Expression and functional implications of CCR2 expression on murine alveolar epithelial cells

Acute lung injury results in damage to the alveolar epithelium, leading to leak of proteins into the alveolar space and impaired gas exchange. Lung function can be restored only if the epithelial layer is restored. The process of reepithelialization requires migration of lung epithelial cells to cover denuded basement membranes. The factors that control the migration of lung epithelial cells are incompletely understood. We examined isolated murine type II alveolar epithelial cells (AECs) for expression of CC chemokine receptor 2 (CCR2) and functional consequences of the binding of the main CCR2 ligand monocyte chemoattractant protein-1 (MCP-1). We found that primary AECs bound MCP-1 and expressed CCR2 mRNA. These cells demonstrated functional consequences of CCR2 expression with migration in response to MCP-1 in chemotaxis/haptotaxis assays. Primary AECs cultured from mice lacking CCR2 did not respond to MCP-1. Monolayers of AECs lacking CCR2 demonstrated delayed closure of mechanical wounds compared with AEC monolayers expressing CCR2. Delayed closure of mechanical wounds of wild-type AECs was also demonstrated in the presence of anti-MCP-1 antibody. These data demonstrate for the first time that AECs express CCR2 and are capable of using this receptor for chemotaxis and healing of wounds. CCR2-MCP-1 interactions may be important in the process of reepithelialization after lung injury.     

Protection from fluorescein isothiocyanate-induced fibrosis in IL-13-deficient, but not IL-4-deficient, mice results from impaired collagen synthesis by fibroblasts

Intratracheal injection of FITC results in acute lung injury and progresses to fibrosis by day 21 postchallenge. In response to FITC, BALB/c mice produce IL-4 and IL-13 in the lung. To investigate whether IL-4 and/or IL-13 were important profibrotic mediators in this model, we examined the fibrotic response to FITC in mice that were genetically deficient in IL-4 (IL-4(-/-)), IL-13 (IL-13(-/-)), or IL-4 and IL-13 combined (IL-4/13(-/-)). Baseline levels of collagen were similar in all mice. In response to FITC, both BALB/c and IL-4(-/-) mice developed fibrosis, whereas the IL-13(-/-) and IL-4/13(-/-) mice were significantly protected, as measured by total lung collagen levels and histology. Total leukocyte recruitment to the lung was similar in all four strains of mice when measured on days 7, 14, and 21 post-FITC. BALB/c mice showed prominent eosinophilia on day 7 that was absent in IL-4(-/-), IL-13(-/-), and IL-4/13(-/-) mice, suggesting that eosinophilia is not necessary for development of a fibrotic response. There were no significant differences in the percentages of any other leukocytes analyzed between the genotypes. Similarly, protection in IL-13(-/-) mice was not associated with alterations in cytokine or eicosanoid profiles. Interestingly, TGF-beta1 production was not reduced in IL-13(-/-) mice. Analyses of fibroblasts isolated from the four genotypes demonstrated that although there were similar numbers of fibroblasts present in cultures of lung minces, fibroblasts from IL-13-deficient strains have reduced basal and stimulated levels of collagen production. IL-13Ralpha1 expression increases on fibroblasts during fibrotic responses in vivo, and IL-13 increases collagen synthesis in fibroblasts. Thus, IL-13 mediates its profibrotic actions through direct effects on fibroblast production of extracellular matrix.     

Monitoring Tribolium castaneum (Coleoptera: Tenebrionidae) in pilot-scale warehouses treated with residual applications of (S)-hydroprene and cyfluthrin

Pilot-scale warehouses, artificially infested with all life stages of Tribolium castaneum (Herbst), were used to evaluate the efficacy of two contact insecticides, (S)-hydroprene and cyfluthrin, and to determine the effect of insecticide treatments on insect captures in food- and pheromone-baited pitfall traps. Two application strategies were compared; insecticides were applied at the labeled rate either around the inside perimeter of the warehouse or in a band around the base of shelf units containing discrete food patches (10 g of wheat flour) infested with T. castaneum. Insect populations were assessed weekly for 6 wk by recording number of dead adults on the warehouse floor; number of larvae and adults captured in pitfall traps; and number of larvae, pupae, and adults recovered from food patch samples. There were significantly more dead adults in warehouses treated with cyfluthrin than with (S)-hydroprene or water (control treatment). However, food patch samples showed no detectable differences in quantity of larvae, pupae, or adults among any treatments. Pitfall traps detected fewer larvae starting the fourth week of the study in the warehouses treated with cyfluthrin around the shelf perimeter. Rate of larval capture in traps increased overall with increasing larval populations, but it was more pronounced in traps located closer to the food patches. Number of adults captured in pitfall traps reflected adult mortality in cyfluthrin-treated warehouses. Capture of larvae and adults was greater near the source of the infestation than elsewhere in the warehouse, suggesting that trapping data should be considered when precision targeting insecticide applications in the field.     

