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Sample records for mouse lung primordium

  1. The lung primordium an outpouching from the foregut! Evidence-based dogma or myth?

    PubMed

    Brown, Ediel; James, Kenneth

    2009-03-01

    This study examines and challenges the "evidence-based legitimacy" of the theory, "the lung primordium is an outpouching from the foregut." A literature review was undertaken using computer database, journals, and relevant anatomical and embryological texts. The independent path of development taken by the tracheobronchial system and the oesophagus once identified as separate entities; the lack of morphologic, molecular, biological, and genetic supportive evidence for the "common-origin" theory; the distinct longitudinal line of demarcation between the nonsegmented muscles of the esophagus and the highly segmented cartilaginous structure of the tracheobronchial tree; the absence of a tracheoesophageal septum in the process of separation; the differences in epithelial lining; and the diametrically opposed mucociliary cascade of the upper airway vs the mucociliary escalator of the tracheobronchial tree all seriously challenge the authenticity of a common origin to these 2 entities. To the extent that the foregut is seen as consisting of 2 separate semitubular splanchnopleuric entities ventrodorsally juxtaposed, it is true that the lung primordium as an outpouching of, and not from, the foregut. This must never be confused with the notion that the esophagus and tracheobronchial tree have a common origin. In fact, they develop from 2 completely separate segments of the trilaminar germ disk, but because of head fold development are brought together to create a common tracheoesophageal chamber that is later separated, facilitated by the prochordal membrane diverticulum.

  2. Development of Mouse Lung Deposition Models

    DTIC Science & Technology

    2015-07-01

    Particle inhalability in mice was lower than that in rats . In contrast, deposition of the same size particle was higher in mice nasal passages than...that in rats . Thus, fewer particles entered the mouse lung in comparison with rat particle inhalation. The penetration was severely limited for...geometry that was previously developed for humans, rats , and rhesus monkeys [6], [7]. Inputs to the model included lung geometry and volumes, and

  3. Antibodies to mouse lung capillary endothelium.

    PubMed

    Rorvik, M C; Allison, D P; Hotchkiss, J A; Witschi, H P; Kennel, S J

    1988-07-01

    We are interested in developing monoclonal antibodies (MoAbs) that recognize specific cell types in the lung of BALB/c mice. Normal mouse lung homogenate was used to immunize F344 rats and hybridomas were produced by fusion of rat spleen cells with mouse myeloma SP 2/0. Two hybridomas were selected which produced MoAbs active in immunohistochemistry of lung cells. MoAb 273-34A and 411-201B both show extensive peroxidase staining of capillary endothelial cells within alveolar walls of lungs at the light microscopic level. To demonstrate cell specificity, immunoelectron microscopy with gold-labeled antibody was performed. Lightly fixed lungs were frozen and thin-sectioned before staining with MoAb and 5-nm gold particles coupled to secondary antibody. Quantitative analyses of these cryosections show that both antibodies, used at optimal concentrations, are specific for binding to capillary endothelial cells. More than 95% of the gold particles are associated with capillary endothelial cells on the thin side of the alveolar wall. When capillaries adjoined thick septa containing interstitial cells, about two thirds of the gold particles were associated with endothelial cells and about one quarter with interstitial cells. These MoAbs should be useful in studying the role of endothelial cells in toxic lung injury.

  4. A mouse model of orthotopic vascularized aerated lung transplantation.

    PubMed

    Okazaki, M; Krupnick, A S; Kornfeld, C G; Lai, J M; Ritter, J H; Richardson, S B; Huang, H J; Das, N A; Patterson, G A; Gelman, A E; Kreisel, D

    2007-06-01

    Outcomes after lung transplantation are markedly inferior to those after other solid organ transplants. A better understanding of cellular and molecular mechanisms contributing to lung graft injury will be critical to improve outcomes. Advances in this field have been hampered by the lack of a mouse model of lung transplantation. Here, we report a mouse model of vascularized aerated single lung transplantation utilizing cuff techniques. We show that syngeneic grafts have normal histological appearance with minimal infiltration of T lymphocytes. Allogeneic grafts show acute cellular rejection with infiltration of T lymphocytes and recipient-type antigen presenting cells. Our data show that we have developed a physiological model of lung transplantation in the mouse, which provides ample opportunity for the study of nonimmune and immune mechanisms that contribute to lung allograft injury.

  5. Micro-imaging of the Mouse Lung via MRI

    NASA Astrophysics Data System (ADS)

    Wang, Wei

    Quantitative measurement of lung microstructure is of great significance in assessment of pulmonary disease, particularly in the earliest stages. Conventional stereological assessment of ex-vivo fixed tissue specimens under the microscope has a long and successful tradition and is regarded as a gold standard, but the invasive nature limits its applications and the practicality of use in longitudinal studies. The technique for diffusion MRI-based 3He lung morphometry was previously developed and validated for human lungs, and was recently extended to ex-vivo mouse lungs. The technique yields accurate, quantitative information about the microstructure and geometry of acinar airways. In this dissertation, the 3He lung morphometry technique is for the first time successfully implemented for in-vivo studies of mice. It can generate spatially-resolved maps of parameters that reveal the microstructure of mouse lung. Results in healthy mice indicate excellent agreement between in-vivo morphometry via 3He MRI and microscopic morphometry after sacrifice. The implementation and validation of 3He morphometry in healthy mice open up new avenues for application of the technique as a precise, noninvasive, in-vivo biomarker of changes in lung microstructure, within various mouse models of lung disease. We have applied 3He morphometry to the Sendai mouse model of lung disease. Specifically, the Sendai-virus model of chronic obstructive lung disease has demonstrated an innate immune response in mouse airways that exhibits similarities to the chronic airway inflammation in human COPD and asthma, but the effect on distal lung parenchyma had not been investigated. We imaged the time course and regional distribution of mouse lung microstructural changes in vivo after Sendai virus (SeV) infection with 1H and 3He diffusion MRI. 1H MR images detected the SeV-induced pulmonary inflammation in vivo and 3He lung morphometry showed modest increase in alveolar duct radius distal to airway

  6. Chemically-induced Mouse Lung Tumors: Applications to ...

    EPA Pesticide Factsheets

    A state-of-the-science workshop on chemically-induced mouse lung tumors was conducted by U.S. Environmental Protection Agency to better understand the mouse lung tumor data’s role in human health assessments. Three environmental chemicals - naphthalene, styrene, and ethylbenzene were chosen for the analysis due to the commonality of mouse lung tumors in all three chemicals. The goals of the workshop were to: identify the evidence, from multiple scientific disciplines, regarding formation of chemically-induced lung tumors in mice; discuss analysis and interpretation of the evidence; discuss how such evidence informs human health assessments; and identify commonalities, linkages, or differences between the evidence from various disciplines and across the chemicals. Evidence informing the association between occupational exposure to styrene, ethylbenzene, or naphthalene and lung cancer; comparative biology of mouse lung tumors, associated pathologic effects, issues related to tissue and species concordance; mode of action analysis and biological mechanisms including pharmacokinetics and pharmacodynamics; and evidence from cellular, genetic and molecular toxicity was discussed. In summary, although consensus was not sought, the panelists agreed that available mouse lung tumor data should be considered for human health risk evaluation on an individual chemical basis. Key data gaps were identified that would assist in further understanding the mechanism and relevan

  7. Chemically-induced mouse lung tumors: applications to ...

    EPA Pesticide Factsheets

    A state-of-the-science workshop on chemically-induced mouse lung tumors was conducted by U.S. Environmental Protection Agency to discuss issues related to the use of mouse lung tumor data in human health assessments. Naphthalene, styrene, and ethylbenzene were chosen for the analysis due to the commonality of mouse lung tumors in all these three environmental chemicals. The goals of the workshop were to: identify the evidence, from multiple scientific disciplines, regarding formation of chemically-induced lung tumors in mice; discuss analysis and interpretation of the evidence; discuss how such evidence informs human health assessments; and identify commonalities, linkages, or differences between the evidence from various disciplines and across the chemicals. Evidence informing the association between occupational exposure to styrene, ethylbenzene, or naphthalene and lung cancer; comparative biology of mouse lung tumors, associated pathologic effects, issues related to tissue and species concordance; mode of action analysis and biological mechanisms including pharmacokinetics and pharmacodynamics; and evidence from cellular, genetic and molecular toxicity was discussed. In summary, although consensus was not sought, the panelists agreed that data showing mouse lung tumors with chemical exposures can be relevant for human health risk evaluation on an individual chemical basis. Key data gaps were identified that would assist in further understanding the mechanism

  8. Chemically-induced Mouse Lung Tumors: Applications to ...

    EPA Pesticide Factsheets

    A state-of-the-science workshop on chemically-induced mouse lung tumors was conducted by U.S. Environmental Protection Agency to better understand the mouse lung tumor data’s role in human health assessments. Three environmental chemicals - naphthalene, styrene, and ethylbenzene were chosen for the analysis due to the commonality of mouse lung tumors in all three chemicals. The goals of the workshop were to: identify the evidence, from multiple scientific disciplines, regarding formation of chemically-induced lung tumors in mice; discuss analysis and interpretation of the evidence; discuss how such evidence informs human health assessments; and identify commonalities, linkages, or differences between the evidence from various disciplines and across the chemicals. Evidence informing the association between occupational exposure to styrene, ethylbenzene, or naphthalene and lung cancer; comparative biology of mouse lung tumors, associated pathologic effects, issues related to tissue and species concordance; mode of action analysis and biological mechanisms including pharmacokinetics and pharmacodynamics; and evidence from cellular, genetic and molecular toxicity was discussed. In summary, although consensus was not sought, the panelists agreed that available mouse lung tumor data should be considered for human health risk evaluation on an individual chemical basis. Key data gaps were identified that would assist in further understanding the mechanism and relevan

  9. ESR measurement of radical clearance in lung of whole mouse

    SciTech Connect

    Takeshita, K.; Utsumi, H.; Hamada, A. )

    1991-06-14

    Clearance of the nitroxide radicals, hydroxy-TEMPO and carboxy-PROxYL, in whole-mouse lung was directly measured by in vivo ESR. After injecting a nitroxide radical, distribution of the nitroxide radical all over the lung was confirmed by ESR imaging. The ESR signal of hydroxy-TEMPO was reduced in the lung and the clearance obeyed first-order kinetics, whereas the signal of carboxy-PROxYL remained constant. Comparison of the clearance rates of live and dead mice indicated the presence of 2 different clearance systems in the lung: loss of its paramagnetism in the lung, and transfer from alveolar to the blood circulation system.

  10. Quantitative analysis of tumor burden in mouse lung via MRI.

    PubMed

    Tidwell, Vanessa K; Garbow, Joel R; Krupnick, Alexander S; Engelbach, John A; Nehorai, Arye

    2012-02-01

    Lung cancer is the leading cause of cancer death in the United States. Despite recent advances in screening protocols, the majority of patients still present with advanced or disseminated disease. Preclinical rodent models provide a unique opportunity to test novel therapeutic drugs for targeting lung cancer. Respiratory-gated MRI is a key tool for quantitatively measuring lung-tumor burden and monitoring the time-course progression of individual tumors in mouse models of primary and metastatic lung cancer. However, quantitative analysis of lung-tumor burden in mice by MRI presents significant challenges. Herein, a method for measuring tumor burden based upon average lung-image intensity is described and validated. The method requires accurate lung segmentation; its efficiency and throughput would be greatly aided by the ability to automatically segment the lungs. A technique for automated lung segmentation in the presence of varying tumor burden levels is presented. The method includes development of a new, two-dimensional parametric model of the mouse lungs and a multi-faceted cost function to optimally fit the model parameters to each image. Results demonstrate a strong correlation (0.93), comparable with that of fully manual expert segmentation, between the automated method's tumor-burden metric and the tumor burden measured by lung weight.

  11. Practical use of advanced mouse models for lung cancer.

    PubMed

    Safari, Roghaiyeh; Meuwissen, Ralph

    2015-01-01

    To date a variety of non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) mouse models have been developed that mimic human lung cancer. Chemically induced or spontaneous lung cancer in susceptible inbred strains has been widely used, but the more recent genetically engineered somatic mouse models recapitulate much better the genotype-phenotype correlations found in human lung cancer. Additionally, improved orthotopic transplantation of primary human cancer tissue fragments or cells into lungs of immune-compromised mice can be valuable tools for preclinical research such as antitumor drug tests. Here we give a short overview of most somatic mouse models for lung cancer that are currently in use. We accompany each different model with a description of its practical use and application for all major lung tumor types, as well as the intratracheal injection or direct injection of fresh or freeze-thawed tumor cells or tumor cell lines into lung parenchyma of recipient mice. All here presented somatic mouse models are based on the ability to (in) activate specific alleles at a time, and in a tissue-specific cell type, of choice. This spatial-temporal controlled induction of genetic lesions allows the selective introduction of main genetic lesions in an adult mouse lung as found in human lung cancer. The resulting conditional somatic mouse models can be used as versatile powerful tools in basic lung cancer research and preclinical translational studies alike. These distinctively advanced lung cancer models permit us to investigate initiation (cell of origin) and progression of lung cancer, along with response and resistance to drug therapy. Cre/lox or FLP/frt recombinase-mediated methods are now well-used techniques to develop tissue-restricted lung cancer in mice with tumor-suppressor gene and/or oncogene (in)activation. Intranasal or intratracheal administration of engineered adenovirus-Cre or lentivirus-Cre has been optimized for introducing Cre

  12. Differential Gene Expression in Chemically Induced Mouse Lung Adenomas1

    PubMed Central

    Yao, Ruisheng; Wang, Yian; Lubet, Ronald A; You, Ming

    2003-01-01

    Abstract Because of similarities in histopathology and tumor progression stages between mouse and human lung adenocarcinomas, the mouse lung tumor model with lung adenomas as the endpoint has been used extensively to evaluate the efficacy of putative lung cancer chemopreventive agents. In this study, a competitive cDNA library screening (CCLS) was employed to determine changes in the expression of mRNA in chemically induced lung adenomas compared with paired normal lung tissues. A total of 2555 clones having altered expression in tumors were observed following competitive hybridization between normal lung and lung adenomas after primary screening of over 160,000 clones from a mouse lung cDNA library. Among the 755 clones confirmed by dot blot hybridization, 240 clones were underexpressed, whereas 515 clones were overexpressed in tumors. Sixty-five clones with the most frequently altered expression in six individual tumors were confirmed by semiquantitative RT-PCR. When examining the 58 known genes, 39 clones had increased expression and 19 had decreased expression, whereas the 7 novel genes showed overexpression. A high percentage (>60%) of overexpressed or underexpressed genes was observed in at least two or three of the lesions. Reproducibly overexpressed genes included ERK-1, JAK-1, surfactant proteins A, B, and C, NFAT1, α-1 protease inhibitor, helix-loop-helix ubiquitous kinase (CHUK), α-adaptin, α-1 PI2, thioether S-methyltransferase, and CYP2C40. Reproducibly underexpressed genes included paroxanase, ALDH II, CC10, von Ebner salivary gland protein, and α- and β-globin. In addition, CCLS identified several novel genes or genes not previously associated with lung carcinogenesis, including a hypothetical protein (FLJ11240) and a guanine nucleotide exchange factor homologue. This study shows the efficacy of this methodology for identifying genes with altered expression. These genes may prove to be helpful in our understanding of the genetic basis of lung

  13. Imaging mouse lung allograft rejection with 1H MRI

    PubMed Central

    Guo, Jinbang; Huang, Howard J.; Wang, Xingan; Wang, Wei; Ellison, Henry; Thomen, Robert P.; Gelman, Andrew E.; Woods, Jason C.

    2014-01-01

    Purpose To demonstrate that longitudinal, non-invasive monitoring via MRI can characterize acute cellular rejection (ACR) in mouse orthotopic lung allografts. Methods Nineteen Balb/c donor to C57BL/6 recipient orthotopic left lung transplants were performed, further divided into control-Ig vs anti-CD4/anti-CD8 treated groups. A two-dimensional multi-slice gradient-echo pulse sequence synchronized with ventilation was used on a small-animal MR scanner to acquire proton images of lung at post-operative days 3, 7 and 14, just before sacrifice. Lung volume and parenchymal signal were measured, and lung compliance was calculated as volume change per pressure difference between high and low pressures. Results Normalized parenchymal signal in the control-Ig allograft increased over time, with statistical significance between day 14 and day 3 post transplantation (0.046→0.789, P < 0.05), despite large inter-mouse variations; this was consistent with histopathologic evidence of rejection. Compliance of the control-Ig allograft decreased significantly over time (0.013→0.003, P < 0.05), but remained constant in mice treated with anti-CD4/anti-CD8 antibodies. Conclusion Lung allograft rejection in individual mice can be monitored by lung parenchymal signal changes and by lung compliance through MRI. Longitudinal imaging can help us better understand the time course of individual lung allograft rejection and response to treatment. PMID:24954886

  14. Preconditioning allows engraftment of mouse and human embryonic lung cells, enabling lung repair in mice.

    PubMed

    Rosen, Chava; Shezen, Elias; Aronovich, Anna; Klionsky, Yael Zlotnikov; Yaakov, Yasmin; Assayag, Miri; Biton, Inbal Eti; Tal, Orna; Shakhar, Guy; Ben-Hur, Herzel; Shneider, David; Vaknin, Zvi; Sadan, Oscar; Evron, Shmuel; Freud, Enrique; Shoseyov, David; Wilschanski, Michael; Berkman, Neville; Fibbe, Willem E; Hagin, David; Hillel-Karniel, Carmit; Krentsis, Irit Milman; Bachar-Lustig, Esther; Reisner, Yair

    2015-08-01

    Repair of injured lungs represents a longstanding therapeutic challenge. We show that human and mouse embryonic lung tissue from the canalicular stage of development (20-22 weeks of gestation for humans, and embryonic day 15-16 (E15-E16) for mouse) are enriched with progenitors residing in distinct niches. On the basis of the marked analogy to progenitor niches in bone marrow (BM), we attempted strategies similar to BM transplantation, employing sublethal radiation to vacate lung progenitor niches and to reduce stem cell competition. Intravenous infusion of a single cell suspension of canalicular lung tissue from GFP-marked mice or human fetal donors into naphthalene-injured and irradiated syngeneic or SCID mice, respectively, induced marked long-term lung chimerism. Donor type structures or 'patches' contained epithelial, mesenchymal and endothelial cells. Transplantation of differentially labeled E16 mouse lung cells indicated that these patches were probably of clonal origin from the donor. Recipients of the single cell suspension transplant exhibited marked improvement in lung compliance and tissue damping reflecting the energy dissipation in the lung tissues. Our study provides proof of concept for lung reconstitution by canalicular-stage human lung cells after preconditioning of the pulmonary niche.

  15. Lung regeneration by fetal lung tissue implantation in a mouse pulmonary emphysema model.

    PubMed

    Uyama, Koh; Sakiyama, Shoji; Yoshida, Mitsuteru; Kenzaki, Koichiro; Toba, Hiroaki; Kawakami, Yukikiyo; Okumura, Kazumasa; Takizawa, Hiromitsu; Kondo, Kazuya; Tangoku, Akira

    2016-01-01

    The mortality and morbidity of chronic obstructive pulmonary disease are high. However, no radical therapy has been developed to date. The purpose of this study was to evaluate whether fetal mouse lung tissue can grow and differentiate in the emphysematous lung. Fetal lung tissue from green fluorescent protein C57BL/6 mice at 16 days' gestation was used as donor material. Twelve-month-old pallid mice were used as recipients. Donor lungs were cut into small pieces and implanted into the recipient left lung by performing thoracotomy under anesthesia. The recipient mice were sacrificed at day 7, 14, and 28 after implantation and used for histological examination. Well-developed spontaneous pulmonary emphysema was seen in 12-month-old pallid mice. Smooth and continuous connection between implanted fetal lung tissue and recipient lung was recognized. Air space expansion and donor tissue differentiation were observed over time. We could clearly distinguish the border zones between injected tissue and native tissue by the green fluorescence of grafts. Fetal mouse lung fragments survived and differentiated in the emphysematous lung of pallid mice. Implantation of fetal lung tissue in pallid mice might lead to further lung regeneration research from the perspective of respiratory and exercise function. J. Med. Invest. 63: 182-186, August, 2016.

  16. Chronic hypercapnia alters lung matrix composition in mouse pups

    PubMed Central

    Heldt, Gregory P.; Nguyen, Mary; Gavrialov, Orit; Haddad, Gabriel G.

    2010-01-01

    Rationale: permissive hypercapnia, a stretch-limiting ventilation strategy, often results in high PaCO2. This strategy is associated with reduced morbidity and mortality in premature infants and its benefits have been attributed to diminished barotrauma. However, little is known about the independent effect of high CO2 levels during the lung development. Methods: mice were exposed to 8% CO2 or room air for 2 wk either from postnatal day 2 through 17 or as adults (∼2 mo of age). Lungs were excised and processed for protein, RNA, histology, and total lung volumes. Results: histologic analysis demonstrated that alveolar walls of CO2-exposed mouse pups were thinner than those of controls and had twice the total lung volume. Molecular analysis revealed that several matrix proteins in the lung were downregulated in mouse pups exposed to hypercapnia. Interstitial collagen type I α1, type III α1, elastin and fibronectin protein, and mRNA levels were less than half of controls while collagen IV α5 was unaffected. This decrease in interstitial collagen could thus account for the thinning of the interstitial matrix and the altered lung biomechanics. Matrix metalloproteinase (MMP)-8, a collagenase that has specificity for collagen types I and III, increased in hypercapnic mouse pups, suggesting increased collagen degradation. Moreover, tissue inhibitor of MMP (TIMP)-1, a potent inhibitor of MMP-8, was significantly decreased. However, unlike pups, adult mice exposed to hypercapnia demonstrated only a mild increase in total lung volumes and did not exhibit similar molecular or histologic changes. Conclusions: although permissive hypercapnia may prevent lung injury from barotrauma, our study revealed that exposure to hypercapnia may be an important factor in lung remodeling and function, especially in early life. PMID:20360436

  17. Surgical technique for lung retransplantation in the mouse

    PubMed Central

    Li, Wenjun; Goldstein, Daniel R.; Bribriesco, Alejandro C.; Nava, Ruben G.; Spahn, Jessica H.; Wang, Xingan; Gelman, Andrew E.; Krupnick, Alexander S.

    2013-01-01

    Microsurgical cuff techniques for orthotopic vascularized murine lung transplantation have allowed for the design of studies that examine mechanisms contributing to the high failure rate of pulmonary grafts. Here, we provide a detailed technical description of orthotopic lung retransplantation in mice, which we have thus far performed in 144 animals. The total time of the retransplantation procedure is approximately 55 minutes, 20 minutes for donor harvest and 35 minutes for the implantation, with a success rate exceeding 95%. The mouse lung retransplantation model represents a novel and powerful tool to examine how cells that reside in or infiltrate pulmonary grafts shape immune responses. PMID:23825768

  18. In vivo compartmental analysis of leukocytes in mouse lungs

    PubMed Central

    Patel, Brijesh V.; Tatham, Kate C.; Wilson, Michael R.; O'Dea, Kieran P.

    2015-01-01

    The lung has a unique structure consisting of three functionally different compartments (alveolar, interstitial, and vascular) situated in an extreme proximity. Current methods to localize lung leukocytes using bronchoalveolar lavage and/or lung perfusion have significant limitations for determination of location and phenotype of leukocytes. Here we present a novel method using in vivo antibody labeling to enable accurate compartmental localization/quantification and phenotyping of mouse lung leukocytes. Anesthetized C57BL/6 mice received combined in vivo intravenous and intratracheal labeling with fluorophore-conjugated anti-CD45 antibodies, and lung single-cell suspensions were analyzed by flow cytometry. The combined in vivo intravenous and intratracheal CD45 labeling enabled robust separation of the alveolar, interstitial, and vascular compartments of the lung. In naive mice, the alveolar compartment consisted predominantly of resident alveolar macrophages. The interstitial compartment, gated by events negative for both intratracheal and intravenous CD45 staining, showed two conventional dendritic cell populations, as well as a Ly6Clo monocyte population. Expression levels of MHCII on these interstitial monocytes were much higher than on the vascular Ly6Clo monocyte populations. In mice exposed to acid aspiration-induced lung injury, this protocol also clearly distinguished the three lung compartments showing the dynamic trafficking of neutrophils and exudative monocytes across the lung compartments during inflammation and resolution. This simple in vivo dual-labeling technique substantially increases the accuracy and depth of lung flow cytometric analysis, facilitates a more comprehensive examination of lung leukocyte pools, and enables the investigation of previously poorly defined “interstitial” leukocyte populations during models of inflammatory lung diseases. PMID:26254421

  19. Methods of in-vivo mouse lung micro-CT

    NASA Astrophysics Data System (ADS)

    Recheis, Wolfgang A.; Nixon, Earl; Thiesse, Jacqueline; McLennan, Geoffrey; Ross, Alan; Hoffman, Eric

    2005-04-01

    Micro-CT will have a profound influence on the accumulation of anatomical and physiological phenotypic changes in natural and transgenetic mouse models. Longitudinal studies will be greatly facilitated, allowing for a more complete and accurate description of events if in-vivo studies are accomplished. The purpose of the ongoing project is to establish a feasible and reproducible setup for in-vivo mouse lung micro-computed tomography (μCT). We seek to use in-vivo respiratory-gated μCT to follow mouse models of lung disease with subsequent recovery of the mouse. Methodologies for optimizing scanning parameters and gating for the in-vivo mouse lung are presented. A Scireq flexiVent ventilated the gas-anesthetized mice at 60 breaths/minute, 30 cm H20 PEEP, 30 ml/kg tidal volume and provided a respiratory signal to gate a Skyscan 1076 μCT. Physiologic monitoring allowed the control of vital functions and quality of anesthesia, e.g. via ECG monitoring. In contrary to longer exposure times with ex-vivo scans, scan times for in-vivo were reduced using 35μm pixel size, 158ms exposure time and 18μm pixel size, 316ms exposure time to reduce motion artifacts. Gating via spontaneous breathing was also tested. Optimal contrast resolution was achieved at 50kVp, 200μA, applying an aluminum filter (0.5mm). There were minimal non-cardiac related motion artifacts. Both 35μm and 1μm voxel size images were suitable for evaluation of the airway lumen and parenchymal density. Total scan times were 30 and 65 minutes respectively. The mice recovered following scanning protocols. In-vivo lung scanning with recovery of the mouse delivered reasonable image quality for longitudinal studies, e.g. mouse asthma models. After examining 10 mice, we conclude μCT is a feasible tool evaluating mouse models of lung pathology in longitudinal studies with increasing anatomic detail available for evaluation as one moves from in-vivo to ex-vivo studies. Further developments include automated

  20. Hyperpolarized helium-3 mouse lung MRI: Studies of lung structure and function

    NASA Astrophysics Data System (ADS)

    Dugas, Joseph Paul

    Hyperpolarized 3He magnetic resonance imaging (MRI) of human and animal lungs has displayed promising and useful applications to studies of lung structure and function in both healthy and diseased lungs. Hyperpolarized 3He MRI allows the visualization of gas in the gas-exchange spaces of the lungs (as opposed to tissue) and has proven especially effective in studying diseases that are characterized by ventilation defects, such as emphysema. In particular, in-vivo measurements of the 3He apparent diffusion coefficient (ADC) can quantify lung structure by measuring its restrictive effects on the motion of 3He spins. This allows for detection and longitudinal tracking of changes in micro-architecture that result from disease destruction of alveolar walls. Due, in part, to the difficulties inherent in administering and imaging hyperpolarized 3He within the small (0.5 cc volume) mouse lung, applications of hyperpolarized 3He MRI techniques to laboratory mice are scarce. We have been able to implement and improve the techniques of hyperpolarized 3He mouse lung MRI and subsequently apply them to studies of several mouse models of disease, including elastase-induced emphysema, smoking-induced emphysema, and lung cancer. Here we detail the design, development, and implementation of a versatile, electronically-controlled, small animal ventilator that is capable of delivering tiny volumes of hyperpolarized 3He, mixed with oxygen, to the mouse and is also compatible with both the easily depolarized 3He gas and the highly magnetic environment within and around an imaging magnet. Also described are NM techniques developed to improve the signal-to-noise ratio of our images and effectively utilize the gas hyperpolarization. Applications of these technologies and techniques to small animal models of disease are presented wherein we have measured up to a 35% increase in 3He ADC in mice with elastase-induced emphysema as compared to healthy mice. We also demonstrate the potential

  1. MALDI imaging MS of phospholipids in the mouse lung[S

    PubMed Central

    Berry, Karin A. Zemski; Li, Bilan; Reynolds, Susan D.; Barkley, Robert M.; Gijón, Miguel A.; Hankin, Joseph A.; Henson, Peter M.; Murphy, Robert C.

    2011-01-01

    Lipid mediators are important in lung biochemistry and are derived from the enzymatic oxidation of arachidonic and docosahexaenoic acids, which are PUFAs that are present in phospholipids in cell membranes. In this study, MALDI imaging MS was used to determine the localization of arachidonate- and docosahexaenoate-containing phospholipids in mouse lung. These PUFA-containing phospholipids were determined to be uniquely abundant at the lining of small and large airways, which were unequivocally identified by immunohistochemistry. In addition, it was found that the blood vessels present in the lung were characterized by sphingomyelin molecular species, and lung surfactant phospholipids appeared evenly distributed throughout the lung parenchyma, indicating alveolar localization. This technique revealed unexpected high concentrations of arachidonate- and docosahexaenoate-containing phospholipids lining the airways in pulmonary tissue, which could serve as precursors of lipid mediators affecting airways biology. PMID:21508254

  2. Measuring the lung function in the mouse: the challenge of size

    PubMed Central

    Irvin, Charles G; Bates, Jason HT

    2003-01-01

    Measurement of the effects of drugs, mediators and infectious agents on various models of lung disease, as well as assessment of lung function in the intact mouse has the potential for significantly advancing our knowledge of lung disease. However, the small size of the mouse presents significant challenges for the assessment of lung function. Because of compromises made between precision and noninvasiveness, data obtained may have an uncertain bearing on the mechanical response of the lung. Nevertheless, considerable recent progress has been made in developing valid and useful measures of mouse lung function. These advances, resulting in our current ability to measure sophisticated indices of lung function in laboratory animals, are likely to lead to important insights into the mechanisms of lung disease. PMID:12783622

  3. A novel SCID mouse model for studying spontaneous metastasis of human lung cancer to human tissue.

    PubMed

    Teraoka, S; Kyoizumi, S; Seyama, T; Yamakido, M; Akiyama, M

    1995-05-01

    We established a novel severe combined immunodeficient (SCID) mouse model for the study of human lung cancer metastasis to human lung. Implantation of both human fetal and adult lung tissue into mammary fat pads of SCID mice showed a 100% rate of engraftment, but only fetal lung implants revealed normal morphology of human lung tissue. Using these chimeric mice, we analyzed human lung cancer metastasis to both mouse and human lungs by subcutaneous inoculation of human squamous cell carcinoma and adenocarcinoma cell lines into the mice. In 60 to 70% of SCID mice injected with human-lung squamous-cell carcinoma, RERF-LC-AI, cancer cells were found to have metastasized to both mouse lungs and human fetal lung implants but not to human adult lung implants 80 days after cancer inoculation. Furthermore, human-lung adenocarcinoma cells, RERF-LC-KJ, metastasized to the human lung implants within 90 days in about 40% of SCID mice, whereas there were no metastases to the lungs of the mice. These results demonstrate the potential of this model for the in vivo study of human lung cancer metastasis.

