Differences in control by UV radiation of inflammatory airways disease in na?ve and allergen pre-sensitised mice

Exposure of skin to UV radiation (UVR) prior to allergen exposure can inhibit inflammatory airways disease in mice by reducing effector CD4+ T cells in both the trachea and the airway draining lymph nodes. This study analysed the immunomodulatory properties of UVR delivered to na?ve versus allergen pre-sensitised mice. In a model of inflammatory airways disease, BALB/c mice were sensitised by peritoneal injection of the allergen, ovalbumin (OVA) (20 μg/mouse), in the adjuvant, alum (4 mg/mouse), on days 0 and 14. On day 21, the mice were exposed to aerosolised OVA and 24 h later, proliferative responses by the cells in the airway draining lymph nodes were examined. UVR (8 kJ m(-2)) was administered 3 days prior to first OVA sensitisation (day -3), or OVA aerosol challenge (day 18). UVR before sensitisation reduced immune responses associated with expression of allergic airways disease; seven days after first OVA sensitisation, regulation of OVA-induced proliferation in vitro but not in vivo by CD4+CD25+ cells from UV-irradiated mice was detected. UVR administered to pre-sensitised mice regulated allergen responsiveness by cells from the airway draining lymph nodes only with a sensitisation protocol involving allergen and adjuvant at 5% strength of the original dose (1 μg OVA in 0.2 mg alum/mouse). These results suggest that UVR may modulate allergic airways disease by two mechanisms. The first, and more potent, is by reducing effector cells in respiratory tissues and requires UV delivery prior to sensitisation. The second, associated with administration to pre-sensitised mice, is weaker and is detected when the mice are sensitised with lower levels of allergen and adjuvant.     

Capacity for resolution of Ras-MAPK-initiated early pathogenic myocardial hypertrophy modeled in mice

Activation of Ras signaling in cardiomyocytes has been linked to pathogenic myocardial hypertrophy progression and subsequent heart failure. Whether cardiomyopathy can regress once initiated needs to be established more fully. A 'tet-off' system was used to regulate expression of H-Ras-G12V in myocardium to examine whether Ras-induced pathogenic myocardial hypertrophy could resolve after removal of Ras signaling in vivo. Ras activation at weaning for 2 wk caused hypertrophy, whereas activation for 4 to 8 wk led to cardiomyopathy and heart failure. Discontinuing H-Ras-G12V transgene expression after cardiomyopathy onset led to improved survival and cardiomyopathy lesion scores, with reduced heart:body weight ratios, demonstrating the reversibility of early pathogenic hypertrophy. Activation of Ras and downstream ERK 1/2 was associated with elevated expression of proliferating cell nuclear antigen and cyclins B1 and D1, indicating cell-cycle activation and reentry. Coordinate elevation of broad-spectrum cyclin-dependent kinase inhibitors (p21, p27, and p57) and Tyr15 phosphorylation of cdc2 signified the activation of cell-cycle checkpoints; absence of cell-cycle completion and cardiomyocyte replication were documented by using immunohistochemistry for mitosis and cytokinesis markers. After resolution of cardiomyopathy, cell-cycle activators and inhibitors examined returned to basal levels, a change that we interpreted as exit from the cell cycle. Cardiac cell-cycle regulation plays a role in recovery from pathogenic hypertrophy. The model we present provides a means to further explore the underlying mechanisms governing cell-cycle capacity in cardiomyocytes, as well as progression and regression of pathogenic cardiomyocyte hypertrophy.     

Quantifying Histological Features of Cancer Biospecimens for Biobanking Quality Assurance Using Automated Morphometric Pattern Recognition Image Analysis Algorithms

Biorepository-supported translational research depends on high-quality, well-annotated specimens. Histopathology assessment contributes insight into how representative lesions are for research objectives. Feasibility of documenting histological proportions of tumor and stroma was studied in an effort to enhance information regarding biorepository tissue heterogeneity. Using commercially available software, unique spatial-spectral algorithms were developed for applying automated pattern recognition morphometric image analysis to quantify histologic tumor and nontumor tissue areas in biospecimen tissue sections. Measurements were acquired successfully for 75/75 (100%) lymphomas, 76/77 (98.7%) osteosarcomas, and 60/70 (85.7%) melanomas. The percentage of tissue area occupied by tumor varied among patients and tumor types and was distributed around medians of 94% [interquartile range (IQR)=14%] for lymphomas, 84% for melanomas (IQR=24%), and 39% for osteosarcomas (IQR=44%). Within-patient comparisons from a subset, including multiple individual patient specimens, revealed ≤12% median coefficient of variation (CV) for lymphomas and melanomas. Phenotypic heterogeneity of osteosarcomas resulted in 33% median CV. Uniformly applied, tumor-specific pattern recognition software permits automated tissue-feature quantification. Furthermore, dispersion analyses of area measurements across collections, as well as of multiple specimens from individual patients, support using limited tissue slices to gauge features for some tumor types. Quantitative image analysis automation is anticipated to minimize variability associated with routine biorepository pathologic evaluations and enhance biomarker discovery by helping to guide the selection of study-appropriate specimens.     

