correlated with CPAt. CONCLUSIONS: Sirius red staining for CPA determination was more accurate and reliable for quantifying hepatic collagen compared with trichrome staining.

Preliminary studies on the development of IgA-loaded chitosan-dextran sulphate nanoparticles as a potential nasal delivery system for protein antigens.

This study describes the development of a biodegradable nanoparticulate system for the intranasal delivery of multiple proteins. Chitosan (CS)-dextran sulphate (DS) nanoparticles were developed and optimised for the incorporation of pertussis toxin (PTX) and a potential targeting ligand (immunoglobulin-A, IgA). In vitro characterization and in vivo uptake studies were performed for the evaluation of developed nanoparticles. The ratio of CS to DS, the order of mixing and pH of nanoparticle suspension were identified as important formulation factors governing the size and zeta potential of nanoparticles. An optimised CS-DS nanoparticle formulation prepared with the CS to DS weight ratio of 3 : 1 was used to load PTX and/or IgA. Entrapment efficiency of >90% was obtained for both. The in vivo uptake of IgA-loaded CS-DS nanoparticles in mice showed a preferential uptake of nanoparticles probably by nasal membranous or microfold cells following intranasal administration. The results of this study indicate the potential application of IgA-loaded CS-DS nanoparticles as a nasal vaccine delivery system.

Molecular exploration of fossil eggshell uncovers hidden lineage of giant extinct bird.

The systematics of Madagascar's extinct elephant birds remains controversial due to large gaps in the fossil record and poor biomolecular preservation of skeletal specimens. Here, a molecular analysis of 1000-year-old fossil eggshells provides the first description of elephant bird phylogeography and offers insight into the ecology and evolution of these flightless giants. Mitochondrial genomes from across Madagascar reveal genetic variation that is correlated with eggshell morphology, stable isotope composition, and geographic distribution. The elephant bird crown is dated to ca. 30 Mya, when Madagascar is estimated to have become less arid as it moved northward. High levels of between-clade genetic variation support reclassifying Mullerornis into a separate family. Low levels of within-clade genetic variation suggest there were only two elephant bird genera existing in southern Madagascar during the Holocene. However, we find an eggshell collection from Madagascar's far north that represents a unique lineage of Aepyornis. Furthermore, divergence within Aepyornis coincides with the aridification of Madagascar during the early Pleistocene ca. 1.5 Ma, and is consistent with the fragmentation of populations in the highlands driving diversification and the evolution of extreme gigantism over shorts timescales. We advocate for a revision of their taxonomy that integrates palaeogenomic and palaeoecological perspectives.

Multi-species bacterial biofilm and intracellular infection in otitis media.

BACKGROUND: Bacteria which are metabolically active yet unable to be cultured and eradicated by antibiotic treatment are present in the middle ear effusion of children with chronic otitis media with effusion (COME) and recurrent acute otitis media (rAOM). These observations are suggestive of biofilm presence or intracellular sequestration of bacteria and may play a role in OM pathogenesis. The aim of this project is to provide evidence for the presence of otopathogenic bacteria intracellularly or within biofilm in the middle ear mucosa of children with COME or rAOM. METHODS: Middle ear mucosal biopsies from 20 children with COME or rAOM were examined for otopathogenic bacteria (either in biofilm or located intracellularly) using transmission electron microscopy (TEM) or species specific fluorescent in situ hybridisation (FISH) and confocal laser scanning microscopy (CLSM). One healthy control biopsy from a child undergoing cochlear implant surgery was also examined. RESULTS: No bacteria were observed in the healthy control sample. In 2 of the 3 biopsies imaged using TEM, bacteria were observed in mucus containing vacuoles within epithelial cells. Bacterial species within these could not be identified and biofilm was not observed. Using FISH with CLSM, bacteria were seen in 15 of the 17 otitis media mucosal specimens. In this group, 11 (65%) of the 17 middle ear mucosal biopsies showed evidence of bacterial biofilm and 12 demonstrated intracellular bacteria. 52% of biopsies were positive for both biofilm and intracellular bacteria. At least one otopathogen was identified in 13 of the 15 samples where bacteria were present. No differences were observed between biopsies from children with COME and those with rAOM. CONCLUSION: Using FISH and CLSM, bacterial biofilm and intracellular infection with known otopathogens are demonstrated on/in the middle ear mucosa of children with COME and/or rAOM. While their role in disease pathogenesis remains to be determined, this previously undescribed infection pattern may help explain the ineffectiveness of current treatment strategies at preventing or resolving COME or rAOM.