  4. Activation of proto-oncogenes in human and mouse lung tumors

    SciTech Connect

    Reynolds, S.H.; Anderson, M.W. )

    1991-06-01

    Lung cancer is a leading cause of cancer-related deaths in several nations. Epidemiological studies have indicated that 85% of all lung cancer deaths and 30% of all cancer deaths in the US are associated with tobacco smoking. Various chemicals in tobacco smoke are thought to react with DNA and to ultimately yield heritable mutations. In an effort to understand the molecular mechanisms involved in lung tumorigenesis, the authors have analyzed proto-oncogene activation in a series of human lung tumors from smokers and spontaneously occurring and chemically induced lung tumors in mice. Approximately 86% of the human lung tumors and > 90% of the mouse lung tumors were found to contain activated oncogenes. ras Oncogenes activated by point mutations were detected in many of the human lung adenocarcinomas and virtually all of the mouse lung adenomas and adenocarcinomas. The mutation profiles of the activated K-ras genes detected in the chemically induced mouse lung tumors suggest that the observed mutations result from genotoxic effects of the chemicals. Comparison of the K-ras mutations observed in the human lung adenocarcinomas with mutation profiles observed in the mouse lung tumors suggest that bulky hydrophobic DNA adducts may be responsible for the majority of the mutations observed in the activated human K-ras genes. Other data indicate that approximately 20% of human lung tumors contain potentially novel transforming genes that may also be targets for mutagens in cigarette smoke.

  5. The laryngeal primordium and epithelial lamina. A new interpretation.

    PubMed Central

    Sañudo, J R; Domenech-Mateu, J M

    1990-01-01

    The laryngeal primordium is present in both the laryngotracheal sulcus (LTS) and the primitive pulmonary sac (PPS). Its early period of development may be subdivided into two phases. The first phase (Stage 11) is represented by what is traditionally referred to as the LTS, located directly beneath the PP4 on the ventral wall of the foregut (primary segment), and by the PPS which is situated at its caudal end. The LTS will represent the primordium of the upper or membranous infraglottic cavity region; whereas the PPS, will give rise not only to the bronchial tree, but also to the primordium of the trachea and the lower or cartilaginous region of the infraglottic cavity. The second phase (Stages 13 and 14) is distinguished by the cranial growth of the LTS above the PP4 and therefore by its absorption into the floor of the primitive pharynx in the mesobranchial area (secondary segment), which will develop into the primordium of the vestibule of the larynx. Similarly, we observed that in the development of the laryngeal cavity there are two temporally and spatially separate epithelial structures: the epithelial septum and the epithelial lamina. In this respect we differ from other authors who are of the opinion that there is a single structure (the epithelial lamina). The epithelial septum is a primary structure responsible for the final configuration of the LTS, as it contributes to the development of the lower end of the primary segment of the LTS and also to the creation of the secondary segment. The epithelial lamina is a secondary structure which appears inside the LTS as a result of pressure exerted by the mesenchyme on its lateral walls, without having any effect on the morphogenesis of the LTS. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:2081706

  6. The laryngeal primordium and epithelial lamina. A new interpretation.

    PubMed

    Sañudo, J R; Domenech-Mateu, J M

    1990-08-01

    The laryngeal primordium is present in both the laryngotracheal sulcus (LTS) and the primitive pulmonary sac (PPS). Its early period of development may be subdivided into two phases. The first phase (Stage 11) is represented by what is traditionally referred to as the LTS, located directly beneath the PP4 on the ventral wall of the foregut (primary segment), and by the PPS which is situated at its caudal end. The LTS will represent the primordium of the upper or membranous infraglottic cavity region; whereas the PPS, will give rise not only to the bronchial tree, but also to the primordium of the trachea and the lower or cartilaginous region of the infraglottic cavity. The second phase (Stages 13 and 14) is distinguished by the cranial growth of the LTS above the PP4 and therefore by its absorption into the floor of the primitive pharynx in the mesobranchial area (secondary segment), which will develop into the primordium of the vestibule of the larynx. Similarly, we observed that in the development of the laryngeal cavity there are two temporally and spatially separate epithelial structures: the epithelial septum and the epithelial lamina. In this respect we differ from other authors who are of the opinion that there is a single structure (the epithelial lamina). The epithelial septum is a primary structure responsible for the final configuration of the LTS, as it contributes to the development of the lower end of the primary segment of the LTS and also to the creation of the secondary segment. The epithelial lamina is a secondary structure which appears inside the LTS as a result of pressure exerted by the mesenchyme on its lateral walls, without having any effect on the morphogenesis of the LTS.

  7. Alterations of lung microbiota in a mouse model of LPS-induced lung injury

    PubMed Central

    Meng, Fanyong; Meliton, Angelo; Afonyushkin, Taras; Ulanov, Alexander; Semenyuk, Ekaterina; Latif, Omar; Tesic, Vera; Birukova, Anna A.; Birukov, Konstantin G.

    2015-01-01

    Acute lung injury (ALI) and the more severe acute respiratory distress syndrome are common responses to a variety of infectious and noninfectious insults. We used a mouse model of ALI induced by intratracheal administration of sterile bacterial wall lipopolysaccharide (LPS) to investigate the changes in innate lung microbiota and study microbial community reaction to lung inflammation and barrier dysfunction induced by endotoxin insult. One group of C57BL/6J mice received LPS via intratracheal injection (n = 6), and another received sterile water (n = 7). Bronchoalveolar lavage (BAL) was performed at 72 h after treatment. Bacterial DNA was extracted and used for qPCR and 16S rRNA gene-tag (V3–V4) sequencing (Illumina). The bacterial load in BAL from ALI mice was increased fivefold (P = 0.03). The community complexity remained unchanged (Simpson index, P = 0.7); the Shannon diversity index indicated the increase of community evenness in response to ALI (P = 0.07). Principal coordinate analysis and analysis of similarity (ANOSIM) test (P = 0.005) revealed a significant difference between microbiota of control and ALI groups. Bacteria from families Xanthomonadaceae and Brucellaceae increased their abundance in the ALI group as determined by Metastats test (P < 0.02). In concordance with the 16s-tag data, Stenotrohomonas maltophilia (Xanthomonadaceae) and Ochrobactrum anthropi (Brucellaceae) were isolated from lungs of mice from both groups. Metabolic profiling of BAL detected the presence of bacterial substrates suitable for both isolates. Additionally, microbiota from LPS-treated mice intensified IL-6-induced lung inflammation in naive mice. We conclude that the morbid transformation of ALI microbiota was attributed to the set of inborn opportunistic pathogens thriving in the environment of inflamed lung, rather than the external infectious agents. PMID:25957290

  8. Alterations of lung microbiota in a mouse model of LPS-induced lung injury.

    PubMed

    Poroyko, Valeriy; Meng, Fanyong; Meliton, Angelo; Afonyushkin, Taras; Ulanov, Alexander; Semenyuk, Ekaterina; Latif, Omar; Tesic, Vera; Birukova, Anna A; Birukov, Konstantin G

    2015-07-01

    Acute lung injury (ALI) and the more severe acute respiratory distress syndrome are common responses to a variety of infectious and noninfectious insults. We used a mouse model of ALI induced by intratracheal administration of sterile bacterial wall lipopolysaccharide (LPS) to investigate the changes in innate lung microbiota and study microbial community reaction to lung inflammation and barrier dysfunction induced by endotoxin insult. One group of C57BL/6J mice received LPS via intratracheal injection (n = 6), and another received sterile water (n = 7). Bronchoalveolar lavage (BAL) was performed at 72 h after treatment. Bacterial DNA was extracted and used for qPCR and 16S rRNA gene-tag (V3-V4) sequencing (Illumina). The bacterial load in BAL from ALI mice was increased fivefold (P = 0.03). The community complexity remained unchanged (Simpson index, P = 0.7); the Shannon diversity index indicated the increase of community evenness in response to ALI (P = 0.07). Principal coordinate analysis and analysis of similarity (ANOSIM) test (P = 0.005) revealed a significant difference between microbiota of control and ALI groups. Bacteria from families Xanthomonadaceae and Brucellaceae increased their abundance in the ALI group as determined by Metastats test (P < 0.02). In concordance with the 16s-tag data, Stenotrohomonas maltophilia (Xanthomonadaceae) and Ochrobactrum anthropi (Brucellaceae) were isolated from lungs of mice from both groups. Metabolic profiling of BAL detected the presence of bacterial substrates suitable for both isolates. Additionally, microbiota from LPS-treated mice intensified IL-6-induced lung inflammation in naive mice. We conclude that the morbid transformation of ALI microbiota was attributed to the set of inborn opportunistic pathogens thriving in the environment of inflamed lung, rather than the external infectious agents.

  9. Lung Cancer Signatures in Plasma Based on Proteome Profiling of Mouse Tumor Models

    PubMed Central

    Taguchi, Ayumu; Politi, Katerina; Pitteri, Sharon J.; Lockwood, William W.; Faça, Vitor M.; Kelly-Spratt, Karen; Wong, Chee-Hong; Zhang, Qing; Chin, Alice; Park, Kwon-Sik; Goodman, Gary; Gazdar, Adi F.; Sage, Julien; Dinulescu, Daniela M.; Kucherlapati, Raju; DePinho, Ronald A.; Kemp, Christopher J.; Varmus, Harold E.; Hanash, Samir M.

    2012-01-01

    SUMMARY We investigated the potential of in-depth quantitative proteomics to reveal plasma protein signatures that reflect lung tumor biology. We compared plasma protein profiles of four mouse models of lung cancer with profiles of models of pancreatic, ovarian, colon, prostate, and breast cancer and two models of inflammation. A protein signature for Titf1/Nkx2-1, a known lineage-survival oncogene in lung cancer, was found in plasmas of mouse models of lung adenocarcinoma. An EGFR signature was found in plasma of an EGFR mutant model, and a distinct plasma signature related to neuroendocrine development was uncovered in the small-cell lung cancer model. We demonstrate relevance to human lung cancer of the protein signatures identified on the basis of mouse models. PMID:21907921

  10. Multi-walled carbon nanotube-induced gene expression in the mouse lung: Association with lung pathology

    SciTech Connect

    Pacurari, M.; Qian, Y.; Porter, D.W.; Wolfarth, M.; Wan, Y.; Luo, D.; Ding, M.; Castranova, V.; Guo, N.L.

    2011-08-15

    Due to the fibrous shape and durability of multi-walled carbon nanotubes (MWCNT), concerns regarding their potential for producing environmental and human health risks, including carcinogenesis, have been raised. This study sought to investigate how previously identified lung cancer prognostic biomarkers and the related cancer signaling pathways are affected in the mouse lung following pharyngeal aspiration of well-dispersed MWCNT. A total of 63 identified lung cancer prognostic biomarker genes and major signaling biomarker genes were analyzed in mouse lungs (n = 80) exposed to 0, 10, 20, 40, or 80 {mu}g of MWCNT by pharyngeal aspiration at 7 and 56 days post-exposure using quantitative PCR assays. At 7 and 56 days post-exposure, a set of 7 genes and a set of 11 genes, respectively, showed differential expression in the lungs of mice exposed to MWCNT vs. the control group. Additionally, these significant genes could separate the control group from the treated group over the time series in a hierarchical gene clustering analysis. Furthermore, 4 genes from these two sets of significant genes, coiled-coil domain containing-99 (Ccdc99), muscle segment homeobox gene-2 (Msx2), nitric oxide synthase-2 (Nos2), and wingless-type inhibitory factor-1 (Wif1), showed significant mRNA expression perturbations at both time points. It was also found that the expression changes of these 4 overlapping genes at 7 days post-exposure were attenuated at 56 days post-exposure. Ingenuity Pathway Analysis (IPA) found that several carcinogenic-related signaling pathways and carcinogenesis itself were associated with both the 7 and 11 gene signatures. Taken together, this study identifies that MWCNT exposure affects a subset of lung cancer biomarkers in mouse lungs. - Research Highlights: > Multi-Walled Carbon Nanotubes affect lung cancer biomarkers in mouse lungs. > The results suggest potentially harmful effects of MWCNT exposure on human lungs. > The results could potentially be used for

  11. Phenotyping mouse pulmonary function in vivo with the lung diffusing capacity.

    PubMed

    Limjunyawong, Nathachit; Fallica, Jonathan; Ramakrishnan, Amritha; Datta, Kausik; Gabrielson, Matthew; Horton, Maureen; Mitzner, Wayne

    2015-01-06

    The mouse is now the primary animal used to model a variety of lung diseases. To study the mechanisms that underlie such pathologies, phenotypic methods are needed that can quantify the pathologic changes. Furthermore, to provide translational relevance to the mouse models, such measurements should be tests that can easily be done in both humans and mice. Unfortunately, in the present literature few phenotypic measurements of lung function have direct application to humans. One exception is the diffusing capacity for carbon monoxide, which is a measurement that is routinely done in humans. In the present report, we describe a means to quickly and simply measure this diffusing capacity in mice. The procedure involves brief lung inflation with tracer gases in an anesthetized mouse, followed by a 1 min gas analysis time. We have tested the ability of this method to detect several lung pathologies, including emphysema, fibrosis, acute lung injury, and influenza and fungal lung infections, as well as monitoring lung maturation in young pups. Results show significant decreases in all the lung pathologies, as well as an increase in the diffusing capacity with lung maturation. This measurement of lung diffusing capacity thus provides a pulmonary function test that has broad application with its ability to detect phenotypic structural changes with most of the existing pathologic lung models.

  12. Oral recombinant human or mouse lactoferrin reduces Mycobacterium tuberculosis TDM induced granulomatous lung pathology.

    PubMed

    Hwang, Shen-An; Kruzel, Marian L; Actor, Jeffrey K

    2017-02-01

    Trehalose 6'6-dimycolate (TDM) is the most abundant glycolipid on the cell wall of Mycobacterium tuberculosis (MTB). TDM is capable of inducing granulomatous pathology in mouse models that resembles those induced by MTB infection. Using the acute TDM model, this work investigates the effect of recombinant human and mouse lactoferrin to reduce granulomatous pathology. C57BL/6 mice were injected intravenously with TDM at a dose of 25 μg·mouse(-1). At day 4 and 6, recombinant human or mouse lactoferrin (1 mg·(100 μL)(-1)·mouse(-1)) were delivered by gavage. At day 7 after TDM injection, mice were evaluated for lung pathology, cytokine production, and leukocyte populations. Mice given human or mouse lactoferrin had reduced production of IL-12p40 in their lungs. Mouse lactoferrin increased IL-6 and KC (CXCL1) in lung tissue. Increased numbers of macrophages were observed in TDM-injected mice given human or mouse lactoferrin. Granulomatous pathology, composed of mainly migrated leukocytes, was visually reduced in mice that received human or mouse lactoferrin. Quantitation of granulomatous pathology demonstrated a significant decrease in mice given human or mouse lactoferrin compared with TDM control mice. This report is the first to directly compare the immune modulatory effects of both heterologous recombinant human and homologous mouse lactoferrin on the development of TDM-induced granulomas.

  13. Refraction-enhanced tomography of mouse and rabbit lungs

    SciTech Connect

    Sera, T.; Uesugi, K.; Yagi, N.

    2005-09-15

    In order to evaluate the effectiveness of edge enhancement by refraction in computed tomography, images of a cross section of a euthanized mouse thorax were recorded at low (20 keV) and high (72 keV) x-ray energies at a spatial resolution of about 40 {mu}m. Compared with the images obtained with the detector at 30 cm from an object, when the object was located at 113 cm from the detector, the contrast between tissues and air was improved at both energies. The improvement was more pronounced at 72 keV where the absorption contrast was weaker. This effect was due to refraction at the surfaces of alveolar membranes and small airways which creates areas with apparently high and low linear attenuation coefficients within tissues. The edge enhancement by refraction was also effective in images of a euthanized rabbit thorax at x-ray energies of 40 and 70 keV at a spatial resolution of about 0.15 mm. These results raise the possibility that the refraction contrast may be utilized to obtain a high-resolution tomographic image of human lung and bone with low dose.

  14. Developing Novel Therapeutic Approaches in Small Cell Lung Carcinoma Using Genetically Engineered Mouse Models and Human Circulating Tumor Cells

    DTIC Science & Technology

    2015-10-01

    Using Genetically Engineered Mouse Models and Human Circulating Tumor Cells PRINCIPAL INVESTIGATOR: Jeffrey Engelman MD PhD CONTRACTING...SUBTITLE Developiing Novel Therapeutic Approaches in Small Cell Lung 5a. CONTRACT NUMBER Carcinoma Using Genetically Engineered Mouse Models and 5b...biomarkers. 15. SUBJECT TERMS Small cell lung cancer (SCLC), Genetically engineered mouse model (GEMM), BH3 mimetic, TORC inhibitor, Apoptosis

  15. Curcumin Inhibits Transforming Growth Factor β Induced Differentiation of Mouse Lung Fibroblasts to Myofibroblasts

    PubMed Central

    Liu, Daishun; Gong, Ling; Zhu, Honglan; Pu, Shenglan; Wu, Yang; Zhang, Wei; Huang, Guichuan

    2016-01-01

    Transforming growth factor β (TGF-β) induced differentiation of lung fibroblasts to myofibroblasts is a key event in the pathogenesis of pulmonary fibrosis. This study aimed to evaluate the effect of curcumin on TGF-β induced differentiation of lung fibroblasts to myofibroblasts and explore the underlying mechanism. Mouse lung fibroblasts were cultured and treated with TGF-β2 and curcumin or rosiglitazone. Cell vitality was examined by MTT assay. The secretion of collagen-1 was assessed by ELISA. α smooth muscle actin (α-SMA) was visualized by immunofluorescence technique. The expression of peroxisome proliferator activated receptor γ (PPAR-γ) and platelet derived growth factor R β (PDGFR-β) was detected by PCR and Western blot analysis. We found that curcumin and rosiglitazone inhibited the proliferation and TGF-β induced differentiation of mouse lung fibroblasts. In addition, curcumin and rosiglitazone inhibited collagen-1 secretion and α-SMA expression in mouse lung fibroblasts. Furthermore, curcumin and rosiglitazone upregulated PPAR-γ and downregulated PDGFR-β expression in mouse lung fibroblasts. In conclusion, our study reveals novel mechanism by which curcumin inhibits TGF-β2 driven differentiation of lung fibroblasts to myofibroblasts. Curcumin could potentially be used for effective treatment of pulmonary fibrosis. PMID:27877129

  16. Regulation of IL-33 by Oncostatin M in Mouse Lung Epithelial Cells

    PubMed Central

    Izakelian, Laura; Dubey, Anisha; Zhang, Grace; Wong, Steven; Kwofie, Karen; Qureshi, Aatif; Botelho, Fernando

    2016-01-01

    IL-33 modulates both innate and adaptive immune responses at tissue sites including lung and may play critical roles in inflammatory lung disease. Although IL-33 expression can be altered upon NF-Kappa B activation, here we examine regulation by Oncostatin M, a gp130 cytokine family member, in mouse lung tissue. Responses were assessed in BALB/c mouse lung at day 7 of transient overexpression using endotracheally administered adenovirus encoding OSM (AdOSM) or empty vector (AdDel70). Whole lung extracts showed induction of IL-33 mRNA (>20-fold) and protein (10-fold increase in immunoblots) by AdOSM relative to AdDel70. Immunohistochemistry for IL-33 indicated a marked induction of nuclear staining in alveolar epithelial cells in vivo. Oncostatin M stimulated IL-33 mRNA and IL-33 full length protein in C10 mouse type 2 alveolar epithelial cells in culture in time-dependent and dose-dependent fashion, whereas IL-6, LIF, IL-31, IL-4, or IL-13 did not, and TGFβ repressed IL-33. IL-33 induction was associated with activation of STAT3, and pharmacological inhibition of STAT3 ameliorated IL-33 levels. These results indicate Oncostatin M as a potent inducer of IL-33 in mouse lung epithelial cells and suggest that an OSM/IL-33 axis may participate in innate immunity and inflammatory conditions in lung. PMID:27703303

  17. Genomics and developmental approaches to an ascidian adenohypophysis primordium.

    PubMed

    Kano, Shungo

    2010-07-01

    Ascidians, which are the closest phylogenetic relatives to vertebrates, lack a distinct pituitary gland, which is the major endocrine gland in vertebrates. Nevertheless, for the past 130 years, it has been debated that the ascidian neural complex (NC) is homologous to the pituitary. Of the three major components of the NC, the neural gland (NG) has mainly been thought to be the ascidian counterpart of the pituitary. Recently, however, the ciliated funnel, and not the NG, was postulated to be the adenohypophysis (AH) primordium because it is likely derived from oral ectoderm, and because the expression of several placodal genes is comparable to their expression in vertebrates. An extensive in silico survey of the Ciona intestinalis genome sequence revealed that genes encoding pituitary hormones are absent in ascidians. Under the circumstances, this thesis attempts to find a path that shows that the AH primordium is recognizable in the ascidian by revisiting molecular and developmental data from recent public resources on C. intestinalis, and through the use of advanced bio-imaging techniques. A putative Ciona genetic pathway, which was constructed by referring to data from mammals, shows that only a patchwork of the genetic network exists to achieve terminal differentiation of the AH endocrine cells in the Ciona genome. Re-annotation on glycoprotein hormone related proteins, a GPA2/ARP and two GPB5/BRP ones previously reported, reveals that the GPA2 locus contains two splicing variants, and one variant likely formed a three-dimensional conformation similar to that of human GPA2. No clone of the GPB5/BRP1 locus has been isolated, and another candidate, BRP2, is unlikely to be a GPB5. Next, I argued a possibility that endocrine activities of Ciona species could be specialized in association with its short generation time, and I suggest that not only Ciona species but also other ascidians should be studied in order to understand ascidian endocrinology. Confocal images

  18. Prenatal exposure to bisphenol A disrupts mouse fetal lung development.

    PubMed

    Hijazi, Ayten; Guan, Haiyan; Cernea, Maria; Yang, Kaiping

    2015-12-01

    Developmental exposure to bisphenol A (BPA) is associated with lung dysfunction and diseases. However, it is unknown if this association has a fetal origin. The present study addressed this important question by examining the effects of BPA on fetal lung development. BPA was administered to pregnant mice via diet from embryonic day (E) 7.5 to E18.5. Fetal lungs were analyzed at E18.5 for changes in structure and expression of key molecular markers of lung maturation. Our main findings were as follows: BPA severely retards fetal lung maturation, as evidenced by diminished alveolar airspace (15% of control) and thickened septa, hallmarks of lung immaturity; this immaturity is characterized by aberrant alveolar epithelial type I cell differentiation because expression of the type I cell marker, aquaporin 5, but not type II cell markers, is dramatically reduced (16% of control); and the effects of BPA are likely mediated through the glucocorticoid signaling pathway because the expression of epithelial sodium channel γ and glutathione peroxidase, 2 well-known glucocorticoid target genes, is down-regulated in BPA-exposed fetal lungs, and, importantly, maternal dexamethasone administration rescues the lung immaturity phenotype. Taken together, these findings demonstrate that BPA disrupts fetal lung maturation, thus suggesting a fetal origin for BPA-induced lung diseases. © FASEB.

  19. Stereotactic Body Radiation Therapy Delivery in a Genetically Engineered Mouse Model of Lung Cancer

    PubMed Central

    Du, Shisuo; Lockamy, Virginia; Zhou, Lin; Xue, Christine; LeBlanc, Justin; Glenn, Shonna; Shukla, Gaurav; Yu, Yan; Dicker, Adam P.; Leeper, Dennis B.; Lu, You; Lu, Bo

    2016-01-01

    Purpose To implement clinical stereotactic body radiation therapy (SBRT) using a small animal radiation research platform (SARRP) in a genetically engineered mouse model of lung cancer. Methods and Materials A murine model of multinodular Kras-driven spontaneous lung tumors was used for this study. High-resolution cone beam computed tomography (CBCT) imaging was used to identify and target peripheral tumor nodules, whereas off-target lung nodules in the contralateral lung were used as a nonirradiated control. CBCT imaging helps localize tumors, facilitate high-precision irradiation, and monitor tumor growth. SBRT planning, prescription dose, and dose limits to normal tissue followed the guidelines set by RTOG protocols. Pathologic changes in the irradiated tumors were investigated using immunohistochemistry. Results The image guided radiation delivery using the SARRP system effectively localized and treated lung cancer with precision in a genetically engineered mouse model of lung cancer. Immunohistochemical data confirmed the precise delivery of SBRT to the targeted lung nodules. The 60 Gy delivered in 3 weekly fractions markedly reduced the proliferation index, Ki-67, and increased apoptosis per staining for cleaved caspase-3 in irradiated lung nodules. Conclusions It is feasible to use the SARRP platform to perform dosimetric planning and delivery of SBRT in mice with lung cancer. This allows for preclinical studies that provide a rationale for clinical trials involving SBRT, especially when combined with immunotherapeutics. PMID:27681749

  20. AKT1E¹⁷K Is Oncogenic in Mouse Lung and Cooperates with Chemical Carcinogens in Inducing Lung Cancer.

    PubMed

    Malanga, Donatella; Belmonte, Stefania; Colelli, Fabiana; Scarfò, Marzia; De Marco, Carmela; Oliveira, Duarte Mendes; Mirante, Teresa; Camastra, Caterina; Gagliardi, Monica; Rizzuto, Antonia; Mignogna, Chiara; Paciello, Orlando; Papparella, Serenella; Fagman, Henrik; Viglietto, Giuseppe

    2016-01-01

    The hotspot AKT1E17K mutation in the pleckstrin homology domain of AKT1 occurs in approximately 0.6-2% of human lung cancers. Recently, we have demonstrated that AKT1E17K transforms immortalized human bronchial cells. Here by use of a transgenic Cre-inducible murine strain in the wild type Rosa26 (R26) locus (R26-AKT1E17K mice) we demonstrate that AKT1E17K is a bona-fide oncogene and plays a role in the development of lung cancer in vivo. In fact, we report that mutant AKT1E17K induces bronchial and/or bronchiolar hyperplastic lesions in murine lung epithelium, which progress to frank carcinoma at very low frequency, and accelerates tumor formation induced by chemical carcinogens. In conclusion, AKT1E17K induces hyperplasia of mouse lung epithelium in vivo and cooperates with urethane to induce the fully malignant phenotype.

  1. Chromosomal changes in high- and low-invasive mouse lung adenocarcinoma cell strains derived from early passage mouse lung adenocarcinoma cell strains

    SciTech Connect

    Sargent, Linda M. Ensell, Mang X.; Ostvold, Anne-Carine; Baldwin, Kimberly T.; Kashon, Michael L.; Lowry, David T.; Senft, Jamie R.; Jefferson, Amy M.; Johnson, Robert C.; Li Zhi; Tyson, Frederick L.; Reynolds, Steven H.

    2008-11-15

    The incidence of adenocarcinoma of the lung is increasing in the United States, however, the difficulties in obtaining lung cancer families and representative samples of early to late stages of the disease have lead to the study of mouse models for lung cancer. We used Spectral Karyotyping (SKY), mapping with fluorescently labeled genomic clones (FISH), comparative genomic hybridization (CGH) arrays, gene expression arrays, Western immunoblot and real time polymerase chain reaction (PCR) to analyze nine pairs of high-invasive and low-invasive tumor cell strains derived from early passage mouse lung adenocarcinoma cells to detect molecular changes associated with tumor invasion. The duplication of chromosomes 1 and 15 and deletion of chromosome 8 were significantly associated with a high-invasive phenotype. The duplication of chromosome 1 at band C4 and E1/2-H1 were the most significant chromosomal changes in the high-invasive cell strains. Mapping with FISH and CGH array further narrowed the minimum region of duplication of chromosome 1 to 71-82 centimorgans (cM). Expression array analysis and confirmation by real time PCR demonstrated increased expression of COX-2, Translin (TB-RBP), DYRK3, NUCKS and Tubulin-{alpha}4 genes in the high-invasive cell strains. Elevated expression and copy number of these genes, which are involved in inflammation, cell movement, proliferation, inhibition of apoptosis and telomere elongation, were associated with an invasive phenotype. Similar linkage groups are altered in invasive human lung adenocarcinoma, implying that the mouse is a valid genetic model for the study of the progression of human lung adenocarcinoma.

  2. Mechanical properties of acellular mouse lungs after sterilization by gamma irradiation.

    PubMed

    Uriarte, Juan J; Nonaka, Paula N; Campillo, Noelia; Palma, Renata K; Melo, Esther; de Oliveira, Luis V F; Navajas, Daniel; Farré, Ramon

    2014-12-01

    Lung bioengineering using decellularized organ scaffolds is a potential alternative for lung transplantation. Clinical application will require donor scaffold sterilization. As gamma-irradiation is a conventional method for sterilizing tissue preparations for clinical application, the aim of this study was to evaluate the effects of lung scaffold sterilization by gamma irradiation on the mechanical properties of the acellular lung when subjected to the artificial ventilation maneuvers typical within bioreactors. Twenty-six mouse lungs were decellularized by a sodium dodecyl sulfate detergent protocol. Eight lungs were used as controls and 18 of them were submitted to a 31kGy gamma irradiation sterilization process (9 kept frozen in dry ice and 9 at room temperature). Mechanical properties of acellular lungs were measured before and after irradiation. Lung resistance (RL) and elastance (EL) were computed by linear regression fitting of recorded signals during mechanical ventilation (tracheal pressure, flow and volume). Static (Est) and dynamic (Edyn) elastances were obtained by the end-inspiratory occlusion method. After irradiation lungs presented higher values of resistance and elastance than before irradiation: RL increased by 41.1% (room temperature irradiation) and 32.8% (frozen irradiation) and EL increased by 41.8% (room temperature irradiation) and 31.8% (frozen irradiation). Similar increases were induced by irradiation in Est and Edyn. Scanning electron microscopy showed slight structural changes after irradiation, particularly those kept frozen. Sterilization by gamma irradiation at a conventional dose to ensure sterilization modifies acellular lung mechanics, with potential implications for lung bioengineering.

  3. TACE in perinatal mouse lung epithelial cells promotes lung saccular formation

    PubMed Central

    Xu, Wei; Liu, Chengyu; Kaartinen, Vesa; Chen, Hui; Lu, Chi-Han; Zhang, Wenming; Luo, Yongfeng

    2013-01-01

    Tumor necrosis factor-α converting enzyme (TACE) is a cell membrane sheddase, expressed in both developmental lung epithelia and mesenchyme. Global abrogation of TACE results in neonatal lethality and multiple organ developmental abnormalities, including dysplastic lung. To further define the roles of TACE in regulating lung development, lung epithelial and/or mesenchymal specific TACE conditional knockout mice were generated. Blockade of TACE function in developing lung epithelial cells caused reduced saccular formation, decreased cell proliferation, and reduced mid-distal lung epithelial cell differentiation. In contrast, mesenchymal TACE knockout did not have any phenotypic change in developing lung. Simultaneous abrogation of TACE in both lung epithelial and mesenchymal cells did not result in a more severe lung abnormality. Interestingly, these lung-specific TACE conditional knockout mice were not neonatal lethal, and their lung structures were essentially normal after alveolarization. In addition, TACE conditional knockout in developing cardiomyocytes resulted in noncompaction of ventricular myocardium, as seen in TACE conventional knockout mice. However, these mice were also not neonatal lethal. In conclusion, lung epithelial TACE is essential for promoting fetal lung saccular formation, but not postnatal lung alveolarization in mice. Because the developmental abnormality of either lung or heart induced by TACE deficiency does not directly lead to neonatal lethality, the neonatal death of TACE conventional knockout mice is likely a result of synergistic effects of multiple organ abnormalities. PMID:24142516

  4. Radiation-enhanced lung cancer progression in a transgenic mouse model of lung cancer is predictive of outcomes in human lung and breast cancer.