Cerebroside synthesis as a measure of the rate of remyelination following cuprizone-induced demyelination in brain

We studied markers of myelin content and of the rate of myelination in brains of mice between 8 and 20 weeks of age. During the 12-week time-course, control animals showed slight increases in the content of oligodendroglial-specific cerebroside, as well as cholesterol (enriched in, but not specific to, myelin). In contrast, synthesis of these lipids, as assayed by in vivo incorporation of (3)H(2)O, was substantial, indicating turnover of 0.4% and 0.7% of total brain cerebroside and cholesterol, respectively, each day. We also studied mice exposed to a diet containing 0.2% of the copper chelator, cuprizone. After 6 weeks 20%, and by 12 weeks, over 30% of brain cerebroside was gone. Demyelination was accompanied by down-regulation of mRNA expression for enzymes controlling myelin lipid synthesis (ceramide galactosyl transferase for cerebroside; hydroxymethylglutaryl-CoA reductase for cholesterol), and for myelin basic protein. Synthesis of myelin lipids was also greatly depressed. The 20% cerebroside deficit consequent to 6 weeks of cuprizone exposure was restored 6 weeks after return to a control diet. During remyelination, expression of myelin-related mRNA species, as well as cerebroside and cholesterol synthesis were restored to normal. However, in contrast to the steady state metabolic turnover in the control situation, all the cerebroside and cholesterol made were accumulated. To the extent that accumulating cerebroside is targeted for eventual inclusion in myelin (discussed) the rate of its synthesis is proportional to remyelination. With our assay, in vivo rates of cerebroside synthesis can be determined for a time window of the order of hours. This offers greater temporal resolution and accuracy relative to classical methods assaying accumulation of myelin components at time intervals of several days. We propose this experimental design, and the reproducible cuprizone model, as appropriate for studies of how to promote remyelination.     

Agonist-induced changes in beta-adrenergic receptors on intact cells

Competition by beta-adrenergic agonists and antagonists for 125I-pindolol binding sites on intact cells (1321N1 human astrocytoma and C62B rat glioma) was measured using short time binding assays as previously described (Toews, M. L., Harden, T. K., and Perkins, J. P. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 3553-3557). Preincubation of cells with agonists converted about half of the cellular beta-adrenergic receptors from a form exhibiting high affinity for the agonists isoproterenol and epinephrine and the antagonist sotalol to a form exhibiting much lower apparent affinity for these ligands in short time assays. Exposure to agonists did not alter the affinity of receptors for the antagonist metoprolol. This change in the ligand binding properties of the receptor was rapid (t1/2 = 1-2 min following a lag of about 0.5 min), reversible (t1/2 = 6-8 min), and dependent on the agonist concentration present during the preincubation (K0.5 = 15 nM for isoproterenol). Both isoproterenol and sotalol attained equilibrium with the high affinity receptors very rapidly but equilibrated only slowly with those receptors exhibiting low apparent affinity in short time assays. These results are interpreted in terms of a model which postulates that both the low apparent affinity in short time assays and the subsequent slow equilibration of hydrophilic ligands with these receptors result from agonist-induced internalization of a fraction of cell surface beta-adrenergic receptors. The relationship of this change in receptor binding properties to other aspects of agonist-induced desensitization of the beta-adrenergic receptor-coupled adenylate cyclase system is discussed.     

High definition bronchoscopy: a randomized exploratory study of diagnostic value compared to standard white light bronchoscopy and autofluorescence bronchoscopy

Background

Videobronchoscopy is an essential diagnostic procedure for evaluation of the central airways and pivotal for the diagnosis and staging of lung cancer. Technological improvements have resulted in high definition (HD) images with advanced real time image enhancement techniques (i-scan).