Coupled porohyperelastic mass transport (PHEXPT) finite element models for soft tissues using ABAQUS.

Finite element models (FEMs) including characteristic large deformations in highly nonlinear materials (hyperelasticity and coupled diffusive/convective transport of neutral mobile species) will allow quantitative study of in vivo tissues. Such FEMs will provide basic understanding of normal and pathological tissue responses and lead to optimization of local drug delivery strategies. We present a coupled porohyperelastic mass transport (PHEXPT) finite element approach developed using a commercially available ABAQUS finite element software. The PHEXPT transient simulations are based on sequential solution of the porohyperelastic (PHE) and mass transport (XPT) problems where an Eulerian PHE FEM is coupled to a Lagrangian XPT FEM using a custom-written FORTRAN program. The PHEXPT theoretical background is derived in the context of porous media transport theory and extended to ABAQUS finite element formulations. The essential assumptions needed in order to use ABAQUS are clearly identified in the derivation. Representative benchmark finite element simulations are provided along with analytical solutions (when appropriate). These simulations demonstrate the differences in transient and steady state responses including finite deformations, total stress, fluid pressure, relative fluid, and mobile species flux. A detailed description of important model considerations (e.g., material property functions and jump discontinuities at material interfaces) is also presented in the context of finite deformations. The ABAQUS-based PHEXPT approach enables the use of the available ABAQUS capabilities (interactive FEM mesh generation, finite element libraries, nonlinear material laws, pre- and postprocessing, etc.). PHEXPT FEMs can be used to simulate the transport of a relatively large neutral species (negligible osmotic fluid flux) in highly deformable hydrated soft tissues and tissue-engineered materials.

Dysregulated Notch Signaling in the Airway Epithelium of Children with Wheeze.

The airway epithelium of children with wheeze is characterized by defective repair that contributes to disease pathobiology. Dysregulation of developmental processes controlled by Notch has been identified in chronic asthma. However, its role in airway epithelial cells of young children with wheeze, particularly during repair, is yet to be determined. We hypothesized that Notch is dysregulated in primary airway epithelial cells (pAEC) of children with wheeze contributing to defective repair. This study investigated transcriptional and protein expression and function of Notch in pAEC isolated from children with and without wheeze. Primary AEC of children with and without wheeze were found to express all known Notch receptors and ligands, although pAEC from children with wheeze expressed significantly lower NOTCH2 (10-fold, p = 0.004) and higher JAG1 (3.5-fold, p = 0.002) mRNA levels. These dysregulations were maintained in vitro and cultures from children with wheeze displayed altered kinetics of both NOTCH2 and JAG1 expression during repair. Following Notch signaling inhibition, pAEC from children without wheeze failed to repair (wound closure rate of 76.9 ± 3.2%). Overexpression of NOTCH2 in pAEC from children with wheeze failed to rescue epithelial repair following wounding. This study illustrates the involvement of the Notch pathway in airway epithelial wound repair in health and disease, where its dysregulation may contribute to asthma development.

Fossil avian eggshell preserves ancient DNA.

Owing to exceptional biomolecule preservation, fossil avian eggshell has been used extensively in geochronology and palaeodietary studies. Here, we show, to our knowledge, for the first time that fossil eggshell is a previously unrecognized source of ancient DNA (aDNA). We describe the successful isolation and amplification of DNA from fossil eggshell up to 19 ka old. aDNA was successfully characterized from eggshell obtained from New Zealand (extinct moa and ducks), Madagascar (extinct elephant birds) and Australia (emu and owl). Our data demonstrate excellent preservation of the nucleic acids, evidenced by retrieval of both mitochondrial and nuclear DNA from many of the samples. Using confocal microscopy and quantitative PCR, this study critically evaluates approaches to maximize DNA recovery from powdered eggshell. Our quantitative PCR experiments also demonstrate that moa eggshell has approximately 125 times lower bacterial load than bone, making it a highly suitable substrate for high-throughput sequencing approaches. Importantly, the preservation of DNA in Pleistocene eggshell from Australia and Holocene deposits from Madagascar indicates that eggshell is an excellent substrate for the long-term preservation of DNA in warmer climates. The successful recovery of DNA from this substrate has implications in a number of scientific disciplines; most notably archaeology and palaeontology, where genotypes and/or DNA-based species identifications can add significantly to our understanding of diets, environments, past biodiversity and evolutionary processes.