    PubMed

    Delgado, Oliver; Batten, Kimberly G; Richardson, James A; Xie, Xian-Jin; Gazdar, Adi F; Kaisani, Aadil A; Girard, Luc; Behrens, Carmen; Suraokar, Milind; Fasciani, Gail; Wright, Woodring E; Story, Michael D; Wistuba, Ignacio I; Minna, John D; Shay, Jerry W

    2014-03-15

    Carcinogenesis is an adaptive process between nascent tumor cells and their microenvironment, including the modification of inflammatory responses from antitumorigenic to protumorigenic. Radiation exposure can stimulate inflammatory responses that inhibit or promote carcinogenesis. The purpose of this study is to determine the impact of radiation exposure on lung cancer progression in vivo and assess the relevance of this knowledge to human carcinogenesis. K-ras(LA1) mice were irradiated with various doses and dose regimens and then monitored until death. Microarray analyses were performed using Illumina BeadChips on whole lung tissue 70 days after irradiation with a fractionated or acute dose of radiation and compared with age-matched unirradiated controls. Unique group classifiers were derived by comparative genomic analysis of three experimental cohorts. Survival analyses were performed using principal component analysis and k-means clustering on three lung adenocarcinoma, three breast adenocarcinoma, and two lung squamous carcinoma annotated microarray datasets. Radiation exposure accelerates lung cancer progression in the K-ras(LA1) lung cancer mouse model with dose fractionation being more permissive for cancer progression. A nonrandom inflammatory signature associated with this progression was elicited from whole lung tissue containing only benign lesions and predicts human lung and breast cancer patient survival across multiple datasets. Immunohistochemical analyses suggest that tumor cells drive predictive signature. These results demonstrate that radiation exposure can cooperate with benign lesions in a transgenic model of cancer by affecting inflammatory pathways, and that clinically relevant similarities exist between human lung and breast carcinogenesis. ©2014 AACR.

  5. Efficacy of a technique for exposing the mouse lung to particles aspirated from the pharynx.

    PubMed

    Rao, G V S; Tinkle, Sally; Weissman, David N; Antonini, James M; Kashon, Michael L; Salmen, Rebecca; Battelli, Lori A; Willard, Patsy A; Hoover, Mark D; Hubbs, Ann F

    2003-08-08

    Recent studies have demonstrated that the mouse lung can be exposed to soluble antigens by aspiration of these antigens from the pharynx. This simple technique avoids the trauma associated with intratracheal instillation. In this study, the pharyngeal aspiration technique was validated for exposing the mouse lung to respirable particles. Using respirable fluorescent amine-modified polystyrene latex beads and beryllium oxide particles, we investigated the localization of aspirated particles within the lung and the relationship between the amount of material placed in the pharynx and the amount deposited in the lung. For exposure, mice were anesthetized with isoflurane in a bell jar, placed on a slant board, and the tongue was gently held in full extension while a 50-microl suspension of particles was pipetted onto the base of the tongue. Tongue restraint was maintained until at least two breaths were completed. Less than a minute after exposure, all mice awoke from anesthesia without visible sequela. There were no significant differences in particle distribution between the left and right side of the lung (p=.16). Particles were widely disseminated in a peribronchiolar pattern within the alveolar region. There was a linear and significant correlation (r2=.99) between the amount administered and the amount deposited in the lung. In beryllium-exposed mice, measurable lung beryllium was 77.5 to 88.2% of the administered beryllium. These findings demonstrate that following aspiration of pharyngeal deposited particles, exposures to the deep lung are repeatable, technically simple, and highly correlated to the administered dose.

  6. Gremlin negatively modulates BMP-4 induction of embryonic mouse lung branching morphogenesis.

    PubMed

    Shi, W; Zhao, J; Anderson, K D; Warburton, D

    2001-05-01

    Bone morphogenetic protein-4 (BMP-4) is a key morphogen for embryonic lung development that is expressed at high levels in the peripheral epithelium, but the mechanisms that modulate BMP-4 function in early mouse lung branching morphogenesis are unclear. Here, we studied the BMP-4 antagonist Gremlin, which is a member of the DAN family of BMP antagonists that can bind and block BMP-2/4 activity. The expression level of gremlin in embryonic mouse lungs is highest in the early embryonic pseudoglandular stage [embryonic days (E) 11.5-14.5] and is reduced during fetal lung maturation (E18.5 to postnatal day 1). In situ hybridization indicates that gremlin is diffusely expressed in peripheral lung mesenchyme and epithelium, but relatively high epithelial expression occurs in branching buds at E11.5 and in large airways after E16.5. In E11.5 lung organ culture, we found that exogenous BMP-4 dramatically enhanced peripheral lung epithelial branching morphogenesis, whereas reduction of endogenous gremlin expression with antisense oligonucleotides achieved the same gain-of-function phenotype as exogenous BMP-4, including increased epithelial cell proliferation and surfactant protein C expression. On the other hand, adenoviral overexpression of gremlin blocked the stimulatory effects of exogenous BMP-4. Therefore, our data support the hypothesis that Gremlin is a physiologically negative regulator of BMP-4 in lung branching morphogenesis.

  7. Lessons learned using different mouse models during space radiation-induced lung tumorigenesis experiments

    NASA Astrophysics Data System (ADS)

    Wang, Jian; Zhang, Xiangming; Wang, Ping; Wang, Xiang; Farris, Alton B.; Wang, Ya

    2016-06-01

    Unlike terrestrial ionizing radiation, space radiation, especially galactic cosmic rays (GCR), contains high energy charged (HZE) particles with high linear energy transfer (LET). Due to a lack of epidemiologic data for high-LET radiation exposure, it is highly uncertain how high the carcinogenesis risk is for astronauts following exposure to space radiation during space missions. Therefore, using mouse models is necessary to evaluate the risk of space radiation-induced tumorigenesis; however, which mouse model is better for these studies remains uncertain. Since lung tumorigenesis is the leading cause of cancer death among both men and women, and low-LET radiation exposure increases human lung carcinogenesis, evaluating space radiation-induced lung tumorigenesis is critical to enable safe Mars missions. Here, by comparing lung tumorigenesis obtained from different mouse strains, as well as miR-21 in lung tissue/tumors and serum, we believe that wild type mice with a low spontaneous tumorigenesis background are ideal for evaluating the risk of space radiation-induced lung tumorigenesis, and circulating miR-21 from such mice model might be used as a biomarker for predicting the risk.

  8. Lessons learned using different mouse models during space radiation-induced lung tumorigenesis experiments.

    PubMed

    Wang, Jian; Zhang, Xiangming; Wang, Ping; Wang, Xiang; Farris, Alton B; Wang, Ya

    2016-06-01

    Unlike terrestrial ionizing radiation, space radiation, especially galactic cosmic rays (GCR), contains high energy charged (HZE) particles with high linear energy transfer (LET). Due to a lack of epidemiologic data for high-LET radiation exposure, it is highly uncertain how high the carcinogenesis risk is for astronauts following exposure to space radiation during space missions. Therefore, using mouse models is necessary to evaluate the risk of space radiation-induced tumorigenesis; however, which mouse model is better for these studies remains uncertain. Since lung tumorigenesis is the leading cause of cancer death among both men and women, and low-LET radiation exposure increases human lung carcinogenesis, evaluating space radiation-induced lung tumorigenesis is critical to enable safe Mars missions. Here, by comparing lung tumorigenesis obtained from different mouse strains, as well as miR-21 in lung tissue/tumors and serum, we believe that wild type mice with a low spontaneous tumorigenesis background are ideal for evaluating the risk of space radiation-induced lung tumorigenesis, and circulating miR-21 from such mice model might be used as a biomarker for predicting the risk.

  9. Asymmetric dimethylarginine potentiates lung inflammation in a mouse model of allergic asthma

    PubMed Central

    Klein, Elizabeth; Weigel, Jason; Buford, Mary C.; Holian, Andrij

    2010-01-01

    Nitric oxide (NO), formed by nitric oxide synthase (NOS), is an important mediator of lung inflammation in allergic asthma. Asymmetric dimethylarginine (ADMA), a competitive endogenous inhibitor of NOS, is metabolized by the enzyme dimethylarginine dimethylaminohydrolase (DDAH). Elevated ADMA has been shown to affect lung function in mice, and by inhibiting NOS it alters NO and reactive oxygen species production in mouse lung epithelial cells. However, the effects of altered ADMA levels during lung inflammation have not been explored. A model of allergen-induced airway inflammation was utilized in combination with the modulation of endogenous circulating ADMA levels in mice. Airway inflammation was assessed by quantifying inflammatory cell infiltrates in lung lavage and by histology. Lung DDAH expression was assessed by quantitative PCR and immunohistochemistry. Nitrite levels were determined in lung lavage fluid as a measure of NO production. iNOS expression was determined by immunohistochemistry, immunofluorescence, Western blot, and quantitative PCR. NF-κB binding activity was assessed by a transcription factor binding assay. Allergen-induced lung inflammation was potentiated in mice with elevated circulating ADMA and was reduced in mice overexpressing DDAH. Elevated ADMA reduced nitrite levels in lung lavage fluid in both allergen-challenged and control animals. ADMA increased iNOS expression in airway epithelial cells in vivo following allergen challenge and in vitro in stimulated mouse lung epithelial cells. ADMA also increased NF-κB binding activity in airway epithelial cells in vitro. These data support that ADMA may play a role in inflammatory airway diseases such as asthma through modulation of iNOS expression in lung epithelial cells. PMID:20889675

  10. Genetically manipulated mouse models of lung disease: potential and pitfalls

    PubMed Central

    Choi, Alexander J. S.; Owen, Caroline A.; Choi, Augustine M. K.

    2012-01-01

    Gene targeting in mice (transgenic and knockout) has provided investigators with an unparalleled armamentarium in recent decades to dissect the cellular and molecular basis of critical pathophysiological states. Fruitful information has been derived from studies using these genetically engineered mice with significant impact on our understanding, not only of specific biological processes spanning cell proliferation to cell death, but also of critical molecular events involved in the pathogenesis of human disease. This review will focus on the use of gene-targeted mice to study various models of lung disease including airways diseases such as asthma and chronic obstructive pulmonary disease, and parenchymal lung diseases including idiopathic pulmonary fibrosis, pulmonary hypertension, pneumonia, and acute lung injury. We will attempt to review the current technological approaches of generating gene-targeted mice and the enormous dataset derived from these studies, providing a template for lung investigators. PMID:22198907

  11. Early primordium morphogenesis during lateral root initiation in Arabidopsis thaliana.

    PubMed

    Dubrovsky, J G; Rost, T L; Colón-Carmona, A; Doerner, P

    2001-11-01

    The first morphogenetic events of lateral root primordium (LRP) formation in the Arabidopsis thaliana (L.) Heynh. pericycle occur soon after cells of the primary root complete elongation. Pericycle cells in direct contact with underlying protoxylem cells participate in LRP formation. Two types of LRP initiation were found, longitudinal uni- and bi-cellular. These occur when a single or two pericycle cells within a file, respectively, become founder cells for the entire longitudinal extent of the LRP. Histochemical and cytological analysis suggests that three is the minimum number of cells required to initiate an LRP. In young primordia comprising less than 32 cells, the average cell-doubling time was 3.7 h, indicating a drastic acceleration of cell cycle progression after lateral root initiation. Early in LRP development, cell growth is limited and therefore cytokinesis leads to a reduction of cell volume, similar to cleavage division cycles during animal and plant embryogenesis. The striking coordination of proliferation between pericycle cells in adjacent files in direct contact with the underlying protoxylem implies that intercellular signaling mechanisms act in the root apical meristem or later in development.

  12. Wnt/β-catenin signaling accelerates mouse lung tumorigenesis by imposing an embryonic distal progenitor phenotype on lung epithelium.

    PubMed

    Pacheco-Pinedo, Eugenia C; Durham, Amy C; Stewart, Kathleen M; Goss, Ashley M; Lu, Min Min; Demayo, Francesco J; Morrisey, Edward E

    2011-05-01

    Although mutations in Kras are present in 21% of lung tumors, there is a high level of heterogeneity in phenotype and outcome among patients with lung cancer bearing similar mutations, suggesting that other pathways are important. Wnt/β-catenin signaling is a known oncogenic pathway that plays a well-defined role in colon and skin cancer; however, its role in lung cancer is unclear. We have shown here that activation of Wnt/β-catenin in the bronchiolar epithelium of the adult mouse lung does not itself promote tumor development. However, concurrent activation of Wnt/β-catenin signaling and expression of a constitutively active Kras mutant (KrasG12D) led to a dramatic increase in both overall tumor number and size compared with KrasG12D alone. Activation of Wnt/β-catenin signaling altered the KrasG12D tumor phenotype, resulting in a phenotypic switch from bronchiolar epithelium to the highly proliferative distal progenitors found in the embryonic lung. This was associated with decreased E-cadherin expression at the cell surface, which may underlie the increased metastasis of tumors with active Wnt/β-catenin signaling. Together, these data suggest that activation of Wnt/β-catenin signaling can combine with other oncogenic pathways in lung epithelium to produce a more aggressive tumor phenotype by imposing an embryonic distal progenitor phenotype and by decreasing E-cadherin expression.

  13. Mechanotransduction via TRPV4 regulates inflammation and differentiation in fetal mouse distal lung epithelial cells.

    PubMed

    Nayak, Pritha S; Wang, Yulian; Najrana, Tanbir; Priolo, Lauren M; Rios, Mayra; Shaw, Sunil K; Sanchez-Esteban, Juan

    2015-05-27

    Mechanical ventilation plays a central role in the injury of premature lungs. However, the mechanisms by which mechanical signals trigger an inflammatory cascade to promote lung injury are not well-characterized. Transient receptor potential vanilloid 4 (TRPV4), a calcium-permeable mechanoreceptor channel has been shown to be a major determinant of ventilator-induced acute lung injury in adult models. However, the role of these channels as modulators of inflammation in immature lungs is unknown. In this study, we tested the hypothesis that TRPV4 channels are important mechanotransducers in fetal lung injury. Expression of TRPV4 in the mouse fetal lung was investigated by immunohistochemistry, Western blot and qRT-PCR. Isolated fetal epithelial cells were exposed to mechanical stimulation using the Flexcell Strain Unit and inflammation and differentiation were analyzed by ELISA and SP-C mRNA, respectively. TRPV4 is developmentally regulated in the fetal mouse lung; it is expressed in the lung epithelium and increases with advanced gestation. In contrast, in isolated epithelial cells, TRPV4 expression is maximal at E17-E18 of gestation. Mechanical stretch increases TRPV4 in isolated fetal epithelial cells only during the canalicular stage of lung development. Using the TRPV4 agonist GSK1016790A, the antagonist HC-067047, and the cytokine IL-6 as a marker of inflammation, we observed that TRPV4 regulates release of IL-6 via p38 and ERK pathways. Interestingly, stretch-induced differentiation of fetal epithelial cells was also modulated by TRPV4. These studies demonstrate that TRPV4 may play an important role in the transduction of mechanical signals in the fetal lung epithelium by modulating not only inflammation but also the differentiation of fetal epithelial cells.

  14. CD11b immunophenotyping identifies inflammatory profiles in the mouse and human lungs.

    PubMed

    Duan, M; Steinfort, D P; Smallwood, D; Hew, M; Chen, W; Ernst, M; Irving, L B; Anderson, G P; Hibbs, M L

    2016-03-01

    The development of easily accessible tools for human immunophenotyping to classify patients into discrete disease endotypes is advancing personalized therapy. However, no systematic approach has been developed for the study of inflammatory lung diseases with often complex and highly heterogeneous disease etiologies. We have devised an internally standardized flow cytometry approach that can identify parallel inflammatory alveolar macrophage phenotypes in both the mouse and human lungs. In mice, lung innate immune cell alterations during endotoxin challenge, influenza virus infection, and in two genetic models of chronic obstructive lung disease could be segregated based on the presence or absence of CD11b alveolar macrophage upregulation and lung eosinophilia. Additionally, heightened alveolar macrophage CD11b expression was a novel feature of acute lung exacerbations in the SHIP-1(-/-) model of chronic obstructive lung disease, and anti-CD11b antibody administration selectively blocked inflammatory CD11b(pos) but not homeostatic CD11b(neg) alveolar macrophages in vivo. The identification of analogous profiles in respiratory disease patients highlights this approach as a translational avenue for lung disease endotyping and suggests that heterogeneous innate immune cell phenotypes are an underappreciated component of the human lung disease microenvironment.

  15. Morphological analysis of mouse lungs after treatment with magnetite-based magnetic fluid stabilized with DMSA

    NASA Astrophysics Data System (ADS)

    Garcia, Mônica Pereira; Miranda Parca, Renata; Braun Chaves, Sacha; Paulino Silva, Luciano; Djalma Santos, Antonio; Guerrero Marques Lacava, Zulmira; César Morais, Paulo; Azevedo, Ricardo Bentes

    2005-05-01

    Mouse lungs injected with magnetic fluids based on magnetite nanoparticles stabilized by 2,3-dimercaptosuccinic acid were studied. We observed clusters of magnetic nanoparticles inside blood vessels, within the organ parenchyma and cells, as well as increased numbers of leukocytes in the organ. Both the particle concentration and organ inflammation diminished in a time-dependent manner.

  16. Longitudinal in vivo microcomputed tomography of mouse lungs: No evidence for radiotoxicity

    PubMed Central

    Vande Velde, Greetje; De Langhe, Ellen; Poelmans, Jennifer; Bruyndonckx, Peter; d'Agostino, Emiliano; Verbeken, Erik; Bogaerts, Ria; Himmelreich, Uwe

    2015-01-01

    Before microcomputed tomography (micro-CT) can be exploited to its full potential for longitudinal monitoring of transgenic and experimental mouse models of lung diseases, radiotoxic side effects such as inflammation or fibrosis must be considered. We evaluated dose and potential radiotoxicity to the lungs for long-term respiratory-gated high-resolution micro-CT protocols. Free-breathing C57Bl/6 mice underwent four different retrospectively respiratory gated micro-CT imaging schedules of repeated scans during 5 or 12 wk, followed by ex vivo micro-CT and detailed histological and biochemical assessment of lung damage. Radiation exposure, dose, and absorbed dose were determined by ionization chamber, thermoluminescent dosimeter measurements and Monte Carlo calculations. Despite the relatively large radiation dose delivered per micro-CT acquisition, mice did not show any signs of radiation-induced lung damage or fibrosis when scanned weekly during 5 and up to 12 wk. Doubling the scanning frequency and once tripling the radiation dose as to mimic the instant repetition of a failed scan also stayed without detectable toxicity after 5 wk of scanning. Histological analyses confirmed the absence of radiotoxic damage to the lungs, thereby demonstrating that long-term monitoring of mouse lungs using high-resolution micro-CT is safe. This opens perspectives for longitudinal monitoring of (transgenic) mouse models of lung diseases and therapeutic response on an individual basis with high spatial and temporal resolution, without concerns for radiation toxicity that could potentially influence the readout of micro-CT-derived lung biomarkers. This work further supports the introduction of micro-CT for routine use in the preclinical pulmonary research field where postmortem histological approaches are still the gold standard. PMID:26024893

  17. Lung tumor promotion by chromium-containing welding particulate matter in a mouse model.

    PubMed

    Zeidler-Erdely, Patti C; Meighan, Terence G; Erdely, Aaron; Battelli, Lori A; Kashon, Michael L; Keane, Michael; Antonini, James M

    2013-09-05

    Epidemiology suggests that occupational exposure to welding particulate matter (PM) may increase lung cancer risk. However, animal studies are lacking to conclusively link welding with an increased risk. PM derived from stainless steel (SS) welding contains carcinogenic metals such as hexavalent chromium and nickel. We hypothesized that welding PM may act as a tumor promoter and increase lung tumor multiplicity in vivo. Therefore, the capacity of chromium-containing gas metal arc (GMA)-SS welding PM to promote lung tumors was evaluated using a two-stage (initiation-promotion) model in lung tumor susceptible A/J mice. Male mice (n = 28-30/group) were treated either with the initiator 3-methylcholanthrene (MCA;10 μg/g; IP) or vehicle (corn oil) followed by 5 weekly pharyngeal aspirations of GMA-SS (340 or 680 μg/exposure) or PBS. Lung tumors were enumerated at 30 weeks post-initiation. MCA initiation followed by GMA-SS welding PM exposure promoted tumor multiplicity in both the low (12.1 ± 1.5 tumors/mouse) and high (14.0 ± 1.8 tumors/mouse) exposure groups significantly above MCA/sham (4.77 ± 0.7 tumors/mouse; p = 0.0001). Multiplicity was also highly significant (p < 0.004) across all individual lung regions of GMA-SS-exposed mice. No exposure effects were found in the corn oil groups at 30 weeks. Histopathology confirmed the gross findings and revealed increased inflammation and a greater number of malignant lesions in the MCA/welding PM-exposed groups. GMA-SS welding PM acts as a lung tumor promoter in vivo. Thus, this study provides animal evidence to support the epidemiological data that show welders have an increased lung cancer risk.

  18. Lung tumor promotion by chromium-containing welding particulate matter in a mouse model

    PubMed Central

    2013-01-01

    Background Epidemiology suggests that occupational exposure to welding particulate matter (PM) may increase lung cancer risk. However, animal studies are lacking to conclusively link welding with an increased risk. PM derived from stainless steel (SS) welding contains carcinogenic metals such as hexavalent chromium and nickel. We hypothesized that welding PM may act as a tumor promoter and increase lung tumor multiplicity in vivo. Therefore, the capacity of chromium-containing gas metal arc (GMA)-SS welding PM to promote lung tumors was evaluated using a two-stage (initiation-promotion) model in lung tumor susceptible A/J mice. Methods Male mice (n = 28-30/group) were treated either with the initiator 3-methylcholanthrene (MCA;10 μg/g; IP) or vehicle (corn oil) followed by 5 weekly pharyngeal aspirations of GMA-SS (340 or 680 μg/exposure) or PBS. Lung tumors were enumerated at 30 weeks post-initiation. Results MCA initiation followed by GMA-SS welding PM exposure promoted tumor multiplicity in both the low (12.1 ± 1.5 tumors/mouse) and high (14.0 ± 1.8 tumors/mouse) exposure groups significantly above MCA/sham (4.77 ± 0.7 tumors/mouse; p = 0.0001). Multiplicity was also highly significant (p < 0.004) across all individual lung regions of GMA-SS-exposed mice. No exposure effects were found in the corn oil groups at 30 weeks. Histopathology confirmed the gross findings and revealed increased inflammation and a greater number of malignant lesions in the MCA/welding PM-exposed groups. Conclusions GMA-SS welding PM acts as a lung tumor promoter in vivo. Thus, this study provides animal evidence to support the epidemiological data that show welders have an increased lung cancer risk. PMID:24107379

  19. Longitudinal in vivo microcomputed tomography of mouse lungs: No evidence for radiotoxicity.

    PubMed

    Vande Velde, Greetje; De Langhe, Ellen; Poelmans, Jennifer; Bruyndonckx, Peter; d'Agostino, Emiliano; Verbeken, Erik; Bogaerts, Ria; Lories, Rik; Himmelreich, Uwe

    2015-08-01

    Before microcomputed tomography (micro-CT) can be exploited to its full potential for longitudinal monitoring of transgenic and experimental mouse models of lung diseases, radiotoxic side effects such as inflammation or fibrosis must be considered. We evaluated dose and potential radiotoxicity to the lungs for long-term respiratory-gated high-resolution micro-CT protocols. Free-breathing C57Bl/6 mice underwent four different retrospectively respiratory gated micro-CT imaging schedules of repeated scans during 5 or 12 wk, followed by ex vivo micro-CT and detailed histological and biochemical assessment of lung damage. Radiation exposure, dose, and absorbed dose were determined by ionization chamber, thermoluminescent dosimeter measurements and Monte Carlo calculations. Despite the relatively large radiation dose delivered per micro-CT acquisition, mice did not show any signs of radiation-induced lung damage or fibrosis when scanned weekly during 5 and up to 12 wk. Doubling the scanning frequency and once tripling the radiation dose as to mimic the instant repetition of a failed scan also stayed without detectable toxicity after 5 wk of scanning. Histological analyses confirmed the absence of radiotoxic damage to the lungs, thereby demonstrating that long-term monitoring of mouse lungs using high-resolution micro-CT is safe. This opens perspectives for longitudinal monitoring of (transgenic) mouse models of lung diseases and therapeutic response on an individual basis with high spatial and temporal resolution, without concerns for radiation toxicity that could potentially influence the readout of micro-CT-derived lung biomarkers. This work further supports the introduction of micro-CT for routine use in the preclinical pulmonary research field where postmortem histological approaches are still the gold standard.

  20. Stable isotope resolved metabolomics of lung cancer in a SCID mouse model.

    PubMed

    Fan, Teresa W-M; Lane, Andrew N; Higashi, Richard M; Yan, Jun

    2011-06-01

    We have determined the time course of [U-(13)C]-glucose utilization and transformations in SCID mice via bolus injection of the tracer in the tail vein. Incorporation of (13)C into metabolites extracted from mouse blood plasma and several tissues (lung, heart, brain, liver, kidney, and skeletal muscle) were profiled by NMR and GC-MS, which helped ascertain optimal sampling times for different target tissues. We found that the time for overall optimal (13)C incorporation into tissue was 15-20 min but with substantial differences in (13)C labeling patterns of various organs that reflected their specific metabolism. Using this stable isotope resolved metabolomics (SIRM) approach, we have compared the (13)C metabolite profile of the lungs in the same mouse with or without an orthotopic lung tumor xenograft established from human PC14PE6 lung adenocarcinoma cells. The (13)C metabolite profile shows considerable differences in [U-(13)C]-glucose transformations between the two lung tissues, demonstrating the feasibility of applying SIRM to investigate metabolic networks of human cancer xenograft in the mouse model.

  1. Chemokine and Fgf signalling act as opposing guidance cues in formation of the lateral line primordium

    PubMed Central

    Breau, Marie A.; Wilson, Duncan; Wilkinson, David G.; Xu, Qiling

    2012-01-01

    The directional migration of many cell populations occurs as a coherent group. An amenable model is provided by the posterior lateral line in zebrafish, which is formed by a cohesive primordium that migrates from head to tail and deposits future neuromasts at intervals. We found that prior to the onset of migration, the compact state of the primordium is not fully established, as isolated cells with lateral line identity are present caudal to the main primordium. These isolated cells are retained in position such that they fuse with the migrating primordium as it advances, and later contribute to the leading zone and terminal neuromasts. We found that the isolated lateral line cells are positioned by two antagonistic cues: Fgf signalling attracts them towards the primordium, which counteracts Sdf1α/Cxcr4b-mediated caudal attraction. These findings reveal a novel chemotactic role for Fgf signalling in which it enables the coalescence of the lateral line primordium from an initial fuzzy pattern into a compact group of migrating cells. PMID:22619392

  2. Chemokine and Fgf signalling act as opposing guidance cues in formation of the lateral line primordium.

    PubMed

    Breau, Marie A; Wilson, Duncan; Wilkinson, David G; Xu, Qiling

    2012-06-01

    The directional migration of many cell populations occurs as a coherent group. An amenable model is provided by the posterior lateral line in zebrafish, which is formed by a cohesive primordium that migrates from head to tail and deposits future neuromasts at intervals. We found that prior to the onset of migration, the compact state of the primordium is not fully established, as isolated cells with lateral line identity are present caudal to the main primordium. These isolated cells are retained in position such that they fuse with the migrating primordium as it advances, and later contribute to the leading zone and terminal neuromasts. We found that the isolated lateral line cells are positioned by two antagonistic cues: Fgf signalling attracts them towards the primordium, which counteracts Sdf1α/Cxcr4b-mediated caudal attraction. These findings reveal a novel chemotactic role for Fgf signalling in which it enables the coalescence of the lateral line primordium from an initial fuzzy pattern into a compact group of migrating cells.

  3. Maternally imprinted microRNAs are differentially expressed during mouse and human lung development

    PubMed Central

    Williams, Andrew E.; Moschos, Sterghios A.; Perry, Mark M.; Barnes, Peter J.; Lindsay, Mark A.

    2008-01-01

    MicroRNAs (miRNAs) are a recently discovered class of non-coding genes that regulate the translation of target mRNA. More than 300 miRNAs have now been discovered in humans, although the function of most is still unknown. A highly sensitive, semi-quantitative RT-PCR method was utilised to reveal the differential expression of a number of miRNAs during the development of both mouse and human lung. Of note was the upregulation in neonatal mouse and fetal human lung of a maternally imprinted miRNA cluster located at human chromosome 14q32.21 (mouse chromosome 12F2), which includes the miR-154 and miR-335 families and is situated within the Gtl2-Dio3 domain. Conversely, several miRNAs were upregulated in adult compared to neonatal/fetal lung including miR-29a and miR-29b. Differences in the spatial expression patterns of miR-154, miR-29a and miR-26a was demonstrated using in situ hybridisation of mouse neonatal and adult tissue using miRNA-specific LNA probes. Interestingly, miR-154 appeared to be localised to the stroma of fetal but not adult lungs. The overall expression profile was similar for mouse and human tissue suggesting evolutionary conservation of miRNA expression during lung development and demonstrating the importance of maternally imprinted miRNAs in the developmental process. PMID:17191223

  4. Slit and robo expression in the developing mouse lung.

    PubMed

    Greenberg, James M; Thompson, Felisa Y; Brooks, Sherry K; Shannon, John M; Akeson, Ann L

    2004-06-01

    Mammalian lung development is mediated through complex interactions between foregut endoderm and surrounding mesenchyme. As airway branching progresses, the mesenchyme undergoes dramatic remodeling and differentiation. Little is understood about the mechanisms that direct mesenchymal organization during lung development. A screen for candidate genes mediating this process identified Slit, a ligand for the Roundabout (Robo) receptor previously associated with guidance of axonal projections during central nervous system development. Here, we demonstrate by in situ hybridization that two Slit genes (Slit-2 and Slit-3) and two Robo genes (Robo-1 and Robo-2) are expressed in fetal lung mesenchyme. Slit-2 and Robo-1 expression is present throughout mesenchyme at midgestation and is not detectable by newborn day 1. Slit-3 and Robo-2 expression is restricted to specific, complementary subsets of mesenchyme. Robo-2 is expressed in mesenchymal cells immediately adjacent to large airways, whereas Slit-3 expression predominates in mesenchyme remote from airway epithelium. The temporal and spatial distribution of Slit and Robo mRNAs indicate that these genes may direct the functional organization and differentiation of fetal lung mesenchyme.