Objectives

In this study we aimed to explore the sensitivity of HD+ i-scan bronchoscopy for detection of epithelial changes like vascular abnormalities and suspicious preinvasive lesions, and tumors.

Methods

In patients scheduled for a therapeutic or diagnostic procedure under general anesthesia videos of the bronchial tree were made using 5 videobronchoscopy modes in random order: normal white light videobronchoscopy (WLB), HD-bronchoscopy (HD), HD bronchoscopy with surface enhancement technique (i-scan1), HD with surface- and tone enhancement technique (i-scan2) and dual mode autofluorescence videobronchoscopy (AFB). The videos were scored in random order by two independent and blinded expert bronchoscopists.

Results

In 29 patients all videos were available for analysis. Vascular abnormalities were scored most frequently in HD + i-scan2 bronchoscopy (1.33 ± 0.29 abnormal or suspicious sites per patient) as compared to 0.12 ± 0.05 site for AFB (P = 0.003). Sites suspicious for preinvasive lesions were most frequently reported using AFB (0.74 ± 0.12 sites per patient) as compared to 0.17 ± 0.06 for both WLB and HD bronchoscopy (P = 0.003). Tumors were detected equally by all modalities. The preferred modality was HD bronchoscopy with i-scan (tone- plus surface and surface enhancement in respectively 38% and 35% of cases P = 0.006).

Conclusions

This study shows that high definition bronchoscopy with image enhancement technique may result in better detection of subtle vascular abnormalities in the airways. Since these abnormalities may be related to preneoplastic lesions and tumors this is of clinical relevance. Further investigations using this technique relating imaging to histology are warranted.     

The BioLexicon: a large-scale terminological resource for biomedical text mining

Background

Due to the rapidly expanding body of biomedical literature, biologists require increasingly sophisticated and efficient systems to help them to search for relevant information. Such systems should account for the multiple written variants used to represent biomedical concepts, and allow the user to search for specific pieces of knowledge (or events) involving these concepts, e.g., protein-protein interactions. Such functionality requires access to detailed information about words used in the biomedical literature. Existing databases and ontologies often have a specific focus and are oriented towards human use. Consequently, biological knowledge is dispersed amongst many resources, which often do not attempt to account for the large and frequently changing set of variants that appear in the literature. Additionally, such resources typically do not provide information about how terms relate to each other in texts to describe events.

Results

This article provides an overview of the design, construction and evaluation of a large-scale lexical and conceptual resource for the biomedical domain, the BioLexicon. The resource can be exploited by text mining tools at several levels, e.g., part-of-speech tagging, recognition of biomedical entities, and the extraction of events in which they are involved. As such, the BioLexicon must account for real usage of words in biomedical texts. In particular, the BioLexicon gathers together different types of terms from several existing data resources into a single, unified repository, and augments them with new term variants automatically extracted from biomedical literature. Extraction of events is facilitated through the inclusion of biologically pertinent verbs (around which events are typically organized) together with information about typical patterns of grammatical and semantic behaviour, which are acquired from domain-specific texts. In order to foster interoperability, the BioLexicon is modelled using the Lexical Markup Framework, an ISO standard.

Conclusions

The BioLexicon contains over 2.2 M lexical entries and over 1.8 M terminological variants, as well as over 3.3 M semantic relations, including over 2 M synonymy relations. Its exploitation can benefit both application developers and users. We demonstrate some such benefits by describing integration of the resource into a number of different tools, and evaluating improvements in performance that this can bring.     

A micro RNA processing defect in rapidly progressing idiopathic pulmonary fibrosis

Background

Idiopathic pulmonary fibrosis exhibits differential progression from the time of diagnosis but the molecular basis for varying progression rates is poorly understood. The aim of the present study was to ascertain whether differential miRNA expression might provide one explanation for rapidly versus slowly progressing forms of IPF.

Methodology and Principal Findings

miRNA and mRNA were isolated from surgical lung biopsies from IPF patients with a clinically documented rapid or slow course of disease over the first year after diagnosis. A quantitative PCR miRNA array containing 88 of the most abundant miRNA in the human genome was used to profile lung biopsies from 9 patients with rapidly progressing IPF, 6 patients with slowly progressing IPF, and 10 normal lung biopsies. Using this approach, 11 miRNA were significantly increased and 36 were significantly decreased in rapid biopsies compared with normal biopsies. Slowly progressive biopsies exhibited 4 significantly increased miRNA and 36 significantly decreased miRNA compared with normal lung. Among the miRNA present in IPF with validated mRNA targets were those with regulatory effects on epithelial-mesenchymal transition (EMT). Five miRNA (miR-302c, miR-423-5p, miR-210, miR-376c, and miR-185) were significantly increased in rapid compared with slow IPF lung biopsies. Additional analyses of rapid biopsies and fibroblasts grown from the same biopsies revealed that the expression of AGO1 and AGO2 (essential components of the miRNA processing RISC complex) were lower compared with either slow or normal lung biopsies and fibroblasts.