Conditioned Contextual Freezing is A Neurobehavioral Biomarker of Axonal Injury Indicated by Reduced Fractional Anisotropy in A Mouse Model of Blast-Induced Mild Traumatic Brain Injury.

Mild traumatic brain injury (TBI) is an important public health problem generated by closed head injury. This study is focused on the impact of blast-induced mild TBI on auditory trace and delay fear conditioning, models of declarative and non-declarative memory, respectively, and the correlation of conditioned freezing and fractional anisotropy, a measure of axonal state. A supersonic helium pressure wave was generated by a shock tube to blast 8-week-old male mice on Day 1 for 1.4 msec with an incident pressure of 16 psi, corresponding to a reflected pressure of 56.9 psi at the mouse head. On Day 3, the mice were subjected to auditory trace- or delay-fear conditioning. On Day 4, contextual freezing in the trained context, and precue and cued freezing in a novel context were determined. After cardiac perfusion on Day 5, ex vivo images were obtained with diffusion tensor imaging at 14.1 Tesla. We observed that delay fear conditioning prevented or reversed the decrease in fractional anisotropy in both the medial and lateral corpus callosum suggesting axonal stabilization of potentially behavioral therapeutic significance. Moderately strong and statistically significant Pearson correlations were found between fractional anisotropy and contextual freezing in the medial and lateral corpus callosum of blasted and sham-blasted delay- or trace-fear conditioned mice. Thus, contextual freezing is a neurobehavioral biomarker for axonal injury in mild TBI and is a reliable and high-throughput behavioral assay for the evaluation of potential therapeutics to treat mild TBI.

Multimodal Imaging and Analysis of the Neuroanatomical Organization of the Primary Olfactory Inputs in the Brownbanded Bamboo Shark, Chiloscyllium punctatum.

There is currently a limited understanding of the morphological and functional organization of the olfactory system in cartilaginous fishes, particularly when compared to bony fishes and terrestrial vertebrates. In this fish group, there is a clear paucity of information on the characterization, density, and distribution of olfactory receptor neurons (ORNs) within the sensory olfactory epithelium lining the paired olfactory rosettes, and their functional implications with respect to the hydrodynamics of incurrent water flow into the nares. This imaging study examines the brownbanded bamboo shark Chiloscyllium punctatum (Elasmobranchii) and combines immunohistochemical labeling using antisera raised against five G-protein α-subunits (Gαs/olf, Gαq/ 11 / 14, Gαi- 1 / 2 / 3, Gαi- 3, Gα o ) with light and electron microscopy, to characterize the morphological ORN types present. Three main ORNs ("long", "microvillous" and "crypt-like") are confirmed and up to three additional microvilli-bearing types are also described; "Kappe-like" (potential or homologous "Kappe" as in teleosts), "pear-shaped" and "teardrop-shaped" cells. These morphotypes will need to be confirmed molecularly in the future. Using X-ray diffusible iodine-based contrast-enhanced computed tomography (diceCT), high-resolution scans of the olfactory rosettes, olfactory bulbs (OBs), peduncles, and telencephalon reveal a lateral segregation of primary olfactory inputs within the OBs, with distinct medial and lateral clusters of glomeruli, suggesting a potential somatotopic organization. However, most ORN morphotypes are found to be ubiquitously distributed within the medial and lateral regions of the olfactory rosette, with at least three microvilli-bearing ORNs labeled with anti-Gα o found in significantly higher densities in lateral lamellae [in lateral lamellae] and on the anterior portion of lamellae (facing the olfactory cavity). These microvilli-bearing ORN morphotypes (microvillous, "Kappe-like," "pear-shaped," and "teardrop-shaped") are the most abundant across the olfactory rosette of this species, while ciliated ORNs are less common and crypt cells are rare. Spatial simulations of the fluid dynamics of the incurrent water flow into the nares and within the olfactory cavities indicate that the high densities of microvilli-bearing ORNs located within the lateral region of the rosette are important for sampling incoming odorants during swimming and may determine subsequent tracking behavior.

Initial Biphasic Fractional Anisotropy Response to Blast-Induced Mild Traumatic Brain Injury in a Mouse Model.