  5. Plasminogen activator inhibitor-1 in acute hyperoxic mouse lung injury.

    PubMed Central

    Barazzone, C; Belin, D; Piguet, P F; Vassalli, J D; Sappino, A P

    1996-01-01

    Hyperoxia-induced lung disease is associated with prominent intraalveolar fibrin deposition. Fibrin turnover is tightly regulated by the concerted action of proteases and antiproteases, and inhibition of plasmin-mediated proteolysis could account for fibrin accumulation in lung alveoli. We show here that lungs of mice exposed to hyperoxia overproduce plasminogen activator inhibitor-1 (PAI-1), and that PAI-1 upregulation impairs fibrinolytic activity in the alveolar compartment. To explore whether increased PAI-1 production is a causal or only a correlative event for impaired intraalveolar fibrinolysis and the development of hyaline membrane disease, we studied mice genetically deficient in PAI-1. We found that these mice fail to develop intraalveolar fibrin deposits in response to hyperoxia and that they are more resistant to the lethal effects of hyperoxic stress. These observations provide clear and novel evidence for the pathogenic contribution of PAI-1 in the development of hyaline membrane disease. They identify PAI-1 as a major deleterious mediator of hyperoxic lung injury. PMID:8981909

  6. Revitalization of a Diastemal Tooth Primordium in Spry2 Null Mice Results From Increased Proliferation and Decreased Apoptosis

    PubMed Central

    Peterkova, Renata; Churava, Svatava; Lesot, Herve; Rothova, Michaela; Prochazka, Jan; Peterka, Miroslav; Klein, Ophir D.

    2010-01-01

    An understanding of the factors that promote or inhibit tooth development is essential for designing biological tooth replacements. The embryonic mouse dentition provides an ideal system for studying such factors because it consists of two types of tooth primordia. One type of primordium will go on to form a functional tooth, whereas the other initiates development but arrests at or before the bud stage. This developmental arrest contributes to the formation of the toothless mouse diastema. It is accompanied by the apoptosis of the rudimentary diastemal buds, which presumably results from the insufficient activity of anti-apoptotic signals such as fibroblast growth factors (FGFs). We have previously shown that the arrest of a rudimentary tooth bud can be rescued by inactivating Spry2, an antagonist of FGF signaling. Here, we studied the role of the epithelial cell death and proliferation in this process by comparing the development of a rudimentary diastemal tooth bud (R2) and the first molar in the mandibles of Spry2−/− and wild-type (WT) embryos using histological sections, image analysis and 3D reconstructions. In the WT R2 at embryonic day 13.5, significantly increased apoptosis and decreased proliferation were found compared with the first molar. In contrast, increased levels of FGF signaling in Spry2−/− embryos led to significantly decreased apoptosis and increased proliferation in the R2 bud. Consequently, the R2 was involved in the formation of a supernumerary tooth primordium. Studies of the revitalization of rudimentary tooth primordia in mutant mice can help to lay the foundation for tooth regeneration by enhancing our knowledge of mechanisms that regulate tooth formation. PMID:19127536

  7. Cadmium stimulates myofibroblast differentiation and mouse lung fibrosis.

    PubMed

    Hu, Xin; Fernandes, Jolyn; Jones, Dean P; Go, Young-Mi

    2017-03-21

    Increasing evidence suggests that Cd at levels found in the human diet can cause oxidative stress and activate redox-sensitive transcription factors in inflammatory signaling. Following inflammation, tissue repair often involves activation of redox-sensitive transcription factors in fibroblasts. In lungs, epithelial barrier remodeling is required to restore gas exchange and barrier function, and aberrant myofibroblast differentiation leads to pulmonary fibrosis. Contributions of exogenous exposures, such as dietary Cd, to pulmonary fibrosis remain incompletely defined. In the current study, we tested whether Cd activates fibrotic signaling in human fetal lung fibroblasts (HFLF) at micromolar and submicromolar Cd concentrations that do not cause cell death. Exposure of HFLF to low-dose Cd (≤1.0μM) caused an increase in stress fibers and increased protein levels of myofibroblast differentiation markers, including α-smooth muscle actin (α-SMA) and extra-domain-A-containing fibronectin (ED-A-FN). Assay of transcription factor (TF) activity using a 45-TF array showed that Cd increased activity of 12 TF, including SMAD2/3/4 (mothers against decapentaplegic homolog) signaling differentiation and fibrosis. Results were confirmed by real-time PCR and supported by increased expression of target genes of SMAD2/3/4. Immunocytochemistry of lungs of mice exposed to low-dose Cd (0.3 and 1.0mg/L in drinking water) showed increased α-SMA protein level with lung Cd accumulation similar to lung Cd in non-smoking humans. Together, the results show that relatively low Cd exposures stimulate pulmonary fibrotic signaling and myofibroblast differentiation by activating SMAD2/3/4-dependent signaling. The results indicate that dietary Cd intake could be an important variable contributing to pulmonary fibrosis in humans.

  8. Mouse lung-tumor assay: a final report

    SciTech Connect

    Smith, L.H.; Witschi, H.P.

    1983-05-01

    The objective was to validate a lung tumor assay. Agents evaluated included 5 complex mixtures from modern synfuel processes, 8 nitrated toluenes and 30 compounds which had been tested previously in a standard 2-year NCI-type carcinogenesis bioassay. Male A/Jax mice were injected 3 times a week, for eight consecutive weeks, with different test substances. After the last injection, the animals were left undisturbed for another 4 months, and then they were killed. The carcinogenic potential of the substances was evaluated by counting the number of visible tumors on the lung surface. The average number of tumors per lung was calculated for each group (tumor multiplicity). Tumor incidence and tumor multiplicity for each treatment group was compared to appropriate vehicle control groups. Statistical tests used were Chi-square for tumor incidence and Student's t-test for tumor multiplicity; a p value of 0.05 or less was considered to be significant. Very few of the substances tested were found to give an unequivocally positive response. Shale oil and two of its derivatives and two tar mixtures from a coal gasifier were clearly positive. No positives were found in a series of nitrated toluenes. Out of 18 compounds known with certainty to be animal or human carcinogens, the lung tumor assay correctly identified only 5 as having carcinogenic potential. In view of these data we concluded that the lung tumor assay, as developed and advocated (Advances in Cancer Research 21, 1 to 58, 1975), was not sensitive or accurate enough to serve as a short-term in vivo screening procedure for carcinogens.

  9. Genotoxicity of 3-nitrobenzanthrone and 3-aminobenzanthrone in MutaMouse and lung epithelial cells derived from MutaMouse.

    PubMed

    Arlt, Volker M; Gingerich, John; Schmeiser, Heinz H; Phillips, David H; Douglas, George R; White, Paul A

    2008-11-01

    FE1 lung epithelial cells derived from MutaMouse are a new model system to provide in vitro mutagenicity data with the potential to predict the outcome of an in vivo MutaMouse test. 3-Nitrobenzanthrone (3-NBA) is a potent mutagen and suspected human carcinogen identified in diesel exhaust and urban air pollution. We investigated the mutagenicity and DNA binding of 3-NBA and its main metabolite 3-aminobenzanthrone (3-ABA) in vitro and in vivo in the MutaMouse assay. Mice were treated with 3-NBA or 3-ABA (0, 2 or 5 mg/kg body weight/day) by gavage for 28 days and 28 days later lacZ mutant frequency (MF) was determined in liver, lung and bone marrow. For both compounds, dose-related increases in MF were seen in liver and bone marrow, but not in lung; mutagenic activity was approximately 2-fold lower for 3-ABA than for 3-NBA. With 3-NBA, highest DNA adduct levels (measured by (32)P-post-labelling) were found in liver (approximately 230 adducts per 10(8) nucleotides) with levels 20- to 40-fold lower in bone marrow and lung. With 3-ABA, DNA adduct levels were again highest in the liver, but approximately 4-fold lower than for 3-NBA. FE1 cells were exposed to up to 10 microg/ml 3-NBA or 3-ABA for 6 h with or without exogenous activation (S9) and harvested after 3 days. For 3-NBA, there was a dose-related increase in MF both with and without S9 mix, which was >10 times higher than observed in vivo. At the highest concentration of 3-ABA (10 microg/ml), we found only around a 2-fold increase in MF relative to controls. DNA adduct formation in FE1 cells was dose-dependent for both compounds, but 10- to 20-fold higher for 3-NBA compared to 3-ABA. Collectively, our data indicate that MutaMouse FE1 cells are well suited for cost-effective testing of suspected mutagens with different metabolic activation pathways as a guide for subsequent in vivo MutaMouse testing.

  10. BIIL 284 reduces neutrophils numbers but increases P. aeruginosa bacteraemia and inflammation in mouse lungs

    PubMed Central

    Döring, Gerd; Bragonzi, Alessandra; Paroni, Moira; Aktürk, Firdevs-Fatma; Cigana, Cristina; Schmidt, Annika; Gilpin, Deirdre; Heyder, Susanne; Born, Torsten; Smaczny, Christina; Kohlhäufl, Martin; Wagner, Thomas O. F.; Loebinger, Michael R.; Bilton, Diana; Tunney, Michael M.; Elborn, J. Stuart; Pier, Gerald B.; Konstan, Michael W.; Ulrich, Martina

    2014-01-01

    Background A clinical study to investigate the leukotriene B4 (LTB4)-receptor antagonist BIIL 284 in cystic fibrosis (CF) patients was prematurely terminated due to a significantly increased risk of adverse pulmonary events. We aimed to establish the effect of BIIL284 in models of Pseudomonas aeruginosa lung infection, thereby contributing to a better understanding of what could have led to adverse pulmonary events in CF patients. Methods P. aeruginosa DNA in the blood of CF patients during and after acute pulmonary exacerbations and in stable patients with non-CF bronchiectasis (NCFB) and healthy individuals was assessed by PCR. The effect of BIIL 284 treatment was tested in an agar beads murine model of Pseudomonas aeruginosa lung infection. Bacterial count and inflammation were evaluated in lung and other organs. Result Most CF patients (98%) and all patients with NCFB and healthy individuals had negative P. aeruginosa DNA in their blood. Similarly, the P. aeruginosa-infected mice showed bacterial counts in the lung but not blood or spleen. BIIL 284 treatment decreased pulmonary neutrophils and increased P. aeruginosa numbers in mouse lungs leading to significantly higher bacteremia rates and lung inflammation compared to placebo treated animals. Conclusions Decreased airway neutrophils induced lung proliferation and severe bacteraemia in a murine model of P. aeruginosa lung infection. These data suggest that caution should be taken when administering anti-inflammatory compounds to patients with bacterial infections. PMID:24183915

  11. BIIL 284 reduces neutrophil numbers but increases P. aeruginosa bacteremia and inflammation in mouse lungs.

    PubMed

    Döring, Gerd; Bragonzi, Alessandra; Paroni, Moira; Aktürk, Firdevs-Fatma; Cigana, Cristina; Schmidt, Annika; Gilpin, Deirdre; Heyder, Susanne; Born, Torsten; Smaczny, Christina; Kohlhäufl, Martin; Wagner, Thomas O F; Loebinger, Michael R; Bilton, Diana; Tunney, Michael M; Elborn, J Stuart; Pier, Gerald B; Konstan, Michael W; Ulrich, Martina

    2014-03-01

    A clinical study to investigate the leukotriene B(4) (LTB(4))-receptor antagonist BIIL 284 in cystic fibrosis (CF) patients was prematurely terminated due to a significantly increased risk of adverse pulmonary events. We aimed to establish the effect of BIIL284 in models of Pseudomonas aeruginosa lung infection, thereby contributing to a better understanding of what could have led to adverse pulmonary events in CF patients. P. aeruginosa DNA in the blood of CF patients during and after acute pulmonary exacerbations and in stable patients with non-CF bronchiectasis (NCFB) and healthy individuals was assessed by PCR. The effect of BIIL 284 treatment was tested in an agar bead murine model of P. aeruginosa lung infection. Bacterial count and inflammation were evaluated in lung and other organs. Most CF patients (98%) and all patients with NCFB and healthy individuals had negative P. aeruginosa DNA in their blood. Similarly, the P. aeruginosa-infected mice showed bacterial counts in the lung but not in the blood or spleen. BIIL 284 treatment decreased pulmonary neutrophils and increased P. aeruginosa numbers in mouse lungs leading to significantly higher bacteremia rates and lung inflammation compared to placebo treated animals. Decreased airway neutrophils induced lung proliferation and severe bacteremia in a murine model of P. aeruginosa lung infection. These data suggest that caution should be taken when administering anti-inflammatory compounds to patients with bacterial infections. © 2013.

  12. Cold stress aggravates inflammatory responses in an LPS-induced mouse model of acute lung injury

    NASA Astrophysics Data System (ADS)

    Joo, Su-Yeon; Park, Mi-Ju; Kim, Kyun-Ha; Choi, Hee-Jung; Chung, Tae-Wook; Kim, Yong Jin; Kim, Joung Hee; Kim, Keuk-Jun; Joo, Myungsoo; Ha, Ki-Tae

    2016-08-01

    Although the relationship between environmental cold temperature and susceptibility to respiratory infection is generally accepted, the effect of ambient cold temperature on host reactivity in lung inflammation has not been fully studied. To examine the function of ambient cold temperature on lung inflammation, mice were exposed to 4 °C for 8 h each day for 14 days. In the lungs of mice exposed to cold stress, inflammatory cells in bronchoalveolar lavage (BAL) fluid and lung tissues were slightly increased by about twofold. However, the structures of pulmonary epithelial cells were kept within normal limits. Next, we examined the effect of cold stress on the inflammatory responses in a lipopolysaccharide (LPS)-induced acute lung injury (ALI) mouse model. The infiltration of neutrophils and inflammation of lung tissue determined by histology were significantly increased by exposure to ambient cold temperature. In addition, the production of pro-inflammatory cytokines including interleukin (IL)-12, IL-17, and monokine induced by gamma interferon (MIG) was elevated by exposure to cold stress. Therefore, we suggest that cold stress is a factor that exacerbates lung inflammation including ALI. To our knowledge, this is the first report on the relationship between cold stress and severity of lung inflammation.

  13. The Fas/FasL pathway impairs the alveolar fluid clearance in mouse lungs

    PubMed Central

    Herrero, Raquel; Tanino, Mishie; Smith, Lincoln S.; Kajikawa, Osamu; Wong, Venus A.; Mongovin, Steve; Matute-Bello, Gustavo

    2013-01-01

    Alveolar epithelial damage is a critical event that leads to protein-rich edema in acute lung injury (ALI), but the mechanisms leading to epithelial damage are not completely understood. Cell death by necrosis and apoptosis occurs in alveolar epithelial cells in the lungs of patients with ALI. Fas activation induces apoptosis of alveolar epithelial cells, but its role in the formation of lung edema is unclear. The main goal of this study was to determine whether activation of the Fas/Fas ligand pathway in the lungs could alter the function of the lung epithelium, and the mechanisms involved. The results show that Fas activation alters the alveolar barrier integrity and impairs the ability of the lung alveolar epithelium to reabsorb fluid from the air spaces. This result was dependent on the presence of a normal Fas receptor and was not affected by inflammation induced by Fas activation. Alteration of the fluid transport properties of the alveolar epithelium was partially restored by β-adrenergic stimulation. Fas activation also caused apoptosis of alveolar endothelial cells, but this effect was less pronounced than the effect on the alveolar epithelium. Thus, activation of the Fas pathway impairs alveolar epithelial function in mouse lungs by mechanisms involving caspase-dependent apoptosis, suggesting that targeting apoptotic pathways could reduce the formation of lung edema in ALI. PMID:23812636

  14. Significance of Stat3 Signaling in Epithelial Cell Differentiation of Fetal Mouse Lungs

    PubMed Central

    Kameyama, Hiroki; Kudoh, Shinji; Hatakeyama, Jun; Matuo, Akira; Ito, Takaaki

    2017-01-01

    To study the significance of signal transducer and activator of transcription (Stat) 3 in lung epithelial development of fetal mice, we examined fetal mouse lungs, focusing on the expression of Clara cell secretory protein (CCSP), Forkhead box protein J1 (Foxj1), calcitonin gene-related peptide (CGRP), phosphorylated Stat3 (Tyr705), and hairy/enhancer of split (Hes) 1, and observed cultured fetal lungs upon treatment with IL-6, a Stat3 activator, or cucurbitacin I, a Stat3 inhibitor. Moreover, the interaction of Stat3 signaling and Hes1 was studied using Hes1 gene-deficient mice. Phosphorylated Stat3 was detected in fetal lungs and, immunohistochemically, phosphorylated Stat3 was found to be co-localized in developing Clara cells, but not in ciliated cells. In the organ culture studies, upon treatment with IL-6, quantitative RT-PCR revealed that CCSP mRNA increased with increasing Stat3 phosphorylation, while cucurbitacin I decreased Hes1, CCSP, Foxj1 and CGRP mRNAs with decreasing Stat3 phosphorylation. In the lungs of Hes1 gene-deficient mice, Stat3 phosphorylation was not markedly different from wild-type mice, the expression of CCSP and CGRP was enhanced, and the treatment of IL-6 or cucurbitacin I induced similar effects on mouse lung epithelial differentiation regardless of Hes1 expression status. Stat3 signaling acts in fetal mouse lung development, and seems to regulate Clara cell differentiation positively. Hes1 could regulate Clara cell differentiation in a manner independent from Stat3 signaling. PMID:28386145

  15. Radiation-enhanced Lung Cancer Progression in a Transgenic Mouse Model of Lung Cancer is Predictive of Outcomes in Human Lung and Breast Cancer

    PubMed Central

    Delgado, Oliver; Batten, Kimberly G.; Richardson, James A.; Xie, Xian-Jin; Gazdar, Adi F.; Kaisani, Aadil A.; Girard, Luc; Behrens, Carmen; Suraokar, Milind; Fasciani, Gail; Wright, Woodring E.; Story, Michael D.; Wistuba, Ignacio I.; Minna, John D.; Shay, Jerry W.

    2014-01-01

    Purpose Carcinogenesis is an adaptive process between nascent tumor cells and their microenvironment including the modification of inflammatory responses from anti-tumorigenic to pro-tumorigenic. Radiation exposure can stimulate inflammatory responses that inhibit or promote carcinogenesis. The purpose of this study is to determine the impact of radiation exposure on lung cancer progression in vivo and assess the relevance of this knowledge to human carcinogenesis. Experimental Design K-rasLA1 mice were irradiated with various doses and dose regimens and then monitored till death. Microarray analyses were performed using Illumina® BeadChips on whole lung tissue 70 days post-irradiation with a fractionated or acute dose of radiation and compared to age-matched unirradiated controls. Unique group classifiers were derived by comparative genomic analysis of three experimental cohorts. Survival analyses were performed using principal component analysis and k-means clustering on three lung adenocarcinoma, three breast adenocarcinoma, and two lung squamous carcinoma annotated microarray datasets. Results Radiation exposure accelerates lung cancer progression in the K-rasLA1 lung cancer mouse model with dose fractionation being more permissive for cancer progression. A non-random inflammatory signature associated with this progression was elicited from whole lung tissue containing only benign lesions and predicts human lung and breast cancer patient survival across multiple datasets. Immunohistochemical analyses suggest that tumor cells drive predictive signature. Conclusions These results demonstrate that radiation exposure can cooperate with benign lesions in a transgenic model of cancer by impacting inflammatory pathways, and that clinically relevant similarities exist between human lung and breast carcinogenesis. PMID:24486591

  16. Effects of microcystin-LR on mouse lungs.

    PubMed

    Soares, Raquel M; Cagido, Viviane R; Ferraro, Rodrigo B; Meyer-Fernandes, José Roberto; Rocco, Patrícia R M; Zin, Walter A; Azevedo, Sandra M F O

    2007-09-01

    Toxic cyanobacteria blooms in drinking water supplies have been an increasing public health concern all over the world. Human populations can be exposed to microcystins, an important family of cyanotoxins, mainly by oral ingestion. However, inhalation from recreational water and hemodialysis can represent other routes. This study investigated changes in respiratory mechanics, histology, protein phosphatase (PP) 1 and 2A activity and microcystin in lung of adult mice injected intraperitoneally (i.p.) with microcystin-LR. Thirty-six mice were divided into control (CTRL) and test (CYANO) groups. CTRL group received an i.p. injection of saline and the CYANO group received 40 microg MCYST-LR/kg i.p. After 2 and 8 h, and 1, 2 and 4 days after toxin injection, six mice from each group were sampled for analyses. Resistive and viscoelastic pressures, static and dynamic elastances augmented at 2 h in CYANO and so remained until day 4. Alveolar collapse and inflammatory cell infiltration were found 2h after the injection, reaching peak values at 8 h. However, no microcystin or inhibition of PPases could be detected in mice lungs. In conclusion, MCYST-LR led to a rapid increase in lung impedance and an inflammatory response with interstitial edema and inflammatory cell recruitment in mice.

  17. Alterations of mouse lung tissue dimensions during processing for morphometry: a comparison of methods.

    PubMed

    Schneider, Jan Philipp; Ochs, Matthias

    2014-02-15

    Preservation of original tissue dimensions is an essential prerequisite for morphometric studies. Shrinkage occurring during tissue processing for histology may severely influence the appearance of structures seen under the microscope and stereological calculations. Therefore, shrinkage has to be avoided so that estimates obtained by application of unbiased stereology are indeed unbiased. The present study investigates the alterations of tissue dimensions of mouse lung samples during processing for histology. Different fixatives as well as embedding protocols are considered. Mouse lungs were fixed by instillation of either 4% formalin or a mixture of 1.5% glutaraldehyde/1.5% formaldehyde. Tissue blocks were sampled according to principles of stereology for embedding in paraffin, glycol methacrylate without treatment with osmium tetroxide and uranyl acetate, and glycol methacrylate including treatment with osmium tetroxide and uranyl acetate before dehydration. Shrinkage was investigated by stereological measurements of dimensional changes of tissue cut faces. Results show a shrinkage of the cut face areas of roughly 40% per lung during paraffin embedding, 30% during "simple" glycol methacrylate embedding, and <3% during osmium tetroxide/uranyl acetate/glycol methacrylate embedding. Furthermore, the superiority of the glutaraldehyde-containing fixative regarding shrinkage is demonstrated. In conclusion, the use of a glutaraldehyde-containing fixative and embedding in glycol methacrylate with previous treatment of the samples with osmium tetroxide and uranyl acetate before dehydration is recommended for stereological studies of the mouse lung.

  18. Lung arginase expression and activity is increased in cystic fibrosis mouse models.

    PubMed

    Jaecklin, Thomas; Duerr, Julia; Huang, Hailu; Rafii, Mahroukh; Bear, Christine E; Ratjen, Felix; Pencharz, Paul; Kavanagh, Brian P; Mall, Marcus A; Grasemann, Hartmut

    2014-08-01

    The activity of arginase is increased in airway secretions of patients with cystic fibrosis (CF). Downstream products of arginase activity may contribute to CF lung disease. We hypothesized that pulmonary arginase expression and activity would be increased in mouse models of CF and disproportionally increased in CF mice with Pseudomonas aeruginosa pneumonia. Expression of arginase isoforms in lung tissue was quantified with reverse transcriptase-PCR in naive cystic fibrosis transmembrane conductance regulator (Cftr)-deficient mice and β-epithelial sodium channel-overexpressing [β-ENaC-transgenic (Tg)] mice. An isolated lung stable isotope perfusion model was used to measure arginase activity in Cftr-deficient mice before and after intratracheal instillation of Pseudomonas aeruginosa. The expression of arginase-2 in lung was increased in adult Cftr-deficient animals and in newborn β-ENaC-Tg. Arginase-1 lung expression was normal in Cftr-deficient and in newborn β-ENaC-Tg mice, but was increased in β-ENaC-Tg mice at age 1, 3, and 6 wk. Arginase activity was significantly higher in lung (5.0 ± 0.7 vs. 3.2 ± 0.3 nmol·(-1)·h(-1), P = 0.016) and airways (204.6 ± 49.8 vs. 79.3 ± 17.2 nmol·(-1)·h(-1), P = 0.045) of naive Cftr-deficient mice compared with sex-matched wild-type littermate controls. Infection with Pseudomonas aeruginosa resulted in a far greater increase in lung arginase activity in Cftr-deficient mice (10-fold) than in wild-type controls (6-fold) (P = 0.01). This is the first ex vivo characterization of arginase expression and activity in CF mouse lung and airways. Our data show that pulmonary arginase expression and activity is increased in CF mice, especially with Pseudomonas aeruginosa infections.

  19. Ablating all three retinoblastoma family members in mouse lung leads to neuroendocrine tumor formation

    PubMed Central

    Lázaro, Sara; Pérez-Crespo, Miriam; Enguita, Ana Belén; Hernández, Pilar; Martínez-Palacio, Jesús; Oteo, Marta; Sage, Julien; Paramio, Jesús M.; Santos, Mirentxu

    2017-01-01

    Lung cancer is a deadly disease with increasing cases diagnosed worldwide and still a very poor prognosis. While mutations in the retinoblastoma (RB1) tumor suppressor have been reported in lung cancer, mainly in small cell lung carcinoma, the tumor suppressive role of its relatives p107 and p130 is still a matter of debate. To begin to investigate the role of these two Rb family proteins in lung tumorigenesis, we have generated a conditional triple knockout mouse model (TKO) in which the three Rb family members can be inactivated in adult mice. We found that ablation of all three family members in the lung of mice induces tumorlets, benign neuroendocrine tumors that are remarkably similar to their human counterparts. Upon chemical carcinogenesis, DHPN and urethane accelerate tumor development; the TKO model displays increased sensitivity to DHPN, and urethane increases malignancy of tumors. All the tumors developing in TKO mice (spontaneous and chemically induced) have neuroendocrine features but do not progress to fully malignant tumors. Thus, loss of Rb and its family members confers partial tumor susceptibility in neuroendocrine lineages in the lungs of mice. Our data also imply the requirement of other oncogenic signaling pathways to achieve full transformation in neuroendocrine lung lesions mutant for the Rb family. PMID:27966456

  20. Identification of candidate lung cancer susceptibility genes in mouse using oligonucleotide arrays

    PubMed Central

    Lemon, W; Bernert, H; Sun, H; Wang, Y; You, M

    2002-01-01

    We applied microarray gene expression profiling to lungs from mouse strains having variable susceptibility to lung tumour development as a means to identify, within known quantitative trait loci (QTLs), candidate genes responsible for susceptibility or resistance to lung cancer. At least eight chromosomal regions of mice have been mapped and verified to be linked with lung tumour susceptibility or resistance. In this study, high density oligonucleotide arrays were used to measure the relative expression levels of >36 000 genes and ESTs in lung tissues of A/J, BALB/cJ, SM/J, C3H/HeJ, and C57BL/6J mice. A number of differentially expressed genes were found in each of the lung cancer susceptibility QTLs. Bioinformatic analysis of the differentially expressed genes located within QTLs produced 28 susceptibility candidates and 22 resistance candidates. These candidates may be extremely helpful in the ultimate identification of the precise genes responsible for lung tumour susceptibility or resistance in mice and, through follow up, humans. Complete data sets are available at http://thinker.med.ohio-state.edu. PMID:12205107

  1. Novel Mouse Model of Chronic Pseudomonas aeruginosa Lung Infection Mimicking Cystic Fibrosis

    PubMed Central

    Hoffmann, Nadine; Rasmussen, Thomas Bovbjerg; Jensen, PeterØstrup; Stub, Charlotte; Hentzer, Morten; Molin, Søren; Ciofu, Oana; Givskov, Michael; Johansen, Helle Krogh; Høiby, Niels

    2005-01-01

    Pseudomonas aeruginosa causes a chronic infection in the lungs of cystic fibrosis (CF) patients by establishing an alginate-containing biofilm. The infection has been studied in several animal models; however, most of the models required artificial embedding of the bacteria. We present here a new pulmonary mouse model without artificial embedding. The model is based on a stable mucoid CF sputum isolate (NH57388A) with hyperproduction of alginate due to a deletion in mucA and functional N-acylhomoserine lactone (AHL)-based quorum-sensing systems. Chronic lung infection could be established in both CF mice (CftrtmlUnc−/−) and BALB/c mice, as reflected by the detection of a high number of P. aeruginosa organisms in the lung homogenates at 7 days postinfection and alginate biofilms, surrounded by polymorphonuclear leukocytes in the alveoli. In comparison, both an AHL-producing nonmucoid revertant (NH57388C) from the mucoid isolate (NH57388A) and a nonmucoid isolate (NH57388B) deficient in AHL were almost cleared from the lungs of the mice. This model, in which P. aeruginosa is protected against the defense system of the lung by alginate, is similar to the clinical situation. Therefore, the mouse model provides an improved method for evaluating the interaction between mucoid P. aeruginosa, the host, and antibacterial therapy. PMID:15784597

  2. Regulation of mouse lung development by the extracellular calcium-sensing receptor, CaR.

    PubMed

    Finney, Brenda A; del Moral, Pierre M; Wilkinson, William J; Cayzac, Sebastien; Cole, Martin; Warburton, David; Kemp, Paul J; Riccardi, Daniela

    2008-12-15

    Postnatal lung function is critically dependent upon optimal embryonic lung development. As the free ionized plasma calcium concentration ([Ca(2+)](o)) of the fetus is higher than that of the adult, the process of lung development occurs in a hypercalcaemic environment. In the adult, [Ca(2+)](o) is monitored by the G-protein coupled, extracellular calcium-sensing receptor (CaR), but neither its ontogeny nor its potential role in lung development are known. Here, we demonstrate that CaR is expressed in the mouse lung epithelium, and that its expression is developmentally regulated, with a peak of expression at embryonic day 12.5 (E12.5) and a subsequent decrease by E18, after which the receptor is absent. Experiments carried out using the lung explant culture model in vitro show that lung branching morphogenesis is sensitive to [Ca(2+)](o), being maximal at physiological adult [Ca(2+)](o) (i.e. 1.0-1.3 mM) and lowest at the higher, fetal (i.e. 1.7 mM) [Ca(2+)](o). Administration of the specific CaR positive allosteric modulator, the calcimimetic R-568, mimics the suppressive effects of high [Ca(2+)](o) on branching morphogenesis while both phospholipase C and PI3 kinase inhibition reverse these effects. CaR activation suppresses cell proliferation while it enhances intracellular calcium signalling, lung distension and fluid secretion. Conditions which are restrictive either to branching or to secretion can be rescued by manipulating [Ca(2+)](o) in the culture medium. In conclusion, fetal Ca(2+)(o), acting through a developmentally regulated CaR, is an important extrinsic factor that modulates the intrinsic lung developmental programme. Our observations support a novel role for the CaR in preventing hyperplastic lung disease in utero.

  3. Quantification of Lung Metastases from In Vivo Mouse Models.

    PubMed

    Chang, Joan; Erler, Janine T

    2016-01-01

    Cancer research has made significant progress in terms of understanding and targeting primary tumors; however, the challenge remains for the successful treatment of metastatic cancers. This highlights the importance to use in vivo models to study the metastatic process, as well as for preclinical testing of compounds that could inhibit metastasis. As a result, proper quantification of metastases from in vivo models is of the utmost significance. Here, we provide a detailed protocol for collecting and handling lung tissues from mice, and guidance for subsequent analysis of metastases, as well as interpretation of data.