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes. More than 700 human miRNAs have been identified so far with each having up to hundreds of unique target mRNAs. Computational tools, expression and proteomics assays, and chromatin-immunoprecipitation-based techniques provide important clues for identifying mRNAs that are direct targets of a particular miRNA. In addition, 3''UTR-reporter assays have become an important component of thorough miRNA target studies because they provide functional evidence for and quantitate the effects of specific miRNA-3''UTR interactions in a cell-based system. To enable more researchers to leverage 3''UTR-reporter assays and to support the scale-up of such assays to high-throughput levels, we have created a genome-wide collection of human 3''UTR luciferase reporters in the highly-optimized LightSwitch Luciferase Assay System. The system also includes synthetic miRNA target reporter constructs for use as positive controls, various endogenous 3''UTR reporter constructs, and a series of standardized experimental protocols.Here we describe a method for co-transfection of individual 3''UTR-reporter constructs along with a miRNA mimic that is efficient, reproducible, and amenable to high-throughput analysis.Download video file.^{(45M, mov)}     

Using time-resolved fluorescence to measure serum venom-specific IgE and IgG

We adapted DELFIA™ (dissociation-enhanced lanthanide fluoroimmunoassay), a time resolved fluorescence method, to quantitate whole venom specific and allergenic peptide-specific IgE (sIgE), sIgG₁ and sIgG₄ in serum from people clinically allergic to Australian native ant venoms, of which the predominant cause of allergy is jack jumper ant venom (JJAV). Intra-assay CV was 6.3% and inter-assay CV was 13.7% for JJAV sIgE. DELFIA and Phadia CAP JJAV sIgE results correlated well and had similar sensitivity and specificity for the detection of JJAV sIgE against intradermal skin testing as the gold standard. DELFIA was easily adapted for detecting sIgE to a panel of other native ant venoms.     

Glutamate production by HIV-1 infected human macrophage is blocked by the inhibition of glutaminase

Mononuclear phagocyte (macrophages and microglia) dysfunction plays a significant role in the pathogenesis of human immunodeficiency virus (HIV) associated dementia (HAD) through the production and release of soluble neurotoxic factors including glutamate. The mechanism of glutamate regulation by HIV-1 infection remains unclear. In this report, we investigated whether the enzyme glutaminase is responsible for glutamate generation by HIV-1 infected monocyte-derived macrophages. We tested the functionality of novel small molecule inhibitors designed to specifically block the activity of glutaminase. Glutaminase inhibitors were first characterized in a kinetic assay with crude glutaminase from rat brain revealing an uncompetitive mechanism of inhibition. The inhibitors were then tested in vitro for their ability to prevent glutamate generation by HIV-infected macrophages, their effect upon macrophage viability, and HIV infection. To validate these findings, glutaminase specific siRNA was tested for its ability to prevent glutamate increase during infection. Our results show that both glutaminase specific small molecule inhibitors and glutaminase specific siRNA were effective at preventing increases in glutamate by HIV-1 infected macrophage. These findings support glutaminase as a potential component of the HAD pathogenic process and identify a possible therapeutic avenue for the treatment of neuroinflammatory states such as HAD.     

Evidence for trichloroethylene bioactivation and adduct formation in the rat epididymis and efferent ducts

Recent studies indicate that trichloroethylene (TCE) may be a male reproductive toxicant. It is metabolized by conjugation with glutathione and cytochrome p450-dependent oxidation. Reactive metabolites produced along both pathways are capable of forming protein adducts and are thought to be involved in TCE-induced liver and kidney damage. Similarly, in situ bioactivation of TCE and subsequent binding of metabolites may be one mechanism by which TCE acts as a reproductive toxicant. Cysteine-conjugate beta-lyase (beta-lyase) bioactivates the TCE metabolite dichlorovinyl cysteine (DCVC) to a reactive intermediate that is capable of binding cellular macromolecules. In the present study, Western blot analysis indicated that the soluble form of beta-lyase, but not the mitochondrial form, was present in the epididymis and efferent ducts. Both forms of beta-lyase were detected in the kidney. When rats were dosed with DCVC, no protein adducts were detected in the epididymis or efferent ducts, although adducts were present in the proximal tubule of the kidney. Trichloroethylene can also be metabolized and form protein adducts through a cytochrome p450-mediated pathway. Western blot analysis detected the presence of cytochrome p450 2E1 (CYP2E1) in the efferent ducts. Immunoreactive proteins were localized to efferent duct and corpus epididymis epithelia. Metabolism of TCE was demonstrated in vitro using microsomes prepared from untreated rats. Metabolism was inhibited 77% when efferent duct microsomes were preincubated with an antibody to CYP2E1. Dichloroacetyl adducts were detected in epididymal and efferent duct microsomes exposed in vitro to TCE. Results from the present study indicate that the cytochrome p450-dependent formation of reactive intermediates and the subsequent covalent binding of cellular proteins may be involved in the male reproductive toxicity of TCE.