INTRODUCTION: Blast-induced mild traumatic brain injury was generated in a mouse model using a shock tube to investigate recovery and axonal injury from single blast. METHODS: A supersonic helium wave hit the head of anesthetized male young adult mice with a reflected pressure of 69 psi for 0.2 ms on Day 1. Subsequently, the mice were cardioperfused on Days 2, 5, or 12. The isolated brains were subjected to diffusion tensor imaging. Reduced fractional anisotropy (FA) indicated axonal injury. RESULTS: After single blast, FA showed a biphasic response in the corpus callosum with decrease on Days 2 and 12 and increase on Day 5. CONCLUSIONS: Blast-induced mild traumatic brain injury in a mouse model follows a biphasic FA response within 12 days after a single blast similar to that reported for human subjects.

Aberrant cell migration contributes to defective airway epithelial repair in childhood wheeze.

Abnormal wound repair has been observed in the airway epithelium of patients with chronic respiratory diseases, including asthma. Therapies focusing on repairing vulnerable airways, particularly in early life, present a potentially novel treatment strategy. We report defective lower airway epithelial cell repair to strongly associate with common pre-school-aged and school-aged wheezing phenotypes, characterized by aberrant migration patterns and reduced integrin α5ß1 expression. Next generation sequencing identified the PI3K/Akt pathway as the top upstream transcriptional regulator of integrin α5ß1, where Akt activation enhanced repair and integrin α5ß1 expression in primary cultures from children with wheeze. Conversely, inhibition of PI3K/Akt signaling in primary cultures from children without wheeze reduced α5ß1 expression and attenuated repair. Importantly, the FDA-approved drug celecoxib - and its non-COX2-inhibiting analogue, dimethyl-celecoxib - stimulated the PI3K/Akt-integrin α5ß1 axis and restored airway epithelial repair in cells from children with wheeze. When compared with published clinical data sets, the identified transcriptomic signature was also associated with viral-induced wheeze exacerbations highlighting the clinical potential of such therapy. Collectively, these results identify airway epithelial restitution via targeting the PI3K-integrin α5ß1 axis as a potentially novel therapeutic avenue for childhood wheeze and asthma. We propose that the next step in the therapeutic development process should be a proof-of-concept clinical trial, since relevant animal models to test the crucial underlying premise are unavailable.

Oxidative stress causes a decline in lysosomal integrity during hypothermic incubation of rat hepatocytes.

Oxidative stress during cold preservation has been identified as a significant cause of cell injury but the process by which injury occurs is poorly understood. We examined loss of lysosomal integrity as a possible cause of cell injury during extended cold storage of isolated rat hepatocytes. After 21 h of hypothermia, there was a marked decline in lysosomal integrity, which was correlated with an increase in lipid peroxidation. When lipid peroxidation was prevented with the antioxidant Trolox (a vitamin E analog) or the iron chelator desferrioxamine, lysosomal integrity was preserved. In contrast, increasing lysosomal iron with ferric chloride caused an increase in lipid peroxidation and decreased lysosomal integrity. Loss of lysosomal integrity during cold preservation in this experimental model was consistent with iron-initiated oxidative stress. The progressive loss of lysosomal integrity during hypothermic incubation has the potential to affect liver function after transplantation.

Expression of kallikrein-related peptidases (KRP/hK5, 7, 6, 8) in subtypes of human lung carcinoma.

Lung cancer is currently the leading cause of cancer mortality worldwide. Expression of kallikrein-related peptidases (KRP/hK/KLK) may be induced during lung carcinogenesis. To test the hypothesis that KRP/hK, previously identified in the skin (KRP/hK5, 7) and brain (KRP/hK6, 8), are expressed in lung tumours, experiments were designed to investigate their localization in four malignant sub-types of human lung cancer. Using specific antibodies, expression of these KRP/hK was determined in archived lung tumour sections of the four subtypes, and in normal skin, brain, lung and submandibular gland tissue sections. Immunoperoxidase labelled sections were visualized by brightfield microscopy. In the squamous cell carcinoma, small cell carcinoma and carcinoid tumour, 40-90% of the malignant cells showed positive cytoplasmic labelling for KRP/hK5, 7, 6 and 8 (intensity grade 2+/3+). In the adenocarcinoma there was no cytoplasmic labelling for any of the KRP/hK, but the nuclei of 20% of the tumour cells were labelled for KRP/hK5, 7 and 8 (intensity grade 2+/3+). Further studies are required to determine the functional significance of the expression of KRP/hK in human lung carcinomas, and whether any of these proteins may be potential biomarkers for specific sub-types of lung cancer.