  4. AKT1E17K Is Oncogenic in Mouse Lung and Cooperates with Chemical Carcinogens in Inducing Lung Cancer

    PubMed Central

    Malanga, Donatella; Belmonte, Stefania; Colelli, Fabiana; Scarfò, Marzia; De Marco, Carmela; Oliveira, Duarte Mendes; Mirante, Teresa; Camastra, Caterina; Gagliardi, Monica; Rizzuto, Antonia; Mignogna, Chiara; Paciello, Orlando; Papparella, Serenella; Fagman, Henrik; Viglietto, Giuseppe

    2016-01-01

    The hotspot AKT1E17K mutation in the pleckstrin homology domain of AKT1 occurs in approximately 0.6–2% of human lung cancers. Recently, we have demonstrated that AKT1E17K transforms immortalized human bronchial cells. Here by use of a transgenic Cre-inducible murine strain in the wild type Rosa26 (R26) locus (R26-AKT1E17K mice) we demonstrate that AKT1E17K is a bona-fide oncogene and plays a role in the development of lung cancer in vivo. In fact, we report that mutant AKT1E17K induces bronchial and/or bronchiolar hyperplastic lesions in murine lung epithelium, which progress to frank carcinoma at very low frequency, and accelerates tumor formation induced by chemical carcinogens. In conclusion, AKT1E17K induces hyperplasia of mouse lung epithelium in vivo and cooperates with urethane to induce the fully malignant phenotype. PMID:26859676

  5. Ozone-related fluorescent compounds in mouse liver and lung

    SciTech Connect

    Csallany, A.S.; Manwaring, J.D.; Menken, B.Z.

    1985-08-01

    Groups of ten female, weanling mice were fed a basal, vitamin E-deficient diet or a basal diet supplemented with RRR-alpha-tocopheryl acetate for 14 months. During the last month one group from each dietary regimen was exposed for 30-60 min/day to 1.5 ppm ozone (25 hr total ozone exposure) and the remaining groups to control ambient air. The liver and lung tissues were homogenized and extracted with 2:1 chloroform:methanol and water. Excitation and emission wavelengths for the eluting fractions were determined by continuous emission scans from 250 to 600 nm for each excitation wavelength between 250 and 500 nm. Ozone exposure did not effect the concentration of any of the fluorescent materials examined in the lung, but it resulted in a significant increase in two of four water-soluble compounds in the liver with excitation wavelength maxima/emission wavelength maxima of 270 nm/310 nm and 275 nm/350 nm (smaller molecular weight material) suggesting in vivo lipid oxidation.

  6. Circadian disruption alters mouse lung clock gene expression and lung mechanics.

    PubMed

    Hadden, Hélène; Soldin, Steven J; Massaro, Donald

    2012-08-01

    Most aspects of human physiology and behavior exhibit 24-h rhythms driven by a master circadian clock in the brain, which synchronizes peripheral clocks. Lung function and ventilation are subject to circadian regulation and exhibit circadian oscillations. Sleep disruption, which causes circadian disruption, is common in those with chronic lung disease, and in the general population; however, little is known about the effect on the lung of circadian disruption. We tested the hypothesis circadian disruption alters expression of clock genes in the lung and that this is associated with altered lung mechanics. Female and male mice were maintained on a 12:12-h light/dark cycle (control) or exposed for 4 wk to a shifting light regimen mimicking chronic jet lag (CJL). Airway resistance (Rn), tissue damping (G), and tissue elastance (H) did not differ between control and CJL females. Rn at positive end-expiratory pressure (PEEP) of 2 and 3 cmH(2)O was lower in CJL males compared with controls. G, H, and G/H did not differ between CJL and control males. Among CJL females, expression of clock genes, Bmal1 and Rev-erb alpha, was decreased; expression of their repressors, Per2 and Cry 2, was increased. Among CJL males, expression of Clock was decreased; Per 2 and Rev-erb alpha expression was increased. We conclude circadian disruption alters lung mechanics and clock gene expression and does so in a sexually dimorphic manner.

  7. Early recognition of lung cancer by integrin targeted imaging in K-ras mouse model.

    PubMed

    Ermolayev, Vladimir; Mohajerani, Pouyan; Ale, Angelique; Sarantopoulos, Athanasios; Aichler, Michaela; Kayser, Gian; Walch, Axel; Ntziachristos, Vasilis

    2015-09-01

    Non-small cell lung cancer is characterized by slow progression and high heterogeneity of tumors. Integrins play an important role in lung cancer development and metastasis and were suggested as a tumor marker; however their role in anticancer therapy remains controversial. In this work, we demonstrate the potential of integrin-targeted imaging to recognize early lesions in transgenic mouse model of lung cancer based on spontaneous introduction of mutated human gene bearing K-ras mutation. We conducted ex vivo and fluorescence molecular tomography-X-ray computed tomography (FMT-XCT) in vivo imaging and analysis for specific targeting of early lung lesions and tumors in rodent preclinical model for lung cancer. The lesions and tumors were characterized by histology, immunofluorescence and immunohistochemistry using a panel of cancer markers. Ex vivo, the integrin-targeted fluorescent signal significantly differed between wild type lung tissue and K-ras pulmonary lesions (PL) at all ages studied. The panel of immunofluorescence experiments demonstrated that PL, which only partially show cancer cell features were detected by αvβ3-integrin targeted imaging. Human patient material analysis confirmed the specificity of target localization in different lung cancer types. Most importantly, small tumors in the lungs of 4-week-old animals could be noninvasively detected in vivo on the fluorescence channel of FMT-XCT. Our findings demonstrated αvβ3-integrin targeted fluorescent imaging to specifically detect premalignant pleural lesions in K-ras mice. Integrin targeted imaging may find application areas in preclinical research and clinical practice, such as early lung cancer diagnostics, intraoperative assistance or therapy monitoring.

  8. Scale dependence of structure-function relationship in the emphysematous mouse lung

    PubMed Central

    Sato, Susumu; Bartolák-Suki, Erzsébet; Parameswaran, Harikrishnan; Hamakawa, Hiroshi; Suki, Béla

    2015-01-01

    The purpose of this study was to determine how the initial distribution of elastase in mouse lungs determines the time course of tissue destruction and how structural heterogeneity at different spatial scales influences lung function. We evaluated lung function and alveolar structure in normal and emphysematous C57BL/6 mice at 2 and 21 days following orotracheal treatment with porcine pancreatic elastase (PPE). Initial distribution of elastase 1 h after treatment was assessed using red fluorescently labeled PPE (f-PPE) by laser scanning confocal microscopy. From measured input impedance of the respiratory system, the global lung compliance, and the variability of regional compliance were obtained. Lungs were fixed and equivalent airspace diameters were measured in four lobes of the right lung and three regions of the left lung. At day 2 and day 21, the mean airspace diameter of each region was significantly enlarged which was accompanied by an increased inter-regional heterogeneity. The deposition of f-PPE on day 0 was much more heterogeneous than the inter-regional diameters at both day 2 and day 21 and, at day 21, this reached statistical significance (p < 0.05). Microscale heterogeneity characterized by the overall variability of airspace diameters correlated significantly better with compliance than macroscale or inter-regional heterogeneity. Furthermore, while the spatial distribution of the inflammatory response does not seem to follow that of the elastase deposition, it correlates with the strongest regional determinant of lung function. These results may help interpret lung function decline in terms of structural deterioration in human patients with emphysema. PMID:26029115

  9. Scale dependence of structure-function relationship in the emphysematous mouse lung.

    PubMed

    Sato, Susumu; Bartolák-Suki, Erzsébet; Parameswaran, Harikrishnan; Hamakawa, Hiroshi; Suki, Béla

    2015-01-01

    The purpose of this study was to determine how the initial distribution of elastase in mouse lungs determines the time course of tissue destruction and how structural heterogeneity at different spatial scales influences lung function. We evaluated lung function and alveolar structure in normal and emphysematous C57BL/6 mice at 2 and 21 days following orotracheal treatment with porcine pancreatic elastase (PPE). Initial distribution of elastase 1 h after treatment was assessed using red fluorescently labeled PPE (f-PPE) by laser scanning confocal microscopy. From measured input impedance of the respiratory system, the global lung compliance, and the variability of regional compliance were obtained. Lungs were fixed and equivalent airspace diameters were measured in four lobes of the right lung and three regions of the left lung. At day 2 and day 21, the mean airspace diameter of each region was significantly enlarged which was accompanied by an increased inter-regional heterogeneity. The deposition of f-PPE on day 0 was much more heterogeneous than the inter-regional diameters at both day 2 and day 21 and, at day 21, this reached statistical significance (p < 0.05). Microscale heterogeneity characterized by the overall variability of airspace diameters correlated significantly better with compliance than macroscale or inter-regional heterogeneity. Furthermore, while the spatial distribution of the inflammatory response does not seem to follow that of the elastase deposition, it correlates with the strongest regional determinant of lung function. These results may help interpret lung function decline in terms of structural deterioration in human patients with emphysema.

  10. The Nicotinic Receptor Alpha7 Impacts the Mouse Lung Response to LPS through Multiple Mechanisms

    PubMed Central

    Enioutina, Elena Y.; Myers, Elizabeth J.; Tvrdik, Petr; Hoidal, John R.; Rogers, Scott W.; Gahring, Lorise C.

    2015-01-01

    The nicotinic acetylcholine receptor alpha7 (α7) is expressed by neuronal and non-neuronal cells throughout the body. We examined the mechanisms of the lung inflammatory response to intranasal (i.n.) lipopolysaccharide (LPS) regulated by α7. This was done in mice using homologous recombination to introduce a point mutation in the α7 receptor that replaces the glutamate residue 260 that lines the pore with alanine (α7E260A), which has been implicated in controlling the exceptional calcium ion conductance of this receptor. The α7E260A mice exhibit normal inflammatory cell recruitment to the blood in response to i.n. LPS administration. This differs from the α7knock-out (α7KO) in which upstream signaling to initiate the recruitment to the blood following i.n. LPS is significantly impaired. While hematopoietic cells are recruited to the bloodstream in the α7E260A mouse, they fail to be recruited efficiently into both the interstitium and alveolar spaces of the lung. Bone marrow reconstitution experiments demonstrate that the responsiveness of both CD45+ and CD45- cells of the α7E260A mouse are impaired. The expression of several pro-inflammatory cytokine and chemokine RNAs including TNFα, IL-1α, Ccl2 and Cxcl10 are decreased in the α7E260A mouse. However, there is a substantial increase in IL-13 expression by CD45- lung interstitial cells in the α7E260A mouse. Our results support the conclusion that α7 functional pleiotropy contributes to modulating the tissue response to an inflammatory insult through impacting upon a variety of mechanisms reflecting the individual cell composition of the lung. PMID:25803612

  11. [Distribution of compact bone mesenchymal stem cells in lung tissue and bone marrow of mouse].

    PubMed

    Wang, Rui-Ping; Wu, Ren-Na; Guo, Yu-Qing; Zhang, Bin; Chen, Hu

    2014-02-01

    This study was aimed to investigate the distribution of compact bone mesenchymal stem cells(MSC) marked with lentiviral plasmid pGC FU-RFP-LV in lung tissue and bone marrow of mouse. The MSC were infected by lentivirus with infection efficiency 78%, the infected MSC were injected into BALB/c mice via tail veins in concentration of 1×10(6) /mouse. The mice were randomly divided into 4 group according to 4 time points as 1, 2, 5 and 7 days. The lung tissue and bone marrow were taken and made of frozen sections and smears respectively in order to observed the distributions of MSC. The results indicated that the lentiviral infected MSC displayed phenotypes and biological characteristics which conformed to MSC by immunophenotyping analysis and induction differentiation detection. After the MSC were infected with optimal viral titer MOI = 50, the cell growth no significantly changed; the fluorescent microscopy revealed that the distributions of MSC in bone marrow on day 1, 2, 5 and 7 were 0.50 ± 0.20, 0.67 ± 0.23, 0.53 ± 0.14, 0.33 ± 0.16; those in lung tissue were 0.55 ± 0.15, 0.47 ± 0.13, 0.29 ± 0.13, 0.26 ± 0.08. It is concluded that the distribution of MSC in lung tissue reaches a peak on day 1, while distribution of MSC in bone marrow reaches a peak on day 2. The distribution of mouse MSC relates with RFP gene expression and implantation of MSC in lung tissue and bone marrow.

  12. Susceptibility to quantum dot induced lung inflammation differs widely among the Collaborative Cross founder mouse strains

    PubMed Central

    Scoville, David K.; White, Collin C.; Botta, Dianne; McConnachie, Lisa A.; Zadworny, Megan E.; Schmuck, Stefanie C.; Hu, Xiaoge; Gao, Xiaohu; Yu, Jianbo; Dills, Russell L.; Sheppard, Lianne; Delaney, Martha A.; Griffith, William C.; Beyer, Richard P.; Zangar, Richard C.; Pounds, Joel G.; Faustman, Elaine M.; Kavanagh, Terrance J.

    2015-01-01

    Quantum dots (QDs) are engineered semiconductor nanoparticles with unique physicochemical properties that make them potentially useful in clinical, research and industrial settings. However, a growing body of evidence indicates that like other engineered nanomaterials, QDs have the potential to be respiratory hazards, especially in the context of the manufacture of QDs and products containing them, as well as exposures to consumers using these products. The overall goal of this study was to investigate the role of mouse strain in determining susceptibility to QD-induced pulmonary inflammation and toxicity. Male mice from 8 genetically diverse inbred strains (the Collaborative Cross founder strains) were exposed to CdSe–ZnS core–shell QDs stabilized with an amphiphilic polymer. QD treatment resulted in significant increases in the percentage of neutrophils and levels of cytokines present in bronchoalveolar lavage fluid (BALF) obtained from NOD/ShiLtJ and NZO/HlLtJ mice relative to their saline (Sal) treated controls. Cadmium measurements in lung tissue indicated strain-dependent differences in disposition of QDs in the lung. Total glutathione levels in lung tissue were significantly correlated with percent neutrophils in BALF as well as with lung tissue Cd levels. Our findings indicate that QD-induced acute lung inflammation is mouse strain dependent, that it is heritable, and that the choice of mouse strain is an important consideration in planning QD toxicity studies. These data also suggest that formal genetic analyses using additional strains or recombinant inbred strains from these mice could be useful for discovering potential QD-induced inflammation susceptibility loci. PMID:26476918

  13. Differential responses in the lungs of newborn mouse pups exposed to 85% or >95% oxygen.

    PubMed

    Rogers, Lynette K; Tipple, Trent E; Nelin, Leif D; Welty, Stephen E

    2009-01-01

    Premature infants often develop serious clinical complications associated with respiratory failure and hyperoxic lung injury that includes lung inflammation and alterations in lung development. The goal of these studies is to test the hypothesis that there are differences in the course of lung injury in newborn mice exposed to 85% or >95% oxygen that provide models to address the differential effects of oxidation and inflammation. Our results indicate differences between the 85% and >95% O2 exposure groups by day 14 in weight gain and lung alveolarization. Inflammation, assessed by neutrophil counts, was observed in both hyperoxia groups by day 3 but was dramatically greater in the >95% O2-exposed groups by day 14 and associated with greater developmental deficits. Cytoplasmic phospholipase A2, cyclooxygenase-2, and 5-lipoxygenase levels were elevated but no patterns of differences were observed between exposure groups. Prostaglandins D2, E2, and F2alpha were increased in the tissues from mouse pups exposed to >95% O2 at 7 d indicating a differential expression of cyclooxygenase-2 products. Our data indicate that there are differences in the models of 85% or >95% O2 exposure and these differences may provide mechanistic insights into hyperoxic lung injury in an immature system.

  14. Two Nested Developmental Waves Demarcate a Compartment Boundary in the Mouse Lung

    PubMed Central

    Alanis, Denise Martinez; Chang, Daniel R.; Akiyama, Haruhiko; Krasnow, Mark A.; Chen, Jichao

    2014-01-01

    The lung is a branched tubular network with two distinct compartments — the proximal conducting airways and the peripheral gas exchange region — separated by a discrete boundary termed the bronchoalveolar duct junction (BADJ). Here we image the developing mouse lung in three dimensions and show that two nested developmental waves demarcate the BADJ under the control of a global hormonal signal. A first wave of branching morphogenesis progresses throughout embryonic development, generating branches for both compartments. A second wave of conducting airway differentiation follows the first wave but terminates earlier, specifying the proximal compartment and setting the BADJ. The second wave is terminated by a glucocorticoid signaling: premature activation or loss of glucocorticoid signaling causes a proximal or distal shift, respectively, in BADJ location. The results demonstrate a novel mechanism of boundary formation in complex, three-dimensional organs and provide new insights into glucocorticoid therapies for lung defects in premature birth. PMID:24879355

  15. GATA2 is epigenetically repressed in human and mouse lung tumors and is not requisite for survival of KRAS mutant lung cancer

    PubMed Central

    Tessema, Mathewos; Yingling, Christin M.; Snider, Amanda M.; Do, Kieu; Juri, Daniel E.; Picchi, Maria A.; Zhang, Xiequn; Liu, Yushi; Leng, Shuguang; Tellez, Carmen S.; Belinsky, Steven A.

    2014-01-01

    Introduction GATA2 was recently described as a critical survival factor and therapeutic target for KRAS mutant non-small cell lung cancer (NSCLC). However, whether this role is affected by epigenetic repression of GATA2 in lung cancer is unclear. Methods GATA2 expression and promoter CpG island methylation were evaluated using human and mouse NSCLC cell lines and tumor-normal pairs. In vitro assays were used to study GATA2 repression on cell survival and during tobacco carcinogen-induced transformation. Results GATA2 expression in KRAS wild-type (n=15) and mutant (n=10) NSCLC cell lines and primary lung tumors (n=24) was significantly lower, 1.3–33.6-fold (p=2.2×10−9), compared to corresponding normal lung. GATA2 promoter was unmethylated in normal lung (0/10) but frequently methylated in lung tumors (96%, 159/165) and NSCLC cell lines (97%, 30/31). This highly prevalent aberrant methylation was independently validated using TCGA data for 369 NSCLC tumor-normal pairs. In vitro studies using an established carcinogen-induced pre-malignancy model revealed that GATA2 expression was initially repressed by chromatin remodeling followed by cytosine methylation during transformation. Similarly, expression of Gata2 in NNK-induced mouse lung tumors (n=6) and cell lines (n=5) was 5-fold and 100-fold lower, respectively, than normal mouse lung. Finally, siRNA-mediated knockdown of GATA2 in KRAS mutant [human (n=4) and murine (n=5)] and wild-type [human (n=4)] NSCLC cell lines showed that further reduction of expression (up to 95%) does not induce cell death. Conclusion GATA2 is epigenetically repressed in human and mouse lung tumors and its further inhibition is not a valid therapeutic strategy for KRAS mutant lung cancer. PMID:24807155

  16. CYCLOPENTA-FUSED POLYCYCLIC AROMATIC HYDROCARBONS IN STRAIN A/J MOUSE LUNG: DNA ADDUCTS, ONCOGENE MUTATIONS, & TUMORIGENESIS

    EPA Science Inventory

    Cyclopenta-fused Polycyclic Aromatic Hydrocarbons in Strain AJJ Mouse Lung: DNA Adducts, Oncogene Mutations, and Tumorigenesis.

    We have examined the relationships between DNA adducts, Ki-ras oncogene mutations, DNA adducts, and adenoma induction in the lungs of strain A/J...

  17. CYCLOPENTA-FUSED POLYCYCLIC AROMATIC HYDROCARBONS IN STRAIN A/J MOUSE LUNG: DNA ADDUCTS, ONCOGENE MUTATIONS, & TUMORIGENESIS

    EPA Science Inventory

    Cyclopenta-fused Polycyclic Aromatic Hydrocarbons in Strain AJJ Mouse Lung: DNA Adducts, Oncogene Mutations, and Tumorigenesis.

    We have examined the relationships between DNA adducts, Ki-ras oncogene mutations, DNA adducts, and adenoma induction in the lungs of strain A/J...

  18. Effect of urethane, dimethylnitrosamine, paraquat, and butylated hydroxytoluene on the activities of glycolytic key enzymes in mouse lung

    SciTech Connect

    Arany, I.; Rady, P.; Bojan, I.; Kertai, P.

    1981-12-01

    Effects of carcinogens and noncarcinogenic pulmonary toxicants on the activities of glycolytic key enzymes in the mouse lung were investigated. The carcinogens urethane (URTH) and dimethylnitrosamine (DMN) permanently enhanced, and the noncarcinogenic pulmonary toxicants paraquat (PAR) and butylated hydroxytoluene (BHT) temporarily, enhanced the activities of hexokinase (HK), phosphofructokinase (PFK), and pyruvate kinase (PK) in the lungs of mice.

  19. Mouse lung infection model to assess Rhodococcus equi virulence and vaccine protection.

    PubMed

    González-Iglesias, Patricia; Scortti, Mariela; MacArthur, Iain; Hapeshi, Alexia; Rodriguez, Héctor; Prescott, John F; Vazquez-Boland, José A

    2014-08-06

    The pathogenic actinomycete Rhodococcus equi causes severe purulent lung infections in foals and immunocompromised people. Although relatively unsusceptible to R. equi, mice are widely used for in vivo studies with this pathogen. The most commonly employed mouse model is based on systemic (intravenous) infection and determination of R. equi burdens in spleen and liver. Here, we investigated the murine lung for experimental infection studies with R. equi. Using a 10(7)CFU intranasal challenge in BALB/c mice, virulent R. equi consistently survived in quantifiable numbers up to 10 days in the lungs whereas virulence-deficient R. equi bacteria were rapidly cleared. An internally controlled virulence assay was developed in which the test R. equi strains are co-inoculated and monitored in the same mouse. Isogenic R. equi bacteria lacking either the plasmid vapA gene or the entire virulence plasmid were compared using this competitive assay. Both strains showed no significant differences in in vivo fitness in the lung, indicating that the single loss of the virulence factor VapA was sufficient to account for the full attenuation seen in the absence of the virulence plasmid. To test the adequacy of the lung infection model for monitoring R. equi vaccine efficacy, BALB/c mice were immunized with live R. equi and challenged intranasally. Vaccination conferred protection against acute pulmonary challenge with virulent R. equi. Our data indicate that the murine lung infection model provides a useful tool for both R. equi virulence and vaccine studies. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Glial Fibrillary Acidic Protein-Expressing Glia in the Mouse Lung

    PubMed Central

    Suarez-Mier, Gabriela B.

    2015-01-01

    Autonomic nerves regulate important functions in visceral organs, including the lung. The postganglionic portion of these nerves is ensheathed by glial cells known as non-myelinating Schwann cells. In the brain, glia play important functional roles in neurotransmission, neuroinflammation, and maintenance of the blood brain barrier. Similarly, enteric glia are now known to have analogous roles in gastrointestinal neurotransmission, inflammatory response, and barrier formation. In contrast to this, very little is known about the function of glia in other visceral organs. Like the gut, the lung forms a barrier between airborne pathogens and the bloodstream, and autonomic lung innervation is known to affect pulmonary inflammation and lung function. Lung glia are described as non-myelinating Schwann cells but their function is not known, and indeed no transgenic tools have been validated to study them in vivo. The primary goal of this research was, therefore, to investigate the relationship between non-myelinating Schwann cells and pulmonary nerves in the airways and vasculature and to validate existing transgenic mouse tools that would be useful for studying their function. We focused on the glial fibrillary acidic protein promoter, which is a cognate marker of astrocytes that is expressed by enteric glia and non-myelinating Schwann cells. We describe the morphology of non-myelinating Schwann cells in the lung and verify that they express glial fibrillary acidic protein and S100, a classic glial marker. Furthermore, we characterize the relationship of non-myelinating Schwann cells to pulmonary nerves. Finally, we report tools for studying their function, including a commercially available transgenic mouse line. PMID:26442852

  1. Preclinical evaluation of human secretoglobin 3A2 in mouse models of lung development and fibrosis

    PubMed Central

    Cai, Yan; Winn, Melissa E.; Zehmer, John K.; Gillette, William K.; Lubkowski, Jacek T.; Pilon, Aprile L.

    2013-01-01

    Secretoglobin (SCGB) 3A2 is a member of the SCGB gene superfamily of small secreted proteins, predominantly expressed in lung airways. We hypothesize that human SCGB3A2 may exhibit anti-inflammatory, growth factor, and antifibrotic activities and be of clinical utility. Recombinant human SCGB3A2 was expressed, purified, and biochemically characterized as a first step to its development as a therapeutic agent in clinical settings. Human SCGB3A2, as well as mouse SCGB3A2, readily formed a dimer in solution and exhibited novel phospholipase A2 inhibitory activity. This is the first demonstration of any quantitative biochemical measurement for the evaluation of SCGB3A2 protein. In the mouse as an experimental animal, human SCGB3A2 exhibited growth factor activity by promoting embryonic lung development in both ex vivo and in vivo systems and antifibrotic activity in the bleomycin-induced lung fibrosis model. The results suggested that human SCGB3A2 can function as a growth factor and an antifibrotic agent in humans. When SCGB3A2 was administered to pregnant female mice through the tail vein, the protein was detected in the dam's serum and lung, as well as the placenta, amniotic fluids, and embryonic lungs at 10 min postadministration, suggesting that SCGB3A2 readily crosses the placenta. The results warrant further development of recombinant SCGB3A2 as a therapeutic agent in treating patients suffering from lung diseases or preterm infants with respiratory distress. PMID:24213919

  2. Lung Pathologies in a Chronic Inflammation Mouse Model Are Independent of Eosinophil Degranulation.

    PubMed

    Jacobsen, Elizabeth A; Ochkur, Sergei I; Doyle, Alfred D; LeSuer, William E; Li, Wen; Protheroe, Cheryl A; Colbert, Dana; Zellner, Katie R; Shen, HuaHao H; Irvin, Charles G; Lee, James J; Lee, Nancy A

    2017-05-15

    The release of eosinophil granule proteins in the lungs of patients with asthma has been dogmatically linked with lung remodeling and airway hyperresponsiveness. However, the demonstrated inability of established mouse models to display the eosinophil degranulation occurring in human subjects has prevented a definitive in vivo test of this hypothesis. To demonstrate in vivo causative links between induced pulmonary histopathologies/lung dysfunction and eosinophil degranulation. A transgenic mouse model of chronic T-helper cell type 2-driven inflammation overexpressing IL-5 from T cells and human eotaxin 2 in the lung (I5/hE2) was used to test the hypothesis that chronic histopathologies and the development of airway hyperresponsiveness occur as a consequence of extensive eosinophil degranulation in the lung parenchyma. Studies targeting specific inflammatory pathways in I5/hE2 mice surprisingly showed that eosinophil-dependent immunoregulative events and not the release of individual secondary granule proteins are the central contributors to T-helper cell type 2-induced pulmonary remodeling and lung dysfunction. Specifically, our studies highlighted a significant role for eosinophil-dependent IL-13 expression. In contrast, extensive degranulation leading to the release of major basic protein-1 or eosinophil peroxidase was not causatively linked to many of the induced pulmonary histopathologies. However, these studies did define a previously unappreciated link between the release of eosinophil peroxidase (but not major basic protein-1) and observed levels of induced airway mucin. These data suggest that improvements observed in patients with asthma responding to therapeutic strategies ablating eosinophils may occur as a consequence of targeting immunoregulatory mechanisms and not by simply eliminating the destructive activities of these purportedly end-stage effector cells.

  3. Cell-Specific Oxidative Stress and Cytotoxicity after Wildfire Coarse Particulate Matter Instillation into Mouse Lung

    PubMed Central

    Williams, Keisha M.; Franzi, Lisa M.; Last, Jerold A.

    2012-01-01

    Our previous work has shown that coarse particulate matter (PM10-2.5) from wildfire smoke is more toxic to lung macrophages on an equal dose (by mass) basis than coarse PM isolated from normal ambient air, as evidenced by decreased numbers of macrophages in lung lavage fluid 6 and 24 hours after PM instillation into mouse lungs in vivo and by cytotoxicity to a macrophage cell line observed directly in vitro. We hypothesized that pulmonary macrophages from mice instilled with wildfire coarse PM would undergo more cytotoxicity than macrophages from controls, and that there would be an increase in oxidative stress in their lungs. Cytotoxicity was quantified as decreased viable macrophages and increased percentages of dead macrophages in the bronchoalveolar lavage fluid (BALF) of mice instilled with wildfire coarse PM. At one hour after PM instillation, we observed both decreased numbers of viable macrophages and increased dead macrophage percentages as compared to controls. An increase in free isoprostanes, an indicator of oxidative stress, from control values of 28.1±3.2 pg/mL to 83.9±12.2 pg/mL was observed a half-hour after PM instillation. By one hour after PM instillation, isoprostane values had returned to 30.4±7.6pg/mL, not significantly different from control concentrations. Lung sections from mice instilled with wildfire coarse PM showed rapid Clara cell responses, with decreased intracellular staining for the Clara cell secretory protein CCSP 1 hour after wildfire PM instillation. In conclusion, very rapid cytotoxicity occurs in pulmonary macrophages and oxidative stress responses are seen 0.5-1 hour after wildfire coarse PM instillation. These results define early cellular and biochemical events occurring in vivo and support the hypothesis that oxidative stress-mediated macrophage toxicity plays a key role in the initial response of the mouse lung to wildfire PM exposure. PMID:23142465

  4. Gene expression profiling in mouse lung following polymeric hexamethylene diisocyanate exposure

    SciTech Connect

    Lee, C.-T.; Ylostalo, Joni; Friedman, Mitchell; Hoyle, Gary W. . E-mail: ghoyle@tulane.edu

    2005-05-15

    Isocyanates are a common cause of occupational lung disease. Hexamethylene diisocyanate (HDI), a component of polyurethane spray paints, can induce respiratory symptoms, inflammation, lung function impairment, and isocyanate asthma. The predominant form of HDI in polyurethane paints is a nonvolatile polyisocyanate known as HDI biuret trimer (HDI-BT). Exposure of mice to aerosolized HDI-BT results in pathological effects, including pulmonary edema, lung inflammation, cellular proliferation, and fibrotic lesions, which occur with distinct time courses following exposure. To identify genes that mediate lung pathology in the distinct temporal phases after exposure, gene expression profiles in HDI-BT-exposed C57BL/6J mouse lungs were analyzed. RNase protection assay (RPA) of genes involved in apoptosis, cell survival, and inflammation revealed increased expression of I{kappa}B{alpha}, Fas, Bcl-X{sub L}, TNF{alpha}, KC, MIP-2, IL-6, and GM-CSF following HDI-BT exposure. Microarray analysis of approximately 10 000 genes was performed on lung RNA collected from mice 6, 18, and 90 h after HDI-BT exposure and from unexposed mice. Classes of genes whose expression was increased 6 h after exposure included those involved in stress responses (particularly oxidative stress and thiol redox balance), growth arrest, apoptosis, signal transduction, and inflammation. Types of genes whose expression was increased at 18 h included proteinases, anti-proteinases, cytoskeletal molecules, and inflammatory mediators. Transcripts increased at 90 h included extracellular matrix components, transcription factors, inflammatory mediators, and cell cycle regulators. This characterization of the gene expression profile in lungs exposed to HDI-BT will provide a basis for investigating injury and repair pathways that are operative during isocyanate-induced lung disease.

  5. Failure of catalase to protect against aflatoxin B{sub 1}-induced mouse lung tumorigenicity

    SciTech Connect

    Guindon, Katherine A.; Foley, Julie F.; Maronpot, Robert R.; Massey, Thomas E.