Conclusion

These findings suggest that the development and/or clinical progression of IPF might be the consequence of aberrant miRNA processing.     

Application of Artificial Intelligence/Machine Vision & Learning for the Development of a Live Single-cell Phenotypic Biomarker Test to Predict Prostate Cancer Tumor Aggressiveness

To assess the usefulness and applications of machine vision (MV) and machine learning (ML) techniques that have been used to develop a single cell-based phenotypic (live and fixed biomarkers) platform that correlates with tumor biological aggressiveness and risk stratification, 100 fresh prostate samples were acquired, and areas of prostate cancer were determined by post-surgery pathology reports logged by an independent pathologist. The prostate samples were dissociated into single-cell suspensions in the presence of an extracellular matrix formulation. These samples were analyzed via live-cell microscopy. Dynamic and fixed phenotypic biomarkers per cell were quantified using objective MV software and ML algorithms. The predictive nature of the ML algorithms was developed in two stages. First, random forest (RF) algorithms were developed using 70% of the samples. The developed algorithms were then tested for their predictive performance using the blinded test dataset that contained 30% of the samples in the second stage. Based on the ROC (receiver operating characteristic) curve analysis, thresholds were set to maximize both sensitivity and specificity. We determined the sensitivity and specificity of the assay by comparing the algorithm-generated predictions with adverse pathologic features in the radical prostatectomy (RP) specimens. Using MV and ML algorithms, the biomarkers predictive of adverse pathology at RP were ranked and a prostate cancer patient risk stratification test was developed that distinguishes patients based on surgical adverse pathology features. The ability to identify and track large numbers of individual cells over the length of the microscopy experimental monitoring cycles, in an automated way, created a large biomarker dataset of primary biomarkers. This biomarker dataset was then interrogated with ML algorithms used to correlate with post-surgical adverse pathology findings. Algorithms were generated that predicted adverse pathology with >0.85 sensitivity and specificity and an AUC (area under the curve) of >0.85. Phenotypic biomarkers provide cellular and molecular details that are informative for predicting post-surgical adverse pathologies when considering tumor biopsy samples. Artificial intelligence ML-based approaches for cancer risk stratification are emerging as important and powerful tools to compliment current measures of risk stratification. These techniques have capabilities to address tumor heterogeneity and the molecular complexity of prostate cancer. Specifically, the phenotypic test is a novel example of leveraging biomarkers and advances in MV and ML for developing a powerful prognostic and risk-stratification tool for prostate cancer patients.     

MIGRATION,MITOCHONDRIA, AND THE YELLOW‐RUMPED WARBLER

Discordance between mitochondrial and nuclear DNA has been noted in many systems. Asymmetric introgression of mitochondria is a common cause of such discordances, although in most cases the drivers of introgression are unknown. In the yellow‐rumped warbler, evidence suggests that mtDNA from the eastern, myrtle warbler, has introgressed across much of the range of the western form, the Audubon's warbler. Within the southwestern United States myrtle mtDNA comes into contact with another clade that occurs in the Mexican black‐fronted warbler. Both northern forms exhibit seasonal migration, whereas black‐fronted warblers are nonmigratory. We investigated the link between mitochondrial introgression, mitochondrial function, and migration using novel genetic, isotopic, biochemical, and phenotypic data obtained from populations in the transition zone. Isotopes suggest the zone is coincident with a shift in migration, with individuals in the south being resident and populations further north becoming increasingly more migratory. Mitochondrial respiration in flight muscles demonstrates that myrtle‐type individuals have a significantly greater acceptor control ratio of mitochondria, suggesting it may be more metabolically efficient. To our knowledge this is the first time this type of intraspecific variation in mitochondrial respiration has been measured in wild birds and we discuss how such mitochondrial adaptations may have facilitated introgression.