    2008-03-01

    The carcinogenic mycotoxin aflatoxin B{sub 1} (AFB{sub 1}) induces 8-hydroxy-2'-deoxyguanosine (8-OHdG) formation in mouse lung, an effect that can be prevented by treatment with polyethylene glycol-conjugated catalase (PEG-CAT). G {yields} T transversion mutation in K-ras, an early event in AFB{sub 1}-induced mouse lung carcinogenesis, is thought to result from AFB{sub 1}-8,9-exo-epoxide binding to DNA to form AFB{sub 1}-N{sup 7}-guanine, but may also result from formation of 8-OHdG. Therefore, oxidative DNA damage may be important in AFB{sub 1} carcinogenicity. The objective of this study was to determine whether PEG-CAT would prevent AFB{sub 1} tumorigenicity. Mouse lung tumorigenesis was assessed following treatment of female A/J mice with 300 kU/kg PEG-CAT ip and/or 50 mg/kg AFB{sub 1}. Mice were killed 7 months post-treatment and tumors greater than 1 mm in diameter were excised. Unexpectedly, the mean number of tumors per mouse in the PEG-CAT + AFB{sub 1} group (8.81 {+-} 3.64, n = 47) was greater than that of the group treated with AFB{sub 1} alone (7.05 {+-} 3.45, n = 42) (P < 0.05). The tumors obtained from mice treated with PEG-CAT + AFB{sub 1} were larger than those from mice treated with AFB{sub 1} alone (P < 0.05). There was no difference in K-ras exon 1 mutation spectrum or in the histological diagnosis of tumors between AFB{sub 1} and PEG-CAT + AFB{sub 1} groups (P > 0.05). In vitro incubation with mouse liver catalase (CAT) resulted in conversion of [{sup 3}H]AFB{sub 1} into a DNA-binding species, a possible explanation for the results observed in vivo. These results demonstrate that PEG-CAT is not protective against AFB{sub 1} carcinogenicity in mouse lung despite preventing DNA oxidation.

  6. Generation of Multipotent Lung and Airway Progenitors from Mouse ESCs and Patient-Specific Cystic Fibrosis iPSCs

    PubMed Central

    Mou, Hongmei; Zhao, Rui; Sherwood, Richard; Ahfeldt, Tim; Lapey, Allen; Wain, John; Sicilian, Leonard; Izvolsky, Konstantin; Lau, Frank H.; Musunuru, Kiran; Cowan, Chad; Rajagopal, Jayaraj

    2012-01-01

    SUMMARY Deriving lung progenitors from patient-specific pluripotent cells is a key step in producing differentiated lung epithelium for disease modeling and transplantation. By mimicking the signaling events that occur during mouse lung development, we generated murine lung progenitors in a series of discrete steps. Definitive endoderm derived from mouse embryonic stem cells (ESCs) was converted into foregut endoderm, then into replicating Nkx2.1+ lung endoderm, and finally into multipotent embryonic lung progenitor and airway progenitor cells. We demonstrated that precisely-timed BMP, FGF, and WNT signaling are required for NKX2.1 induction. Mouse ESC-derived Nkx2.1+ progenitor cells formed respiratory epithelium (tracheospheres) when transplanted subcutaneously into mice. We then adapted this strategy to produce disease-specific lung progenitor cells from human Cystic Fibrosis induced pluripotent stem cells (iPSCs), creating a platform for dissecting human lung disease. These disease-specific human lung progenitors formed respiratory epithelium when subcutaneously engrafted into immunodeficient mice. PMID:22482504

  7. Expression and function of the ion channel TRPA1 in vagal afferent nerves innervating mouse lungs.

    PubMed

    Nassenstein, Christina; Kwong, Kevin; Taylor-Clark, Thomas; Kollarik, Marian; Macglashan, Donald M; Braun, Armin; Undem, Bradley J

    2008-03-15

    Transient receptor potential (TRP) A1 and TRPM8 are ion channels that have been localized to afferent nociceptive nerves. These TRP channels may be of particular relevance to respiratory nociceptors in that they can be activated by various inhaled irritants and/or cold air. We addressed the hypothesis that mouse vagal sensory nerves projecting to the airways express TRPA1 and TRPM8 and that they can be activated via these receptors. Single cell RT-PCR analysis revealed that TRPA1 mRNA, but not TRPM8, is uniformly expressed in lung-labelled TRPV1-expressing vagal sensory neurons. Neither TRPA1 nor TRPM8 mRNA was expressed in TRPV1-negative neurons. Capsaicin-sensitive, but not capsaicin-insensitive, lung-specific neurons responded to cinnamaldehyde, a TRPA1 agonist, with increases in intracellular calcium. Menthol, a TRPM8 agonist, was ineffective at increasing cellular calcium in lung-specific vagal sensory neurons. Cinnamaldehyde also induced TRPA1-like inward currents (as measured by means of whole cell patch clamp recordings) in capsaicin-sensitive neurons. In an ex vivo vagal innervated mouse lung preparation, cinnamaldehyde evoked action potential discharge in mouse vagal C-fibres with a peak frequency similar to that observed with capsaicin. Cinnamaldehyde inhalation in vivo mimicked capsaicin in eliciting strong central-reflex changes in breathing pattern. Taken together, our results support the hypothesis that TRPA1, but not TRPM8, is expressed in vagal sensory nerves innervating the airways. TRPA1 activation provides a mechanism by which certain environmental stimuli may elicit action potential discharge in airway afferent C-fibres and the consequent nocifensor reflexes.

  8. Antibodies against benzo[a]pyrene in immunized mouse and in lung cancer patients.

    PubMed

    Ustinov, V A; Matveeva, V A; Kostyanko, M A; Glushkov, A N

    2013-09-01

    To evaluate the production of antibodies against benzo[a]pyrene (BP) (Ab1) and corresponding antiidiotypic antibodies (Ab2) in mice after immunization with BP-protein conjugate and in lung cancer patients. The Ab1 and Ab2 levels were measured by non-competitive ELISA in blood serum of 10 mice immunized with BP-protein conjugate, and in blood serum of 288 healthy persons and 165 lung cancer patients. The Ab1 level of was 2-fold higher than Ab2 level in blood serum of BP-immunized mice. In lung cancer patients the Ab1 level was almost 3 times higher and the Ab2 level was by 30% higher than these indexes in healthy individuals. The Ab1/Ab2 ratio was 2 in BP-immunized mice and healthy individuals and 1 in lung cancer patients. Our data have shown that the Ab1/Ab2 ratio in lung cancer patients differ from that in healthy individuals and is close to the Ab1/Ab2 ratio in BP-immunized mouse.

  9. CpG-ODN increases the release of VEGF in a mouse model of lung carcinoma.

    PubMed

    Sorrentino, Rosalinda; Morello, Silvana; Giordano, Maria Grazia; Arra, Claudio; Maiolino, Piera; Adcock, Ian M; Pinto, Aldo

    2011-06-15

    Vascular endothelial-derived growth factor (VEGF) plays a fundamental role in the formation of new vessels within the tumour mass. Increasing evidence has highlighted the involvement of Toll-like receptors (TLRs) in cancer. Of interest, TLR9 is over-expressed in human lung carcinoma tissues. The aim of our study was to determine whether TLR9 activation could alter VEGF release in a mouse model of lung carcinoma. Lewis lung carcinoma cells were intravenously (i.v.) inoculated and 10 days later, tumour-bearing mice were treated with CpG-ODN (CpG, a TLR9 ligand) or PBS. CpG administration enhanced VEGF release, which was associated with increased tumour lesions in the lung. CpG induced high levels of IL-6 expression and activation of STAT3 in tumour-bearing mice. Moreover, CpG induced VEGF release from primary fibroblasts and endothelial cells, which correlated with IL-6 and TGFβ production. This may explain the large influx of fibroblasts and the production of basic fibroblast growth factor (bFGF) in the tumour mass. The administration of a monoclonal antibody against VEGF A arrested tumour progression and induced a Th1-like response in CpG-treated tumour-bearing mice. In conclusion, our study demonstrates that the combination of CpG with anti-VEGF monoclonal antibody could be of potential therapeutic in lung carcinoma.

  10. Ezh2 restricts the smooth muscle lineage during mouse lung mesothelial development.

    PubMed

    Snitow, Melinda; Lu, MinMin; Cheng, Lan; Zhou, Su; Morrisey, Edward E

    2016-10-15

    During development, the lung mesoderm generates a variety of cell lineages, including airway and vascular smooth muscle. Epigenetic changes in adult lung mesodermal lineages are thought to contribute towards diseases such as idiopathic pulmonary fibrosis and chronic obstructive pulmonary disease, although the factors that regulate early lung mesoderm development are unknown. We show in mouse that the PRC2 component Ezh2 is required to restrict smooth muscle differentiation in the developing lung mesothelium. Mesodermal loss of Ezh2 leads to the formation of ectopic smooth muscle in the submesothelial region of the developing lung mesoderm. Loss of Ezh2 specifically in the developing mesothelium reveals a mesothelial cell-autonomous role for Ezh2 in repression of the smooth muscle differentiation program. Loss of Ezh2 derepresses expression of myocardin and Tbx18, which are important regulators of smooth muscle differentiation from the mesothelium and related cell lineages. Together, these findings uncover an Ezh2-dependent mechanism to restrict the smooth muscle gene expression program in the developing mesothelium and allow appropriate cell fate decisions to occur in this multipotent mesoderm lineage. © 2016. Published by The Company of Biologists Ltd.

  11. Pharmacokinetic and Genomic Effects of Arsenite in Drinking Water on Mouse Lung in a 30-Day Exposure

    PubMed Central

    Chilakapati, Jaya; Wallace, Kathleen; Hernandez-Zavala, Araceli; Moore, Tanya; Ren, Hongzu

    2015-01-01

    The 2 objectives of this subchronic study were to determine the arsenite drinking water exposure dependent increases in female C3H mouse liver and lung tissue arsenicals and to characterize the dose response (to 0, 0.05, 0.25, 1, 10, and 85 ppm arsenite in drinking water for 30 days and a purified AIN-93M diet) for genomic mouse lung expression patterns. Mouse lungs were analyzed for inorganic arsenic, monomethylated, and dimethylated arsenicals by hydride generation atomic absorption spectroscopy. The total lung mean arsenical levels were 1.4, 22.5, 30.1, 50.9, 105.3, and 316.4 ng/g lung tissue after 0, 0.05, 0.25, 1, 10, and 85 ppm, respectively. At 85 ppm, the total mean lung arsenical levels increased 14-fold and 131-fold when compared to either the lowest noncontrol dose (0.05 ppm) or the control dose, respectively. We found that arsenic exposure elicited minimal numbers of differentially expressed genes (DEGs; 77, 38, 90, 87, and 87 DEGs) after 0.05, 0.25, 1, 10, and 85 ppm, respectively, which were associated with cardiovascular disease, development, differentiation, apoptosis, proliferation, and stress response. After 30 days of arsenite exposure, this study showed monotonic increases in mouse lung arsenical (total arsenic and dimethylarsinic acid) concentrations but no clear dose-related increases in DEG numbers. PMID:26674514

  12. Pharmacokinetic and Genomic Effects of Arsenite in Drinking Water on Mouse Lung in a 30-Day Exposure.

    PubMed

    Chilakapati, Jaya; Wallace, Kathleen; Hernandez-Zavala, Araceli; Moore, Tanya; Ren, Hongzu; Kitchin, Kirk T

    2015-01-01

    The 2 objectives of this subchronic study were to determine the arsenite drinking water exposure dependent increases in female C3H mouse liver and lung tissue arsenicals and to characterize the dose response (to 0, 0.05, 0.25, 1, 10, and 85 ppm arsenite in drinking water for 30 days and a purified AIN-93M diet) for genomic mouse lung expression patterns. Mouse lungs were analyzed for inorganic arsenic, monomethylated, and dimethylated arsenicals by hydride generation atomic absorption spectroscopy. The total lung mean arsenical levels were 1.4, 22.5, 30.1, 50.9, 105.3, and 316.4 ng/g lung tissue after 0, 0.05, 0.25, 1, 10, and 85 ppm, respectively. At 85 ppm, the total mean lung arsenical levels increased 14-fold and 131-fold when compared to either the lowest noncontrol dose (0.05 ppm) or the control dose, respectively. We found that arsenic exposure elicited minimal numbers of differentially expressed genes (DEGs; 77, 38, 90, 87, and 87 DEGs) after 0.05, 0.25, 1, 10, and 85 ppm, respectively, which were associated with cardiovascular disease, development, differentiation, apoptosis, proliferation, and stress response. After 30 days of arsenite exposure, this study showed monotonic increases in mouse lung arsenical (total arsenic and dimethylarsinic acid) concentrations but no clear dose-related increases in DEG numbers.

  13. Impact of Cigarette Smoke on the Human and Mouse Lungs: A Gene-Expression Comparison Study

    PubMed Central

    Morissette, Mathieu C.; Lamontagne, Maxime; Bérubé, Jean-Christophe; Gaschler, Gordon; Williams, Andrew; Yauk, Carole; Couture, Christian; Laviolette, Michel; Hogg, James C.; Timens, Wim; Halappanavar, Sabina; Stampfli, Martin R.; Bossé, Yohan

    2014-01-01

    Cigarette smoke is well known for its adverse effects on human health, especially on the lungs. Basic research is essential to identify the mechanisms involved in the development of cigarette smoke-related diseases, but translation of new findings from pre-clinical models to the clinic remains difficult. In the present study, we aimed at comparing the gene expression signature between the lungs of human smokers and mice exposed to cigarette smoke to identify the similarities and differences. Using human and mouse whole-genome gene expression arrays, changes in gene expression, signaling pathways and biological functions were assessed. We found that genes significantly modulated by cigarette smoke in humans were enriched for genes modulated by cigarette smoke in mice, suggesting a similar response of both species. Sixteen smoking-induced genes were in common between humans and mice including six newly reported to be modulated by cigarette smoke. In addition, we identified a new conserved pulmonary response to cigarette smoke in the induction of phospholipid metabolism/degradation pathways. Finally, the majority of biological functions modulated by cigarette smoke in humans were also affected in mice. Altogether, the present study provides information on similarities and differences in lung gene expression response to cigarette smoke that exist between human and mouse. Our results foster the idea that animal models should be used to study the involvement of pathways rather than single genes in human diseases. PMID:24663285

  14. Cell kinetics in mouse lung following administration of carcinogens and butylated hydroxytoluene

    SciTech Connect

    Witschi, H.P.; Morse, C.C.

    1985-01-01

    A series of experiments is described which was designed to test the hypothesis that, in mouse lung, enhancement of tumor development could occur independently of overall alveolar cell hyperplasia. Male A/J mice were given 1000 mg/kg of urethane or 10 mg/kg of 3-methylcholanthrene (MCA). Alveolar cells were labeled through continuous infusion of (TH)thymidine for 6 weeks after administration of the carcinogen. Urethane produced a significant hyperplasia of the type II alveolar cell population, whereas MCA had no such effect. Five repeated injections of 300 mg/kg of butylated hydroxytoluene (BHT), a procedure known to enhance lung tumor development, produced cell hyperplasia only during the first 2 weeks; later the mice became resistant to the action of BHT. In animals treated with piperonyl butoxide prior to BHT, cell proliferation was abolished. BHT still had a small but significant enhancing effect on tumor development. However, this effect was dwarfed by the observation that piperonyl butoxide alone greatly inhibited tumor development. The data do not allow exclusion of alveolar cell hyperplasia as a mechanism in BHT-mediated enhancement of mouse lung tumor development. 19 references, 4 figures, 3 tables.

  15. Protective effect of 4,4'-diaminodiphenylsulfone against paraquat-induced mouse lung injury.

    PubMed

    Cho, Sung Chun; Rhim, Ji Heon; Choi, Hae Ri; Son, Young Hoon; Lee, Seok Jin; Song, Kye Yong; Park, Sang Chul

    2011-09-30

    Although 4,4'-diaminodiphenylsulfone (DDS, dapsone) has been used to treat several dermatologic conditions, including Hansen disease, for the past several decades, its mode of action has remained a topic of debate. We recently reported that DDS treatment significantly extends the lifespan of the nematode C. elegans by decreasing the generation of reactive oxygen species. Additionally, in in vitro experiments using non-phagocytic human fibroblasts, we found that DDS effectively counteracted the toxicity of paraquat (PQ). In the present study, we extended our work to test the protective effect of DDS against PQ in vivo using a mouse lung injury model. Oral administration of DDS to mice significantly attenuated the lung tissue damage caused by subsequent administration of PQ. Moreover, DDS reduced the local expression of mRNA transcripts encoding inflammation-related molecules, including endothelin-1 (ET-1), macrophage inflammatory protein-1α (MIP-1α), and transforming growth factor-β (TGF-β). In addition, DDS decreased the PQ-induced expression of NADPH oxidase mRNA and activation of protein kinase Cμ (PKCμ). DDS treatment also decreased the PQ-induced generation of superoxide anions in mouse lung fibroblasts. Taken together, these data suggest the novel efficacy of DDS as an effective protective agent against oxidative stress-induced tissue damages.

  16. Morphological and Cytochemical Characterization of Cells Infiltrating Mouse Lungs After Influenza Infection

    PubMed Central

    Wyde, Philip R.; Peavy, Duane L.; Cate, Thomas R.

    1978-01-01

    To initiate evaluation of the cell-mediated immunological response to influenza virus in a major site of disease, lung cells were obtained by transpleural lavage from lungs of uninfected mice and from those infected 3 or 6 days previously with 5 50% mouse infectious doses (MID50) of avirulent (P3) or virulent (P9) influenza A Hong Kong (H3N2) virus. The number of cells recovered by lavage was dependent on the dose, time after inoculation, and the type of virus used for inoculation. Although lavage pools were shown to contain peripheral blood leukocytes, this contamination was shown to be consistently less than 5% of the total leukocytes harvested. Among the ca. 0.75 × 106 lavage cells obtained from each uninfected mouse, about 90% were macrophages or lymphocytes in approximately equal proportion. T, B, and null (lyphocytes lacking theta or surface immunoglobulin markers) lymphocytes averaged 23, 9, and 7% of cells in these suspensions, respectively. After infection with either P3 or P9 virus, increased numbers of activated macrophages and lymphoblasts were observed. The major change during P3 infection was an increase in absolute numbers of null lymphocytes. In contrast, during P9 infection, T and B lymphocytes and macrophages progressively increased in absolute numbers while null cells decreased. These data suggest that cell-mediated immunological responses to influenza virus occur in the lung during infection, but that the responses to virulent and avirulent variants may differ both qualitatively and quantitatively. PMID:711312

  17. The "Goldilocks Effect" in Cystic Fibrosis: identification of a lung phenotype in the cftr knockout and heterozygous mouse

    PubMed Central

    Craig Cohen, J; Lundblad, Lennart KA; Bates, Jason HT; Levitzky, Michael; Larson, Janet E

    2004-01-01

    Background Cystic Fibrosis is a pleiotropic disease in humans with primary morbidity and mortality associated with a lung disease phenotype. However, knockout in the mouse of cftr, the gene whose mutant alleles are responsible for cystic fibrosis, has previously failed to produce a readily, quantifiable lung phenotype. Results Using measurements of pulmonary mechanics, a definitive lung phenotype was demonstrated in the cftr-/- mouse. Lungs showed decreased compliance and increased airway resistance in young animals as compared to cftr+/+ littermates. These changes were noted in animals less than 60 days old, prior to any long term inflammatory effects that might occur, and are consistent with structural differences in the cftr-/- lungs. Surprisingly, the cftr+/- animals exhibited a lung phenotype distinct from either the homozygous normal or knockout genotypes. The heterozygous mice showed increased lung compliance and decreased airway resistance when compared to either homozygous phenotype, suggesting a heterozygous advantage that might explain the high frequency of this mutation in certain populations. Conclusions In the mouse the gene dosage of cftr results in distinct differences in pulmonary mechanics of the adult. Distinct phenotypes were demonstrated in each genotype, cftr-/-, cftr +/-, and cftr+/+. These results are consistent with a developmental role for CFTR in the lung. PMID:15279681

  18. Plasmacytoid dendritic cells alter the antitumor activity of CpG-oligodeoxynucleotides in a mouse model of lung carcinoma.

    PubMed

    Sorrentino, Rosalinda; Morello, Silvana; Luciano, Antonio; Crother, Timothy R; Maiolino, Piera; Bonavita, Eduardo; Arra, Claudio; Adcock, Ian M; Arditi, Moshe; Pinto, Aldo

    2010-10-15

    The effect of CpG-oligodeoxynucleotides (CpG) has been studied on a number of tumors. Although CpG may facilitate tumor regression in mouse models of melanoma, its activity in lung cancer is unclear. The aim of our study was to elucidate the effect of CpG (0.5-50 μg/mouse) in a mouse model of Lewis lung carcinoma cell-induced lung cancer. Lung tumor growth increased at 3 and 7 d after a single administration of CpG. This was associated with a greater influx of plasmacytoid dendritic cells (pDCs), immature myeloid dendritic cells, and greater recruitment of regulatory T cells. Depletion of pDCs using a specific Ab (m927) reversed the immune-suppressive environment and resulted in a decreased lung tumor burden, accompanied by a greater influx of active myeloid dendritic cells and CD8(+) T cells, and a higher production of Th1- and Th17-like cytokines. Furthermore, the rate of apoptosis in the lungs of mice treated with CpG increased following the depletion of pDCs. CpG treatment alone does not lead to tumor regression in the lung. However, ablation of pDCs renders CpG a good adjuvant for lung cancer chemotherapy in this experimental model.

  19. A new monoclonal antibody to study mouse macrophage antigen during BHT-induced lung injury and repair.

    PubMed

    Kennel, S J; Lankford, T; Galloway, P; Witschi, H P

    1989-04-01

    A rat monoclonal antibody 133-13A to a mouse lung carcinoma cell line was found to react with macrophages in mouse lung [1]. This monoclonal antibody is different from previously described antibodies to macrophages. Immunogold electron-microscopy and immunoperoxidase light microscopy have been used to show that MoAb 133-13A binds specifically to macrophages in normal and in BHT treated mouse lungs. This MoAb recognizes a protein of approximately 100 kDa (P100) on cultured lung carcinoma cells and a 87 kDa protein on macrophages from lung or the peritoneal cavity which is different from other macrophage antigens. The surface glycoprotein has been purified from cultured cells using immunoaffinity chromatography. The purified protein was radioiodinated and MoAb 133-13A was used to develop a competition radioimmunoassay to quantitate P100. Spleen, intestines, lung, skin and uterus all have high levels of P100. P100 on peritoneal macrophages has been determined to be about 94,000 molecules/cell. Analyses of lung lavage and whole lung homogenates from mice treated with BHT, BHT plus 70% O2, and 70% O2 alone show that treated animals have elevated P100 content compared to corn oil treated mice.

  20. Cell-specific oxidative stress and cytotoxicity after wildfire coarse particulate matter instillation into mouse lung

    SciTech Connect

    Williams, Keisha M.; Franzi, Lisa M.; Last, Jerold A.

    2013-01-01

    Our previous work has shown that coarse particulate matter (PM{sub 10-2.5}) from wildfire smoke is more toxic to lung macrophages on an equal dose (by mass) basis than coarse PM isolated from normal ambient air, as evidenced by decreased numbers of macrophages in lung lavage fluid 6 and 24 hours after PM instillation into mouse lungs in vivo and by cytotoxicity to a macrophage cell line observed directly in vitro. We hypothesized that pulmonary macrophages from mice instilled with wildfire coarse PM would undergo more cytotoxicity than macrophages from controls, and that there would be an increase in oxidative stress in their lungs. Cytotoxicity was quantified as decreased viable macrophages and increased percentages of dead macrophages in the bronchoalveolar lavage fluid (BALF) of mice instilled with wildfire coarse PM. At 1 hour after PM instillation, we observed both decreased numbers of viable macrophages and increased dead macrophage percentages as compared to controls. An increase in free isoprostanes, an indicator of oxidative stress, from control values of 28.1 ± 3.2 pg/mL to 83.9 ± 12.2 pg/mL was observed a half-hour after PM instillation. By 1 hour after PM instillation, isoprostane values had returned to 30.4 ± 7.6 pg/mL, not significantly different from control concentrations. Lung sections from mice instilled with wildfire coarse PM showed rapid Clara cell responses, with decreased intracellular staining for the Clara cell secretory protein CCSP 1 hour after wildfire PM instillation. In conclusion, very rapid cytotoxicity occurs in pulmonary macrophages and oxidative stress responses are seen 0.5–1 hour after wildfire coarse PM instillation. These results define early cellular and biochemical events occurring in vivo and support the hypothesis that oxidative stress-mediated macrophage toxicity plays a key role in the initial response of the mouse lung to wildfire PM exposure. -- Highlights: ► We studied very early events (0.5–1 hour) after

  1. Vasodilator-Stimulated Phosphoprotein Deficiency Potentiates PAR-1-induced Increase in Endothelial Permeability in Mouse Lungs

    PubMed Central

    Profirovic, Jasmina; Han, Jingyan; Andreeva, Alexandra V.; Neamu, Radu F.; Pavlovic, Sasha; Vogel, Stephen M.; Walter, Ulrich; Voyno-Yasenetskaya, Tatyana A.

    2010-01-01

    Vasodilator-stimulated phosphoprotein (VASP) is implicated in the protection of the endothelial barrier in vitro and in vivo. VASP function in thrombin signaling in the endothelial cells (ECs) is not known. For the first time we studied the effects of VASP deficiency on EC permeability and pulmonary vascular permeability in response to thrombin receptor stimulation. We provided the evidence that VASP deficiency potentiates the increase in endothelial permeability induced by activation of thrombin receptor in cultured human umbilical vein endothelial cells (HUVECs) and isolated mouse lungs. Using transendothelial resistance measurement, we showed that siRNA-mediated VASP downregulation in HUVECs leads to a potentiation of thrombin- and protease-activated receptor 1 (PAR-1) agonist-induced increase in endothelial permeability. Compared to control cells, VASP-deficient HUVECs had delayed endothelial junctional reassembly and abrogated VE-cadherin cytoskeletal anchoring in the recovery phase after thrombin stimulation, as demonstrated by immunofluorescence studies and cell fractionation analysis, respectively. Measurement of the capillary filtration coefficient in isolated mouse lungs demonstrated that VASP−/− mice have increased microvascular permeability in response to infusion with PAR-1 agonist compared to wild type mice. Lack of VASP led to decreased Rac1 activation both in VASP-deficient HUVECs after thrombin stimulation and VASP−/− mouse lungs after PAR-1 agonist infusion, indicating that VASP effects on thrombin signaling may correlated with changes in Rac1 activity. This study demonstrates that VASP may play critical and complex role in the regulation of thrombin-dependent disruption of the endothelial barrier function. PMID:20945373

  2. Impaired Pulmonary Defense Against Pseudomonas aeruginosa in VEGF Gene Inactivated Mouse Lung

    PubMed Central

    Breen, Ellen C.; Malloy, Jaret L.; Tang, Kechun; Xia, Feng; Fu, Zhenxing; Hancock, Robert E. W.; Overhage, Joerg; Wagner, Peter D.; Spragg, Roger G.

    2012-01-01

    Repeated bacterial and viral infections are known to contribute to worsening lung function in several respiratory diseases, including asthma, cystic fibrosis and chronic obstructive pulmonary disease (COPD). Previous studies have reported alveolar wall cell apoptosis and parenchymal damage in adult pulmonary VEGF gene ablated mice. We hypothesized that VEGF expressed by type II cells is also necessary to provide an effective host defense against bacteria in part by maintaining surfactant homeostasis. Therefore, Pseudomonas aeruginosa (PAO1) levels were evaluated in mice following lung-targeted VEGF gene inactivation, and alterations in VEGF-dependent type II cell function were evaluated by measuring surfactant homeostasis in mouse lungs and isolated type II cells. In VEGF-deficient lungs increased PAO1 levels and pro-inflammatory cytokines, TNFα and IL-6, were detected 24 hours after bacterial instillation compared to control lungs. In vivo lung-targeted VEGF gene deletion (57% decrease in total pulmonary VEGF) did not alter alveolar surfactant or tissue disaturated phosphatidylcholine (DSPC) levels. However, sphingomyelin content, choline phosphate cytidylyltransferase (CCT) mRNA and SP-D expression were decreased. In isolated type II cells an 80% reduction of VEGF protein resulted in decreases in total phospholipids (PL), DSPC, DSPC synthesis, surfactant associated proteins (SP)-B and -D, and the lipid transporters, ABCA1 and Rab3D. TPA-induced DSPC secretion and apoptosis were elevated in VEGF-deficient type II cells. These results suggest a potential protective role for type II cell-expressed VEGF against bacterial initiated infection. PMID:22718316

  3. High Inorganic Phosphate Intake Promotes Tumorigenesis at Early Stages in a Mouse Model of Lung Cancer

    PubMed Central

    Lee, Somin; Kim, Ji-Eun; Hong, Seong-Ho; Lee, Ah-Young; Park, Eun-Jung; Seo, Hwi Won; Chae, Chanhee; Doble, Philip; Bishop, David; Cho, Myung-Haing

    2015-01-01

    Inorganic phosphate (Pi) is required by all living organisms for the development of organs such as bone, muscle, brain, and lungs, regulating the expression of several critical genes as well as signal transduction. However, little is known about the effects of prolonged dietary Pi consumption on lung cancer progression. This study investigated the effects of a high-phosphate diet (HPD) in a mouse model of adenocarcinoma. K-rasLA1 mice were fed a normal diet (0.3% Pi) or an HPD (1% Pi) for 1, 2, or 4 months. Mice were then sacrificed and subjected to inductively coupled plasma mass/optical emission spectrometry and laser ablation inductively coupled plasma mass-spectrometry analyses, western blot analysis, histopathological, immunohistochemical, and immunocytochemical analyses to evaluate tumor formation and progression (including cell proliferation, angiogenesis, and apoptosis), changes in ion levels and metabolism, autophagy, epithelial-to-mesenchymal transition, and protein translation in the lungs. An HPD accelerated tumorigenesis, as evidenced by increased adenoma and adenocarcinoma rates as well as tumor size. However, after 4 months of the HPD, cell proliferation was arrested, and marked increases in liver and lung ion levels and in energy production via the tricarboxylic acid cycle in the liver were observed, which were accompanied by increased autophagy and decreased angiogenesis and apoptosis. These results indicate that an HPD initially promotes but later inhibits lung cancer progression because of metabolic adaptation leading to tumor cell quiescence. Moreover, the results suggest that carefully regulated Pi consumption are effective in lung cancer prevention. PMID:26285136

  4. Proteomic Study of Differential Protein Expression in Mouse Lung Tissues after Aerosolized Ricin Poisoning

    PubMed Central

    Guo, Zhendong; Han, Chao; Du, Jiajun; Zhao, Siyan; Fu, Yingying; Zheng, Guanyu; Sun, Yucheng; Zhang, Yi; Liu, Wensen; Wan, Jiayu; Qian, Jun; Liu, Linna

    2014-01-01

    Ricin is one of the most poisonous natural toxins from plants and is classified as a Class B biological threat pathogen by the Centers for Disease Control and Prevention (CDC) of U.S.A. Ricin exposure can occur through oral or aerosol routes. Ricin poisoning has a rapid onset and a short incubation period. There is no effective treatment for ricin poisoning. In this study, an aerosolized ricin-exposed mouse model was developed and the pathology was investigated. The protein expression profile in the ricin-poisoned mouse lung tissue was analyzed using proteomic techniques to determine the proteins that were closely related to the toxicity of ricin. 2D gel electrophoresis, mass spectrometry and subsequent biological functional analysis revealed that six proteins including Apoa1 apolipoprotein, Ywhaz 14-3-3 protein, Prdx6 Uncharacterized Protein, Selenium-binding protein 1, HMGB1, and DPYL-2, were highly related to ricin poisoning. PMID:24786090

  5. Proteomic study of differential protein expression in mouse lung tissues after aerosolized ricin poisoning.

    PubMed

    Guo, Zhendong; Han, Chao; Du, Jiajun; Zhao, Siyan; Fu, Yingying; Zheng, Guanyu; Sun, Yucheng; Zhang, Yi; Liu, Wensen; Wan, Jiayu; Qian, Jun; Liu, Linna

    2014-04-28

    Ricin is one of the most poisonous natural toxins from plants and is classified as a Class B biological threat pathogen by the Centers for Disease Control and Prevention (CDC) of U.S.A. Ricin exposure can occur through oral or aerosol routes. Ricin poisoning has a rapid onset and a short incubation period. There is no effective treatment for ricin poisoning. In this study, an aerosolized ricin-exposed mouse model was developed and the pathology was investigated. The protein expression profile in the ricin-poisoned mouse lung tissue was analyzed using proteomic techniques to determine the proteins that were closely related to the toxicity of ricin. 2D gel electrophoresis, mass spectrometry and subsequent biological functional analysis revealed that six proteins including Apoa1 apolipoprotein, Ywhaz 14-3-3 protein, Prdx6 Uncharacterized Protein, Selenium-binding protein 1, HMGB1, and DPYL-2, were highly related to ricin poisoning.

  6. The Effect of Different Doses of Cigarette Smoke in a Mouse Lung Tumor Model

    PubMed Central

    Santiago, Ludmilla Nadir; de Camargo Fenley, Juliana; Braga, Lúcia Campanario; Cordeiro, José Antônio; Cury, Patrícia M.

    2009-01-01

    Few studies have used Balb/c mice as an animal model for lung carcinogenesis. In this study, we investigated the effect of different doses of cigarette smoking in the urethane-induced Balb/c mouse lung cancer model. After injection of 3mg/kg urethane intraperitoneally, the mice were then exposed to tobacco smoke once or twice a day, five times a week, in a closed chamber. The animals were randomly divided into four groups. The control group (G0) received urethane only. The experimental groups (G1, G2 and G3) received urethane and exposure to the smoke of 3 cigarettes for 10 minutes once a day, 3 cigarettes for 10 minutes twice a day, and 6 cigarettes for 10 minutes twice a day, respectively. The mice were sacrificed after 16 weeks of exposure, and the number of nodules and hyperplasia in the lungs was counted. The results showed no statistically significant difference in the mean number of nodules and hyperplasia among the different groups, suggesting that the Balb/c mice are not suitable to study the pathogenesis of tobacco smoking-induced tumor progression in the lungs. PMID:19079653

  7. Autocrine growth factors are involved in branching morphogenesis of mouse lung epithelium.

    PubMed

    Okada, Kimiko; Noda, Masatsugu; Nogawa, Hiroyuki

    2013-01-01

    The current model for branching morphogenesis of mouse lung proposes that the epithelium bifurcates as cells pursue separate sources of fibroblast growth factor (FGF) 10, secreted from mesenchymal tissue through interactions with epithelial tissue. If so, it may be assumed that the lung epithelium will grow into a uniform, expanding ball (without branching) when uniformly exposed to a constant concentration of FGF10. To test this hypothesis, we cultured Matrigel-embedded lung epithelium explants in FGF10-supplemented medium while shaking the culture dishes. Shaking cultures with FGF10 resulted in inferior epithelial branching compared to control cultures at rest. However, this effect was unexpectedly accompanied by poor growth rather than by ball-like expansion. When using FGF1, epithelial cultures grew and branched similarly well under either culture condition. Thus, we hypothesized that FGF10 signaling must be mediated by autocrine FGFs, such as FGF1, which might easily diffuse through the culture medium in the shaking culture. Reverse transcription-polymerase chain reaction analyses showed that FGF9 as well as FGF1 were expressed in the epithelium in vivo and in FGF10-stimulated epithelium in vitro, and FGF9 induced epithelial branching at a much lower concentration than FGF10. These results suggest that FGF1 and FGF9 may mediate FGF10 signaling and induce branching in the lung epithelium via autocrine signaling.

  8. Thyroid hormone drives the expression of mouse carbonic anhydrase Car4 in kidney, lung and brain.

    PubMed

    Vujovic, Milica; Dudazy-Gralla, Susi; Hård, Joanna; Solsjö, Peter; Warner, Amy; Vennström, Björn; Mittag, Jens

    2015-11-15

    Thyroid hormone is a well-known regulator of brain, lung and kidney development and function. However, the molecular mechanisms by which the hormone exerts its function have remained largely enigmatic, and only a limited set of target genes have been identified in these tissues. Using a mouse model with a mutation in thyroid hormone receptor α1 (TRα1), we here demonstrate that the expression of carbonic anhydrase 4 in lung and brain of the adult animal depends on intact TRα1 signaling. In the kidney, carbonic anhydrase 4 mRNA and protein are not affected by the mutant TRα1, but are acutely repressed by thyroid hormone. However, neither lung function--as measured by respiration rate and oxygen saturation--nor urine pH levels were affected by altered carbonic anhydrase 4 levels, suggesting that other carbonic anhydrases are likely to compensate. Taken together, our findings identify a previously unknown marker of TRα1 action in brain and lung, and provide a novel negatively regulated target gene to assess renal thyroid hormone status. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  9. Mechanical ventilation enhances lung inflammation and caspase activity in a model of mouse pneumovirus infection.

    PubMed

    Bem, Reinout A; van Woensel, Job B M; Bos, Albert P; Koski, Amy; Farnand, Alex W; Domachowske, Joseph B; Rosenberg, Helene F; Martin, Thomas R; Matute-Bello, Gustavo

    2009-01-01

    Severe infection with respiratory syncytial virus (RSV) in children can progress to respiratory distress and acute lung injury (ALI). Accumulating evidence suggests that mechanical ventilation (MV) is an important cofactor in the development of ALI by modulating the host immune responses to bacteria. This study investigates whether MV enhances the host response to pneumonia virus of mice (PVM), a mouse pneumovirus that has been used as a model for RSV infection in humans. BALB/c mice were inoculated intranasally with diluted clarified lung homogenates from mice infected with PVM strain J3666 or uninfected controls. Four days after inoculation, the mice were subjected to 4 h of MV (tidal volume, 10 ml/kg) or allowed to breathe spontaneously. When compared with that of mice inoculated with PVM only, the administration of MV to PVM-infected mice resulted in increased bronchoalveolar lavage fluid concentrations of the cytokines macrophage inflammatory protein (MIP)-2, MIP-1alpha (CCL3), and IL-6; increased alveolar-capillary permeability to high molecular weight proteins; and increased caspase-3 activity in lung homogenates. We conclude that MV enhances the activation of inflammatory and caspase cell death pathways in response to pneumovirus infection. We speculate that MV potentially contributes to the development of lung injury in patients with RSV infection.

  10. Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

    PubMed Central

    Abt, Melissa A.; Grek, Christina L.; Ghatnekar, Gautam S.; Yeh, Elizabeth S.

    2016-01-01

    Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3–6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue. PMID:26862835

  11. Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR.

    PubMed

    Abt, Melissa A; Grek, Christina L; Ghatnekar, Gautam S; Yeh, Elizabeth S

    2016-01-29

    Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death. Common sites of metastatic spread include lung, lymph node, brain, and bone. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

  12. Curcumin reduces lung inflammation via Wnt/β-catenin signaling in mouse model of asthma.

    PubMed

    Yang, Xia; Lv, Jian-Ning; Li, Hui; Jiao, Bo; Zhang, Qiu-Hong; Zhang, Yong; Zhang, Jie; Liu, Yan-Qin; Zhang, Ming; Shan, Hu; Zhang, Jin-Zhao; Wu, Run-Miao; Li, Ya-Li

    2017-05-01

    Asthma is a chronic inflammatory, heterogeneous airway disease affecting millions of people around the world. Curcumin has been found to have anti-inflammatory and antifibrosis effects. Researchers reported that curcumin regulated Wnt/β-catenin signaling in lots of cells. However, whether curcumin regulates the levels of Wnt/β-Catenin signaling in lung tissues and DCs (dendritic cells) remains unclear. In this study, we assessed the effects of curcumin on DCs and asthma. C57BL/6 mice immunized with OVA (ovalbumin) were challenged thrice with an aerosol of OVA every second day for 8 days. Dexamethasone or curcumin was administered intraperitoneally to OVA-immunized C57BL/6 mice on day 24 once a day for 9 days. Mice were analyzed for effects of curcumin on asthma, inflammatory cell infiltration and cytokine levels in lung tissue. DCs were isolated from mouse bone morrow. The surface markers CD40, CD86 and CD11c of DCs was detected by FACS (fluorescence activated cell sorting) and the function of DCs was detected by mixed lymphocyte reaction. The expression of GSK-3β and β-catenin was detected by Western Blot. Results showed that OVA increased the number of inflammatory factors in BALF (bronchoalveolar lavage fluid), elevated lung inflammation scores in mice. Curcumin dose-dependently reversed the alterations induced by OVA in the asthmatic mice. Curcumin activated Wnt/β-catenin signaling pathway in DCs and asthmatic mouse lungs. Curcumin could influence the morphology and function of DCs, ease asthma symptom and inflammatory reaction through the activation of Wnt/β-catenin signaling. These results provide new evidence new evidence for application of curcumin on asthma.

  13. Acute inflammation decreases the expression of connexin 40 in mouse lung.

    PubMed

    Rignault, Stéphanie; Haefliger, Jacques-Antoine; Waeber, Bernard; Liaudet, Lucas; Feihl, François

    2007-07-01

    Transmigration of neutrophil polymorphonuclear leukocytes through the microvascular endothelium is a cardinal event of acute inflammation. In vitro, this process can be restricted by gap junctional intercellular communication, but whether it also occurs in vivo is unknown. Connexin 40 (Cx40) is a gap junctional protein abundantly present in the lung, notably in vascular endothelium. We hypothesized that acute lung inflammation would be aggravated in knockout mice genetically deficient in Cx40. This hypothesis was tested in two different models: 1) intranasal instillation of LPS at either supramaximal (50 microg/mouse) or inframaximal dose (0.01 microg/mouse) and 2) pulmonary inflammation as a distant consequence of an abdominal infection caused by cecal ligation and perforation. Pulmonary transmigration of neutrophils was assessed by counting these cells in bronchoalveolar lavage fluid (LPS model) or with the myeloperoxidase assay in homogenates of blood-free tissue (cecal ligation and perforation model). Pulmonary content in Cx40 and Cx43 was evaluated with immunoblots. In wild-type mice, there was a time-dependent decrease of Cx40 expression in both models. The time points for studies with the knockout mice were chosen in such a manner that inflammation was clearly present and Cx40 still largely expressed in wild-type animals. In either model, the development of lung inflammation did not differ between wild-type and Cx40-deficient mice. In conclusion, the pulmonary expression of the Cx40 protein is progressively and markedly decreased in two different murine models of acute lung inflammation, but there is no causal relationship between this process and the pulmonary transmigration of neutrophils.

  14. Carbon black nanoparticle instillation induces sustained inflammation and genotoxicity in mouse lung and liver

    PubMed Central

    2012-01-01

    Background Widespread occupational exposure to carbon black nanoparticles (CBNPs) raises concerns over their safety. CBNPs are genotoxic in vitro but less is known about their genotoxicity in various organs in vivo. Methods We investigated inflammatory and acute phase responses, DNA strand breaks (SB) and oxidatively damaged DNA in C57BL/6 mice 1, 3 and 28 days after a single instillation of 0.018, 0.054 or 0.162 mg Printex 90 CBNPs, alongside sham controls. Bronchoalveolar lavage (BAL) fluid was analyzed for cellular composition. SB in BAL cells, whole lung and liver were assessed using the alkaline comet assay. Formamidopyrimidine DNA glycosylase (FPG) sensitive sites were assessed as an indicator of oxidatively damaged DNA. Pulmonary and hepatic acute phase response was evaluated by Saa3 mRNA real-time quantitative PCR. Results Inflammation was strongest 1 and 3 days post-exposure, and remained elevated for the two highest doses (i.e., 0.054 and 0.162 mg) 28 days post-exposure (P < 0.001). SB were detected in lung at all doses on post-exposure day 1 (P < 0.001) and remained elevated at the two highest doses until day 28 (P < 0.05). BAL cell DNA SB were elevated relative to controls at least at the highest dose on all post-exposure days (P < 0.05). The level of FPG sensitive sites in lung was increased throughout with significant increases occurring on post-exposure days 1 and 3, in comparison to controls (P < 0.001-0.05). SB in liver were detected on post-exposure days 1 (P < 0.001) and 28 (P < 0.001). Polymorphonuclear (PMN) cell counts in BAL correlated strongly with FPG sensitive sites in lung (r = 0.88, P < 0.001), whereas no such correlation was observed with SB (r = 0.52, P = 0.08). CBNP increased the expression of Saa3 mRNA in lung tissue on day 1 (all doses), 3 (all doses) and 28 (0.054 and 0.162 mg), but not in liver. Conclusions Deposition of CBNPs in lung induces inflammatory and genotoxic effects in mouse lung that persist considerably after the

  15. Longitudinal assessment of lung cancer progression in the mouse using in vivo micro-CT imaging

    PubMed Central

    Namati, Eman; Thiesse, Jacqueline; Sieren, Jessica C.; Ross, Alan; Hoffman, Eric A.; McLennan, Geoffrey

    2010-01-01

    Purpose: Small animal micro-CT imaging is being used increasingly in preclinical biomedical research to provide phenotypic descriptions of genomic models. Most of this imaging is coincident with animal death and is used to show the extent of disease as an end point. Longitudinal imaging overcomes the limitation of single time-point imaging because it enables tracking of the natural history of disease and provides qualitative and, where possible, quantitative assessments of the effects of an intervention. The pulmonary system is affected by many disease conditions, such as lung cancer, chronic obstructive pulmonary disease, asthma, and granulomatous disorders. Noninvasive imaging can accurately assess the lung phenotype within the living animal, evaluating not only global lung measures, but also regional pathology. However, imaging the lung in the living animal is complicated by rapid respiratory motion, which leads to image based artifacts. Furthermore, no standard mouse lung imaging protocols exist for longitudinal assessment, with each group needing to develop their own systematic approach. Methods: In this article, the authors present an outline for performing longitudinal breath-hold gated micro-CT imaging for the assessment of lung nodules in a mouse model of lung cancer. The authors describe modifications to the previously published intermittent isopressure breath-hold technique including a new animal preparation and anesthesia protocol, implementation of a ring artifact reduction, variable scanner geometry, and polynomial beam hardening correction. In addition, the authors describe a multitime-point data set registration and tumor labeling and tracking strategy. Results:In vivo micro-CT data sets were acquired at months 2, 3, and 4 posturethane administration in cancer mice (n=5) and simultaneously in control mice (n=3). 137 unique lung nodules were identified from the cancer mice while no nodules were detected in the control mice. A total of 411 nodules

  16. New Role of Adult Lung c-kit+ Cells in a Mouse Model of Airway Hyperresponsiveness

    PubMed Central

    Cappetta, Donato; Urbanek, Konrad; Esposito, Grazia; Matteis, Maria; Sgambato, Manuela; Tartaglione, Gioia; Rossi, Francesco

    2016-01-01

    Structural changes contribute to airway hyperresponsiveness and airflow obstruction in asthma. Emerging evidence points to the involvement of c-kit+ cells in lung homeostasis, although their potential role in asthma is unknown. Our aim was to isolate c-kit+ cells from normal mouse lungs and to test whether these cells can interfere with hallmarks of asthma in an animal model. Adult mouse GFP-tagged c-kit+ cells, intratracheally delivered in the ovalbumin-induced airway hyperresponsiveness, positively affected airway remodeling and improved airway function. In bronchoalveolar lavage fluid of cell-treated animals, a reduction in the number of inflammatory cells and in IL-4, IL-5, and IL-13 release, along with an increase of IL-10, was observed. In MSC-treated mice, the macrophage polarization to M2-like subset may explain, at least in part, the increment in the level of anti-inflammatory cytokine IL-10. After in vitro stimulation of c-kit+ cells with proinflammatory cytokines, the indoleamine 2,3-dioxygenase and TGFβ were upregulated. These data, together with the increased apoptosis of inflammatory cells in vivo, indicate that c-kit+ cells downregulate immune response in asthma by influencing local environment, possibly by cell-to-cell contact combined to paracrine action. In conclusion, intratracheally administered c-kit+ cells reduce inflammation, positively modulate airway remodeling, and improve function. These data document previously unrecognized properties of c-kit+ cells, able to impede pathophysiological features of experimental airway hyperresponsiveness. PMID:28090152

  17. Phase-contrast zoom tomography reveals precise locations of macrophages in mouse lungs

    NASA Astrophysics Data System (ADS)

    Krenkel, Martin; Markus, Andrea; Bartels, Matthias; Dullin, Christian; Alves, Frauke; Salditt, Tim

    2015-05-01

    We have performed x-ray phase-contrast tomography on mouse lung tissue. Using a divergent x-ray beam generated by nanoscale focusing, we used zoom tomography to produce three-dimensional reconstructions with selectable magnification, resolution, and field of view. Thus, macroscopic tissue samples extending over several mm can be studied in sub-cellular-level structural detail. The zoom capability and, in particular, the high dose efficiency are enabled by the near-perfect exit wavefront of an optimized x-ray waveguide channel. In combination with suitable phase-retrieval algorithms, challenging radiation-sensitive and low-contrast samples can be reconstructed with minimal artefacts. The dose efficiency of the method is demonstrated by the reconstruction of living macrophages both with and without phagocytized contrast agents. We also used zoom tomography to visualize barium-labelled macrophages in the context of morphological structures in asthmatic and healthy mouse lung tissue one day after intratracheal application. The three-dimensional reconstructions showed that the macrophages predominantly localized to the alveoli, but they were also found in bronchial walls, indicating that these cells might be able to migrate from the lumen of the bronchi through the epithelium.

  18. Phase-contrast zoom tomography reveals precise locations of macrophages in mouse lungs

    PubMed Central

    Krenkel, Martin; Markus, Andrea; Bartels, Matthias; Dullin, Christian; Alves, Frauke; Salditt, Tim

    2015-01-01

    We have performed x-ray phase-contrast tomography on mouse lung tissue. Using a divergent x-ray beam generated by nanoscale focusing, we used zoom tomography to produce three-dimensional reconstructions with selectable magnification, resolution, and field of view. Thus, macroscopic tissue samples extending over several mm can be studied in sub-cellular-level structural detail. The zoom capability and, in particular, the high dose efficiency are enabled by the near-perfect exit wavefront of an optimized x-ray waveguide channel. In combination with suitable phase-retrieval algorithms, challenging radiation-sensitive and low-contrast samples can be reconstructed with minimal artefacts. The dose efficiency of the method is demonstrated by the reconstruction of living macrophages both with and without phagocytized contrast agents. We also used zoom tomography to visualize barium-labelled macrophages in the context of morphological structures in asthmatic and healthy mouse lung tissue one day after intratracheal application. The three-dimensional reconstructions showed that the macrophages predominantly localized to the alveoli, but they were also found in bronchial walls, indicating that these cells might be able to migrate from the lumen of the bronchi through the epithelium. PMID:25966338

  19. Phase-contrast zoom tomography reveals precise locations of macrophages in mouse lungs.

    PubMed

    Krenkel, Martin; Markus, Andrea; Bartels, Matthias; Dullin, Christian; Alves, Frauke; Salditt, Tim

    2015-05-12

    We have performed x-ray phase-contrast tomography on mouse lung tissue. Using a divergent x-ray beam generated by nanoscale focusing, we used zoom tomography to produce three-dimensional reconstructions with selectable magnification, resolution, and field of view. Thus, macroscopic tissue samples extending over several mm can be studied in sub-cellular-level structural detail. The zoom capability and, in particular, the high dose efficiency are enabled by the near-perfect exit wavefront of an optimized x-ray waveguide channel. In combination with suitable phase-retrieval algorithms, challenging radiation-sensitive and low-contrast samples can be reconstructed with minimal artefacts. The dose efficiency of the method is demonstrated by the reconstruction of living macrophages both with and without phagocytized contrast agents. We also used zoom tomography to visualize barium-labelled macrophages in the context of morphological structures in asthmatic and healthy mouse lung tissue one day after intratracheal application. The three-dimensional reconstructions showed that the macrophages predominantly localized to the alveoli, but they were also found in bronchial walls, indicating that these cells might be able to migrate from the lumen of the bronchi through the epithelium.

  20. INDUCTION OF DNA ADDUCTS, TUMORS, AND KI-RAS ONCOGENE MUTATIONS IN STRAIN A/J MOUSE LUNG BY IP. ADMINISTRATION OF DIBENZ[A,H]ANTHRACENE

    EPA Science Inventory

    Induction of DNA adducts, tumors, and Ki-ras oncogene mutations in strain AlJ mouse lung by ip. administration of dibenz[a,h]anthracene

    Previous studies of polycyclic aromatic hydrocarbon (P AH) induced lung tumors in the strain NJ mouse model system have demonstrated qua...

  1. INDUCTION OF DNA ADDUCTS, TUMORS, AND KI-RAS ONCOGENE MUTATIONS IN STRAIN A/J MOUSE LUNG BY IP. ADMINISTRATION OF DIBENZ[A,H]ANTHRACENE

    EPA Science Inventory

    Induction of DNA adducts, tumors, and Ki-ras oncogene mutations in strain AlJ mouse lung by ip. administration of dibenz[a,h]anthracene

    Previous studies of polycyclic aromatic hydrocarbon (P AH) induced lung tumors in the strain NJ mouse model system have demonstrated qua...

  2. Fetal calcium regulates branching morphogenesis in the developing human and mouse lung: involvement of voltage-gated calcium channels.

    PubMed

    Brennan, Sarah C; Finney, Brenda A; Lazarou, Maria; Rosser, Anne E; Scherf, Caroline; Adriaensen, Dirk; Kemp, Paul J; Riccardi, Daniela

    2013-01-01

    Airway branching morphogenesis in utero is essential for optimal postnatal lung function. In the fetus, branching morphogenesis occurs during the pseudoglandular stage (weeks 9-17 of human gestation, embryonic days (E)11.5-16.5 in mouse) in a hypercalcaemic environment (~1.7 in the fetus vs. ~1.1-1.3 mM for an adult). Previously we have shown that fetal hypercalcemia exerts an inhibitory brake on branching morphogenesis via the calcium-sensing receptor. In addition, earlier studies have shown that nifedipine, a selective blocker of L-type voltage-gated Ca(2+) channels (VGCC), inhibits fetal lung growth, suggesting a role for VGCC in lung development. The aim of this work was to investigate the expression of VGCC in the pseudoglandular human and mouse lung, and their role in branching morphogenesis. Expression of L-type (CaV1.2 and CaV1.3), P/Q type (CaV2.1), N-type (CaV2.2), R-type (CaV2.3), and T-type (CaV3.2 and CaV3.3) VGCC was investigated in paraffin sections from week 9 human fetal lungs and E12.5 mouse embryos. Here we show, for the first time, that Cav1.2 and Cav1.3 are expressed in both the smooth muscle and epithelium of the developing human and mouse lung. Additionally, Cav2.3 was expressed in the lung epithelium of both species. Incubating E12.5 mouse lung rudiments in the presence of nifedipine doubled the amount of branching, an effect which was partly mimicked by the Cav2.3 inhibitor, SNX-482. Direct measurements of changes in epithelial cell membrane potential, using the voltage-sensitive fluorescent dye DiSBAC2(3), demonstrated that cyclic depolarisations occur within the developing epithelium and coincide with rhythmic occlusions of the lumen, driven by the naturally occurring airway peristalsis. We conclude that VGCC are expressed and functional in the fetal human and mouse lung, where they play a role in branching morphogenesis. Furthermore, rhythmic epithelial depolarisations evoked by airway peristalsis would allow for branching to match

  3. Deuterium depleted water effects on survival of lung cancer patients and expression of Kras, Bcl2, and Myc genes in mouse lung.

    PubMed

    Gyöngyi, Zoltán; Budán, Ferenc; Szabó, István; Ember, István; Kiss, István; Krempels, Krisztina; Somlyai, Ildikó; Somlyai, Gábor

    2013-01-01

    Although advances in cancer therapies continue to develop, the shortness of the survival of lung cancer patients is still disappointing. Therefore, finding new adjuvant strategies is within the focus of cancer cure. Based on observations that deuterium depletion inhibits the growth of cancer cell lines and suppresses certain proto-oncogenes, we have conducted a clinical study in 129 patients with small cell and nonsmall cell lung cancers who consumed deuterium-depleted drinking water (DDW) as a nontoxic agent in addition to conventional chemotherapy and radiotherapy. Median survival time (MST) was 25.9 mo in males and 74.1 mo in female patients; the difference between genders was statistically significant (p < 0.05). Median survival of subjects with brain metastasis was 27.1 mo. Cumulative 5-yr survival probabilities were 19%, 52%, and 33% in males, females, and all patients with brain metastasis, respectively. Gene expression analysis in mouse lung indicated that DDW attenuates 7,12-dimethylbenz(a)anthracene (DMBA)-induced expression of Bcl2, Kras, and Myc in females. In conclusion, DDW counteracts the DMBA-induced overexpression of Bcl2, Kras and Myc genes in mouse lung, and it may extend survival of lung cancer patients as a nontoxic anticancer dietary supplement, especially for women with tumors overexpressing cancer-related genes, because MST of DDW-consuming group was 2-4 times longer than it is generally observed in lung cancer patients.

  4. Deuterium Depleted Water Effects on Survival of Lung Cancer Patients and Expression of Kras, Bcl2, and Myc Genes in Mouse Lung

    PubMed Central

    Gyöngyi, Zoltán; Budán, Ferenc; Szabó, István; Ember, István; Kiss, István; Krempels, Krisztina; Somlyai, Ildikó; Somlyai, Gábor

    2013-01-01

    Although advances in cancer therapies continue to develop, the shortness of the survival of lung cancer patients is still disappointing. Therefore, finding new adjuvant strategies is within the focus of cancer cure. Based on observations that deuterium depletion inhibits the growth of cancer cell lines and suppresses certain proto-oncogenes, we have conducted a clinical study in 129 patients with small cell and nonsmall cell lung cancers who consumed deuterium-depleted drinking water (DDW) as a nontoxic agent in addition to conventional chemotherapy and radiotherapy. Median survival time (MST) was 25.9 mo in males and 74.1 mo in female patients; the difference between genders was statistically significant (p < 0.05). Median survival of subjects with brain metastasis was 27.1 mo. Cumulative 5-yr survival probabilities were 19%, 52%, and 33% in males, females, and all patients with brain metastasis, respectively. Gene expression analysis in mouse lung indicated that DDW attenuates 7,12-dimethylbenz(a)anthracene (DMBA)-induced expression of Bcl2, Kras, and Myc in females. In conclusion, DDW counteracts the DMBA-induced overexpression of Bcl2, Kras and Myc genes in mouse lung, and it may extend survival of lung cancer patients as a nontoxic anticancer dietary supplement, especially for women with tumors overexpressing cancer-related genes, because MST of DDW-consuming group was 2–4 times longer than it is generally observed in lung cancer patients. PMID:23441611

  5. Enhanced reseeding of decellularized rodent lungs with mouse embryonic stem cells

    PubMed Central

    Lecht, Shimon; Stabler, Collin T.; Rylander, Alexis L.; Chiaverelli, Rachel; Schulman, Edward S.; Marcinkiewicz, Cezary; Lelkes, Peter I.

    2016-01-01

    Repopulation of decellularized lung scaffolds (DLS) is limited due to alterations in the repertoire and ratios of the residual extracellular matrix (ECM) proteins, characterized by e.g., the retention of type I collagen and loss of glycoproteins. We hypothesized that pre-treatment of decellularized matrices with defined ECM proteins, which match the repertoire of integrin receptors expressed by the cells to be seeded (e.g., embryonic stem cells) can increase the efficacy of the reseeding process. To test this hypothesis, we first determined the integrin receptors profile of mouse embryonic stem cells (mESCs). Mouse ESCs express α3, α5, α6, α9 and β1, but not α1, α2 and α4 integrin subunits, as established by Western blotting and adhesion to laminin and fibronectin, but not to collagens type I and IV. Reseeding of DLS with mESCs was inefficient (6.9 ± 0.5%), but was significantly enhanced (2.3 ± 0.1 fold) by pre-treating the scaffolds with media conditioned by A549 human lung adenocarcinoma cells, which we found to contain ~5 μg/ml laminin. Furthermore, pre-treatment with A549-conditioned media resulted in a significantly more uniform distribution of the seeded mESCs throughout the engineered organ as compared to untreated DLS. Our study may advance whole lung engineering by stressing the importance of matching the integrin receptor repertoire of the seeded cells and the cell binding motifs of DLS. PMID:24439414

  6. Gene expression profiling in the lung and liver of PFOA-exposed mouse fetuses.

    PubMed

    Rosen, Mitchell B; Thibodeaux, Julie R; Wood, Carmen R; Zehr, Robert D; Schmid, Judith E; Lau, Christopher

    2007-09-24

    Perfluorooctanoic acid (PFOA) is a stable perfluoroalkyl acid used to synthesize fluoropolymers during the manufacture of a wide variety of products. Concerns have been raised over the potential health effects of PFOA because it is persistent in the environment and can be detected in blood and other tissues of many animal species, including humans. PFOA has also been shown to induce growth deficits and mortality in murine neonates. To better understand the mechanism of PFOA induced developmental toxicity, lung and liver gene expression profiling was conducted in PFOA-exposed full-term mouse fetuses. Thirty timed-pregnant CD-1 mice were orally dosed from gestation days 1-17 with either 0, 1, 3, 5, or 10mg/(kgday) PFOA in water. At term, fetal lung and liver were collected, total RNA prepared, and samples pooled from three fetuses per litter. Five biological replicates consisting of individual litter samples were then evaluated for each treatment group using Affymetrix mouse 430_2 microarrays. The expression of genes related to fatty acid catabolism was altered in both the fetal liver and lung. In the fetal liver, the effects of PFOA were robust and also included genes associated with lipid transport, ketogenesis, glucose metabolism, lipoprotein metabolism, cholesterol biosynthesis, steroid metabolism, bile acid biosynthesis, phospholipid metabolism, retinol metabolism, proteosome activation, and inflammation. These changes are consistent with transactivation of PPARalpha, although, with regard to bile acid biosynthesis and glucose metabolism, non-PPARalpha related effects were suggested as well. Additional studies will be needed to more thoroughly address the role of PPARalpha, and other nuclear receptors, in PFOA mediated developmental toxicity.

  7. In Vivo Measurements of T2 Relaxation Time of Mouse Lungs during Inspiration and Expiration

    PubMed Central

    Hockings, Paul D.

    2016-01-01

    Purpose The interest in measurements of magnetic resonance imaging relaxation times, T1, T2, T2*, with intention to characterize healthy and diseased lungs has increased recently. Animal studies play an important role in this context providing models for understanding and linking the measured relaxation time changes to the underlying physiology or disease. The aim of this work was to study how the measured transversal relaxation time (T2) in healthy lungs is affected by normal respiration in mouse. Method T2 of lung was measured in anaesthetized freely breathing mice. Image acquisition was performed on a 4.7 T, Bruker BioSpec with a multi spin-echo sequence (Car-Purcell-Meiboom-Gill) in both end-expiration and end-inspiration. The echo trains consisted of ten echoes of inter echo time 3.5 ms or 4.0 ms. The proton density, T2 and noise floor were fitted to the measured signals of the lung parenchyma with a Levenberg-Marquardt least-squares three-parameter fit. Results T2 in the lungs was longer (p<0.01) at end-expiration (9.7±0.7 ms) than at end-inspiration (9.0±0.8 ms) measured with inter-echo time 3.5 ms. The corresponding relative proton density (lung/muscle tissue) was higher (p<0.001) during end-expiration, (0.61±0.06) than during end-inspiration (0.48±0.05). The ratio of relative proton density at end-inspiration to that at end-expiration was 0.78±0.09. Similar results were found for inter-echo time 4.0 ms and there was no significant difference between the T2 values or proton densities acquired with different interecho times. The T2 value increased linearly (p< 0.001) with proton density. Conclusion The measured T2 in-vivo is affected by diffusion across internal magnetic susceptibility gradients. In the lungs these gradients are modulated by respiration, as verified by calculations. In conclusion the measured T2 was found to be dependent on the size of the alveoli. PMID:27936061

  8. Strain-dependent Damage in Mouse Lung After Carbon Ion Irradiation

    SciTech Connect

    Moritake, Takashi; Fujita, Hidetoshi; Yanagisawa, Mitsuru; Nakawatari, Miyako; Imadome, Kaori; Nakamura, Etsuko; Iwakawa, Mayumi; Imai, Takashi

    2012-09-01

    Purpose: To examine whether inherent factors produce differences in lung morbidity in response to carbon ion (C-ion) irradiation, and to identify the molecules that have a key role in strain-dependent adverse effects in the lung. Methods and Materials: Three strains of female mice (C3H/He Slc, C57BL/6J Jms Slc, and A/J Jms Slc) were locally irradiated in the thorax with either C-ion beams (290 MeV/n, in 6 cm spread-out Bragg peak) or with {sup 137}Cs {gamma}-rays as a reference beam. We performed survival assays and histologic examination of the lung with hematoxylin-eosin and Masson's trichrome staining. In addition, we performed immunohistochemical staining for hyaluronic acid (HA), CD44, and Mac3 and assayed for gene expression. Results: The survival data in mice showed a between-strain variance after C-ion irradiation with 10 Gy. The median survival time of C3H/He was significantly shortened after C-ion irradiation at the higher dose of 12.5 Gy. Histologic examination revealed early-phase hemorrhagic pneumonitis in C3H/He and late-phase focal fibrotic lesions in C57BL/6J after C-ion irradiation with 10 Gy. Pleural effusion was apparent in C57BL/6J and A/J mice, 168 days after C-ion irradiation with 10 Gy. Microarray analysis of irradiated lung tissue in the three mouse strains identified differential expression changes in growth differentiation factor 15 (Gdf15), which regulates macrophage function, and hyaluronan synthase 1 (Has1), which plays a role in HA metabolism. Immunohistochemistry showed that the number of CD44-positive cells, a surrogate marker for HA accumulation, and Mac3-positive cells, a marker for macrophage infiltration in irradiated lung, varied significantly among the three mouse strains during the early phase. Conclusions: This study demonstrated a strain-dependent differential response in mice to C-ion thoracic irradiation. Our findings identified candidate molecules that could be implicated in the between-strain variance to early

  9. Mutational landscape of EGFR-, MYC-, and Kras-driven genetically engineered mouse models of lung adenocarcinoma

    PubMed Central

    McFadden, David G.; Politi, Katerina; Bhutkar, Arjun; Chen, Frances K.; Song, Xiaoling; Pirun, Mono; Santiago, Philip M.; Kim-Kiselak, Caroline; Platt, James T.; Lee, Emily; Hodges, Emily; Rosebrock, Adam P.; Bronson, Roderick T.; Socci, Nicholas D.; Hannon, Gregory J.; Jacks, Tyler; Varmus, Harold

    2016-01-01

    Genetically engineered mouse models (GEMMs) of cancer are increasingly being used to assess putative driver mutations identified by large-scale sequencing of human cancer genomes. To accurately interpret experiments that introduce additional mutations, an understanding of the somatic genetic profile and evolution of GEMM tumors is necessary. Here, we performed whole-exome sequencing of tumors from three GEMMs of lung adenocarcinoma driven by mutant epidermal growth factor receptor (EGFR), mutant Kirsten rat sarcoma viral oncogene homolog (Kras), or overexpression of MYC proto-oncogene. Tumors from EGFR- and Kras-driven models exhibited, respectively, 0.02 and 0.07 nonsynonymous mutations per megabase, a dramatically lower average mutational frequency than observed in human lung adenocarcinomas. Tumors from models driven by strong cancer drivers (mutant EGFR and Kras) harbored few mutations in known cancer genes, whereas tumors driven by MYC, a weaker initiating oncogene in the murine lung, acquired recurrent clonal oncogenic Kras mutations. In addition, although EGFR- and Kras-driven models both exhibited recurrent whole-chromosome DNA copy number alterations, the specific chromosomes altered by gain or loss were different in each model. These data demonstrate that GEMM tumors exhibit relatively simple somatic genotypes compared with human cancers of a similar type, making these autochthonous model systems useful for additive engineering approaches to assess the potential of novel mutations on tumorigenesis, cancer progression, and drug sensitivity. PMID:27702896

  10. Characterization of FGF family growth factors concerning branching morphogenesis of mouse lung epithelium.

    PubMed

    Goto, Asami; Yamazaki, Naohiro; Nogawa, Hiroyuki

    2014-05-01

    Mouse lung rudiments express eight members of fibroblast growth factor (FGF) family genes from embryonic day 10 (E10) to E13. Some of these are expressed in either the epithelium or mesenchyme, while others are expressed in both. Incorporating the results of our previous study, we characterized the branch-inducing activities of all of FGFs expressed in the early lung rudiment. Of these, FGF1, FGF2, FGF7, FGF9 and FGF10 induced branching morphogenesis in Matrigel-embedded E11 epithelium, and their effective concentrations varied (10 nM, 10 nM, 3 nM, 1 nM, and 100 nM, respectively). Whereas shaking culture dishes containing medium supplemented with FGF7 or FGF10 showed reduced branching morphogenesis, those supplemented with FGF1, FGF2, or FGF9 did not, suggesting the involvement of autocrine growth factor(s) in branching morphogenesis induced by FGF7 or FGF10. In the presence of heparin, a well-known activator of FGF signaling, cystic morphology with lumen expansion was observed in cultures containing FGF1, FGF7, or FGF10, but growth arrest was observed in cultures containing FGF2 or FGF9. These results indicate that several paracrine and autocrine FGFs function during branching morphogenesis of lung epithelium.

  11. Caffeine induces alveolar apoptosis in the hyperoxia-exposed developing mouse lung

    PubMed Central

    Dayanim, Sara; Lopez, Benjamin; Maisonet, Tiffany M.; Grewal, Sungat; Londhe, Vedang A.

    2014-01-01

    Background Caffeine is a nonspecific adenosine receptor antagonist used in premature neonates to treat apnea of prematurity. While its use may reduce the incidence of bronchopulmonary dysplasia (BPD), the precise mechanisms remain unknown. Evidence of increased adenosine levels are noted in chronic lung diseases including tracheal aspirates of infants with BPD. Utilizing a well characterized newborn mouse model of alveolar hypoplasia, we hypothesized that hyperoxia-induced alveolar inflammation and hypoplasia is associated with alterations in the adenosine signaling pathway. Methods Newborn murine pups were exposed to a 14-day period of hyperoxia and daily caffeine administration followed by a 14-day recovery period in room air. Lungs were collected at both time points for bronchoalveolar fluid (BAL) analysis as well as histopathology and mRNA and protein expression. Results Caffeine treatment increased inflammation and worsened alveolar hypoplasia in hyperoxia exposed newborn mice. These changes were associated with decreased alveolar type II cell numbers, increased cell apoptosis, and decreased expression of A2A receptors. Following discontinuation of caffeine and hyperoxia, lung histology returned to baseline levels comparable to hyperoxia exposure alone. Conclusion Results of this study suggest a potentially adverse role of caffeine on alveolar development in a murine model of hyperoxia-induced alveolar hypoplasia. PMID:24321990

  12. Atorvastatin and Simvastatin Promoted Mouse Lung Repair After Cigarette Smoke-Induced Emphysema.

    PubMed

    Pinho-Ribeiro, Vanessa; Melo, Adriana Correa; Kennedy-Feitosa, Emanuel; Graca-Reis, Adriane; Barroso, Marina Valente; Cattani-Cavalieri, Isabella; Carvalho, Giovanna Marcella Cavalcante; Zin, Walter Araújo; Porto, Luis Cristóvão; Gitirana, Lycia Brito; Lanzetti, Manuella; Valença, Samuel Santos

    2017-03-01

    Cigarette smoke (CS) induces pulmonary emphysema by inflammation, oxidative stress, and metalloproteinase (MMP) activation. Pharmacological research studies have not focused on tissue repair after the establishment of emphysema but have instead focused on inflammatory stimulation. The aim of our study was to analyze the effects of atorvastatin and simvastatin on mouse lung repair after emphysema caused by CS. Male mice (C57BL/6, n = 45) were divided into the following groups: control (sham-exposed), CSr (mice exposed to 12 cigarettes a day for 60 days and then treated for another 60 days with the vehicle), CSr+A (CSr mice treated with atorvastatin for 60 days), and CSr+S (CSr mice treated with simvastatin for 60 days). The treatment with atorvastatin and simvastatin was administered via inhalation (15 min with 1 mg/mL once a day). Mice were sacrificed 24 h after the completion of the 120-day experimental procedure. We performed biochemical, morphological, and physiological analyses. We observed decreased levels of leukocytes and cytokines in statin-treated mice, accompanied by a reduction in oxidative stress markers. We also observed a morphological improvement confirmed by a mean linear intercept counting in statin-treated mice. Finally, statins also ameliorated lung function. We conclude that inhaled atorvastatin and simvastatin improved lung repair after cigarette smoke-induced emphysema in mice.

  13. Toxoplasma gondii tachyzoite-infected peripheral blood mononuclear cells are enriched in mouse lungs and liver.

    PubMed

    Unno, Akihiro; Kachi, Seira; Batanova, Tatiana A; Ohno, Tamio; Elhawary, Nagwa; Kitoh, Katsuya; Takashima, Yasuhiro

    2013-06-01

    The intracellular parasite Toxoplasma gondii is thought to disseminate throughout the host by circulation of tachyzoite-infected leukocytes in the blood, and adherence and migration of such leukocytes into solid tissues. However, it is unclear whether T. gondii-infected leukocytes can migrate to solid organs via the general circulation. In this study, we developed a real-time quantitative PCR (qRT-PCR) method to determine the rate of infection of peripheral blood mononuclear cells (PBMCs) flowing into and remaining within solid organs in mice. A transgenic T. gondii parasite line derived from the PLK strain that expresses DsRed Express, and transgenic green fluorescent protein-positive PBMCs, were used for these experiments. Tachyzoite-infected PBMCs were injected into mouse tail veins and qRT-PCR was used to measure the infection rates of the PBMCs remaining in the lungs, liver, spleen and brain. We found that the PBMCs in the lungs and liver had statistically higher infection rates than that of the original inoculum; this difference was statistically significant. However, the PBMC infection rate in the spleen showed no such enhancement. These results show that tachyzoite-infected PBMCs in the general circulation remain in the lungs and liver more effectively than non-infected PBMCs.

  14. Continuity versus split and reconstitution: exploring the molecular developmental corollaries of insect eye primordium evolution.

    PubMed

    Friedrich, Markus

    2006-11-15

    Holometabolous insects like Drosophila proceed through two phases of visual system development. The embryonic phase generates simple eyes of the larva. The postembryonic phase produces the adult specific compound eyes during late larval development and pupation. In primitive insects, by contrast, eye development persists seemingly continuously from embryogenesis through the end of postembryogenesis. Comparative literature suggests that the evolutionary transition from continuous to biphasic eye development occurred via transient developmental arrest. This review investigates how the developmental arrest model relates to the gene networks regulating larval and adult eye development in Drosophila, and embryonic compound eye development in primitive insects. Consistent with the developmental arrest model, the available data suggest that the determination of the anlage of the rudimentary Drosophila larval eye is homologous to the embryonic specification of the juvenile compound eye in directly developing insects while the Drosophila compound eye primordium is evolutionarily related to the yet little studied stem cell based postembryonic eye primordium of primitive insects.

  15. Longitudinal micro-CT provides biomarkers of lung disease that can be used to assess the effect of therapy in preclinical mouse models, and reveal compensatory changes in lung volume.

    PubMed

    Vande Velde, Greetje; Poelmans, Jennifer; De Langhe, Ellen; Hillen, Amy; Vanoirbeek, Jeroen; Himmelreich, Uwe; Lories, Rik J

    2016-01-01

    In vivo lung micro-computed tomography (micro-CT) is being increasingly embraced in pulmonary research because it provides longitudinal information on dynamic disease processes in a field in which ex vivo assessment of experimental disease models is still the gold standard. To optimize the quantitative monitoring of progression and therapy of lung diseases, we evaluated longitudinal changes in four different micro-CT-derived biomarkers [aerated lung volume, lung tissue (including lesions) volume, total lung volume and mean lung density], describing normal development, lung infections, inflammation, fibrosis and therapy. Free-breathing mice underwent micro-CT before and repeatedly after induction of lung disease (bleomycin-induced fibrosis, invasive pulmonary aspergillosis, pulmonary cryptococcosis) and therapy (imatinib). The four lung biomarkers were quantified. After the last time point, we performed pulmonary function tests and isolated the lungs for histology. None of the biomarkers remained stable during longitudinal follow-up of adult healthy mouse lungs, implying that biomarkers should be compared with age-matched controls upon intervention. Early inflammation and progressive fibrosis led to a substantial increase in total lung volume, which affects the interpretation of aerated lung volume, tissue volume and mean lung density measures. Upon treatment of fibrotic lung disease, the improvement in aerated lung volume and function was not accompanied by a normalization of the increased total lung volume. Significantly enlarged lungs were also present in models of rapidly and slowly progressing lung infections. The data suggest that total lung volume changes could partly reflect a compensatory mechanism that occurs during disease progression in mice. Our findings underscore the importance of quantifying total lung volume in addition to aerated lung or lesion volumes to accurately document growth and potential compensatory mechanisms in mouse models of lung

  16. Effect of aerosol particles generated by ultrasonic humidifiers on the lung in mouse

    PubMed Central

    2013-01-01

    Background Ultrasonic humidifiers silently generate water droplets as a cool fog and produce most of the dissolved minerals in the fog in the form of an aerosolized “white dust.” However, the health effect of these airborne particles is largely unknown. This study aimed to characterize the aerosol particles generated by ultrasonic humidifiers and to investigate their effect on the lung tissue of mice. Methods An ultrasonic humidifier was operated with tap water, high-silica water, ultrapure water, or other water types. In a chamber (0.765 m3, ventilation ratio 11.5 m3/hr), male ICR mice (10-week-old) were exposed by inhalation to an aerosol-containing vapor generated by the humidifier. After exposure for 7 or 14 days, lung tissues and bronchoalveolar lavage fluid (BALF) were collected from each mouse and examined by microarray, quantitative reverse transcription-polymerase chain reaction, and light and electron microscopy. Results Particles generated from the humidifier operated with tap water had a mass concentration of 0.46 ± 0.03 mg/m3, number concentration of (5.0 ± 1.1) × 104/cm3, and peak size distribution of 183 nm. The particles were phagocytosed by alveolar macrophages in the lung of mice. Inhalation of particles caused dysregulation of genes related to mitosis, cell adhesion molecules, MHC molecules and endocytosis, but did not induce any signs of inflammation or tissue injury in the lung. Conclusion These results indicate that aerosol particles released from ultrasonic humidifiers operated with tap water initiated a cellular response but did not cause severe acute inflammation in pulmonary tissue. Additionally, high mineral content tap water is not recommended and de-mineralized water should be recommended in order to exclude any adverse effects. PMID:24359587

  17. Light cigarette smoke-induced emphysema and NFκB activation in mouse lung

    PubMed Central

    Santos Valenca, Samuel; Castro, Paulo; Alves Pimenta, Wagner; Lanzetti, Manuella; Vargas Silva, Simone; Barja-Fidalgo, Cristina; Gonçalves Koatz, Vera Lúcia; Porto, Luís Cristóvão

    2006-01-01

    Light cigarette (LC) exposure is supposed to be less hazardous with a decreased incidence of cancer and tobacco-associated diseases. C57BL/6 mouse groups were subjected to smoke from 3, 6 or 12 LC for 60 days and compared with mice exposed to ambient air (EAA) in order to study lung injury by morphometrical and biochemical methods. Bronchoalveolar lavage (BAL) analysis and histology and stereology were performed. Tissue from the right lung was used for measuring thiobarbituric acid reactive substances (TBARS) and Western blot analysis. One way anova was performed followed by the Student–Newman Keuls post-test (P < 0.05). The cellular content of BAL was 95% alveolar macrophages in all groups except in mice exposed to 3 LC, where 23% neutrophils were observed. Emphysema was not observed in three and 6 LC, but it was found in 12 LC parallel to increased volume density (Vv) of airspaces from 61.0 ± 0.6 (EAA) to 80.9 ± 1.0 (12 LC) and decreased Vv of elastic fibres from 17.8 ± 0.9 (EAA) to 11.8 ± 0.6 (12 LC). All exposed groups to LC showed low TBARS levels compared with mice EAA. Lung tissue from animals exposed to 12 LC showed decreased tissue inhibitor of metalloprotease-2 and increased matrix metalloprotease-12 detection, which suggests an imbalance in extracellular matrix (ECM). Increased tumour necrosis factor-α and nuclear factor-κB detection were observed in exposed groups to LC when compared with mice EAA. The data suggest that LC is so dangerous to lungs as full-flavour cigarettes inducing ECM imbalance and emphysema. PMID:16965565

  18. Comparison of inflammatory responses in mouse lungs exposed to atranones A and C from Stachybotrys chartarum.

    PubMed

    Rand, Thomas G; Flemming, J; David Miller, J; Womiloju, Taiwo O

    2006-07-01

    Stachybotrys chartarum isolates can be separated into two distinct chemotypes based on the toxins they produce. One chemotype produces macrocyclic trichothecenes; the other produces atranones (and sometimes simple trichothecenes, e.g., trichodermol and trichodermin). Studies using in vivo models of lung disease revealed that exposure to spores of the atranone producing S. chartarum isolates led to a variety of immunotoxic, inflammatory, and other pathological changes. However, it is unclear from these studies what role the pure atranone toxins sequestered in spores of these isolates exert on lung disease onset. This study examined dose-response (0.2, 1.0, 2.0, 5.0, or 20 microg atranone/animal) and time-course (3, 6, 24, and 48 h postinstillation [PI]) relationships associated with inflammatory cell and proinflammatory chemokine/cytokine responses in mouse lungs intratracheally instilled with two pure atranones (either A or C) isolated from S. chartarum. High doses (2.0 to 20 microg toxin/animal) of atranone A and C induced significant inflammatory responses manifested as differentially elevated macrophage, neutrophil, macrophage inflammatory protein (MIP)-2, tumor necrosis factor (TNF) and interleukin (IL)-6 concentrations in the bronchioalveolar lavage fluid (BALF) of intratracheally exposed mice. Compared to controls, BALF macrophage and neutrophil numbers were increased to significant levels from 6 to 48 h (PI). Except for macrophage numbers in atranone A treatment animals, cells exhibited significant dose dependent-like responses. The chemokine/cytokine marker responses were significantly and dose-dependently increased from 3 to 24 h PI and declined to nonsignificant levels at 48 h PI. The results suggest not only that atranones are inflammatory but also that they exhibit different inflammatory potency with different toxicokinetics. Data also suggest that exposure to these toxins in spores of S. chartarum in contaminated building environments could contribute

  19. CD8(+)IL-17(+) T Cells Mediate Neutrophilic Airway Obliteration in T-bet-Deficient Mouse Lung Allograft Recipients.

    PubMed

    Lendermon, Elizabeth A; Dodd-o, Jeffrey M; Coon, Tiffany A; Miller, Hannah L; Ganguly, Sudipto; Popescu, Iulia; O'Donnell, Christopher P; Cardenes, Nayra; Levine, Melanie; Rojas, Mauricio; Weathington, Nathaniel M; Zhao, Jing; Zhao, Yutong; McDyer, John F

    2015-05-01

    Acute cellular rejection is a known risk factor for the development of obliterative bronchiolitis, which limits the long-term survival of lung transplant recipients. However, the T cell effector mechanisms in both of these processes remain incompletely understood. Using the mouse orthotopic lung transplant model, we investigated whether C57BL/6 T-bet(-/-) recipients of major histocompatibility complex (MHC)-mismatched BALB/c lung grafts develop rejection pathology and allospecific cytokine responses that differ from wild-type mice. T-bet(-/-) recipients demonstrated vigorous allograft rejection at 10 days, characterized by neutrophilic inflammation and predominantly CD8(+) T cells producing allospecific IL-17 and/or IFN-γ, in contrast to IFN-γ-dominant responses in WT mice. CD4(+) T cells produced IL-17 but not IFN-γ responses in T-bet(-/-) recipients, in contrast to WT controls. Costimulation blockade using anti-CD154 Ab significantly reduced allospecific CD8(+)IFN-γ(+) responses in both T-bet(-/-) and WT mice but had no attenuating effect on lung rejection pathology in T-bet(-/-) recipients or on the development of obliterative airway inflammation that occurred only in T-bet(-/-) recipients. However, neutralization of IL-17A significantly attenuated costimulation blockade-resistant rejection pathology and airway inflammation in T-bet(-/-) recipients. In addition, CXCL1 (neutrophil chemokine) was increased in T-bet(-/-) allografts, and IL-17 induced CXCL1 from mouse lung epithelial cells in vitro. Taken together, our data show that T-bet-deficient recipients of complete MHC-mismatched lung allografts develop costimulation blockade-resistant rejection characterized by neutrophilia and obliterative airway inflammation that is predominantly mediated by CD8(+)IL-17(+) T cells. Our data support T-bet-deficient mouse recipients of lung allografts as a viable animal model to study the immunopathogenesis of small airway injury in lung transplantation.

  20. Relationships of microRNA expression in mouse lung with age and exposure to cigarette smoke and light

    PubMed Central

    Izzotti, Alberto; Calin, George A.; Steele, Vernon E.; Croce, Carlo M.; De Flora, Silvio

    2009-01-01

    MicroRNAs provide a formidable tool not only in cancer research but also to investigate physiological mechanisms and to assess the effect of environmental exposures in healthy tissues. Collectively, cigarette smoke and sunlight have been estimated to account for 40% of all human cancers, and not only smoke but also, surprisingly, UV light induced genomic and postgenomic alterations in mouse lung. Here we evaluated by microarray the expression of 484 microRNAs in the lungs of CD-1 mice, including newborns, postweanling males and females, and their dams, either untreated or exposed to environmental cigarette smoke and/or UV-containing light. The results obtained highlighted age-related variations in microRNA profiles, especially during the weanling period, due to perinatal stress and postnatal maturation of the lung. UV light alone did not affect pulmonary microRNAs, whereas smoke produced dramatic changes, mostly in the sense of down-regulation, reflecting both adaptive mechanisms and activation of pathways involved in the pathogenesis of pulmonary diseases. Both gender and age affected smoke-related microRNA dysregulation in mice. The data presented provide supporting evidence that microRNAs play a fundamental role in both physiological and pathological changes occurring in mouse lung.—Izzotti, A., Calin, G. A., Vernon E. St., Croce, G. M., De Flora, S. Relationships of microRNA expression in mouse lung with age and exposure to cigarette smoke and light. PMID:19465468

  1. Phase-contrast computed tomography for quantification of structural changes in lungs of asthma mouse models of different severity.

    PubMed

    Dullin, Christian; Larsson, Emanuel; Tromba, Giuliana; Markus, Andrea M; Alves, Frauke

    2015-07-01

    Lung imaging in mouse disease models is crucial for the assessment of the severity of airway disease but remains challenging due to the small size and the high porosity of the organ. Synchrotron inline free-propagation phase-contrast computed tomography (CT) with its intrinsic high soft-tissue contrast provides the necessary sensitivity and spatial resolution to analyse the mouse lung structure in great detail. Here, this technique has been applied in combination with single-distance phase retrieval to quantify alterations of the lung structure in experimental asthma mouse models of different severity. In order to mimic an in vivo situation as close as possible, the lungs were inflated with air at a constant physiological pressure. Entire mice were embedded in agarose gel and imaged using inline free-propagation phase-contrast CT at the SYRMEP beamline (Synchrotron Light Source, `Elettra', Trieste, Italy). The quantification of the obtained phase-contrast CT data sets revealed an increasing lung soft-tissue content in mice correlating with the degree of the severity of experimental allergic airways disease. In this way, it was possible to successfully discriminate between healthy controls and mice with either mild or severe allergic airway disease. It is believed that this approach may have the potential to evaluate the efficacy of novel therapeutic strategies that target airway remodelling processes in asthma.

  2. Phase-contrast computed tomography for quantification of structural changes in lungs of asthma mouse models of different severity

    PubMed Central

    Dullin, Christian; Larsson, Emanuel; Tromba, Giuliana; Markus, Andrea M.; Alves, Frauke

    2015-01-01

    Lung imaging in mouse disease models is crucial for the assessment of the severity of airway disease but remains challenging due to the small size and the high porosity of the organ. Synchrotron inline free-propagation phase-contrast computed tomography (CT) with its intrinsic high soft-tissue contrast provides the necessary sensitivity and spatial resolution to analyse the mouse lung structure in great detail. Here, this technique has been applied in combination with single-distance phase retrieval to quantify alterations of the lung structure in experimental asthma mouse models of different severity. In order to mimic an in vivo situation as close as possible, the lungs were inflated with air at a constant physiological pressure. Entire mice were embedded in agarose gel and imaged using inline free-propagation phase-contrast CT at the SYRMEP beamline (Synchrotron Light Source, ‘Elettra’, Trieste, Italy). The quantification of the obtained phase-contrast CT data sets revealed an increasing lung soft-tissue content in mice correlating with the degree of the severity of experimental allergic airways disease. In this way, it was possible to successfully discriminate between healthy controls and mice with either mild or severe allergic airway disease. It is believed that this approach may have the potential to evaluate the efficacy of novel therapeutic strategies that target airway remodelling processes in asthma. PMID:26134818

  3. Hyperinsulinemia Promotes Metastasis to the Lung in a Mouse Model of Her2-mediated Breast Cancer

    PubMed Central

    Ferguson, Rosalyn; Gallagher, Emily; Cohen, Dara; Tobin-Hess, Aviva; Alikhani, Nyosha; Novosyadlyy, Ruslan; Haddad, Nadine; Yakar, Shoshana; LeRoith, Derek

    2014-01-01

    The Her2 oncogene is expressed in approximately 25% of human breast cancers and is associated with metastatic progression and poor outcome. Epidemiological studies report that breast cancer incidence and mortality rates are higher in women with type 2 diabetes. Here we use a mouse model of Her2-mediated breast cancer on a background of hyperinsulinemia to determine how elevated circulating insulin levels affect Her2-mediated primary tumor growth and lung metastasis. Hyperinsulinemic (MKR+/+) mice were crossed with doxycycline-inducible NeuNT (MTB/TAN) mice to produce the MTB/TAN/MKR+/+ mouse model. Both MTB/TAN and MTB/TAN/MKR+/+ mice were administered doxycycline in drinking water to induce NeuNT mammary tumor formation. In tumor tissues removed at two, four and six weeks of Neu-NT overexpression, we observed increased tumor mass and higher phosphorylation of the insulin receptor (IR)/insulin-like growth factor receptor 1 (IGF-1R), suggesting that activation of these receptors in conditions of hyperinsulinemia could contribute to the increased growth of mammary tumors. After 12 weeks on doxycycline, although no significant further increase in tumor weight was observed in MTB/TAN/MKR+/+ compared to MTB/TAN mice, the number of lung metastases was significantly higher in MTB/TAN/MKR+/+ mice compared to controls (MTB/TAN/MKR+/+ 16.41 ± 4.18 vs. MTB/TAN 5.36 ± 2.72). In tumors at the six week time-point, we observed an increase in vimentin, a cytoskeletal protein and marker of mesenchymal cells, associated with epithelial-to-mesenchymal transition and cancer associated fibroblasts. We conclude that hyperinsulinemia in MTB/TAN/MKR+/+ mice resulted in larger primary tumors, with more mesenchymal cells and therefore, more aggressive tumors with more numerous pulmonary metastases. PMID:23572162

  4. Interactions between ethanol and cigarette smoke in a mouse lung carcinogenesis model.

    PubMed

    Balansky, Roumen; Ganchev, Gancho; Iltcheva, Marietta; Nikolov, Manasi; La Maestra, S; Micale, Rosanna T; Steele, Vernon E; De Flora, Silvio

    2016-12-12

    Both ethanol and cigarette smoke are classified as human carcinogens. They can synergize, especially in tissues of the upper aerodigestive tract that are targeted by both agents. The main objective of the present study was to evaluate the individual and combined effects of ethanol and smoke in the respiratory tract, either following transplacental exposure and/or postnatal exposure. We designed two consecutive studies in mouse models by exposing Swiss H mice to oral ethanol and/or inhaled mainstream cigarette smoke for up to 4 months, at various prenatal and postnatal life stages. Clastogenic effects and histopathological alterations were evaluated after 4 and 8 months, respectively. Ethanol was per se devoid of clastogenic effects in mouse peripheral blood erythrocytes. However, especially in mice exposed both transplacentally throughout pregnancy and in the postnatal life, ethanol administration was associated not only with liver damage but also with pro-angiogenetic effects in the lung by stimulating the proliferation of blood vessels. In addition, these mice developed pulmonary emphysema, alveolar epithelial hyperplasias, microadenomas, and benign tumors. On the other hand, ethanol interfered in the lung carcinogenesis process resulting from the concomitant exposure of mice to smoke. In fact, ethanol significantly attenuated some smoke-related preneoplastic and neoplastic lesions in the respiratory tract, such as alveolar epithelial hyperplasia, microadenomas, and even malignant tumors. In addition, ethanol attenuated cigarette smoke clastogenicity. In conclusion, preclinical studies provide evidence that, in spite of its pulmonary toxicity, ethanol may mitigate some noxious effects of cigarette smoke in the respiratory tract. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  5. Systems biology-based identification of Mycobacterium tuberculosis persistence genes in mouse lungs.

    PubMed

    Dutta, Noton K; Bandyopadhyay, Nirmalya; Veeramani, Balaji; Lamichhane, Gyanu; Karakousis, Petros C; Bader, Joel S

    2014-02-18

    Identifying Mycobacterium tuberculosis persistence genes is important for developing novel drugs to shorten the duration of tuberculosis (TB) treatment. We developed computational algorithms that predict M. tuberculosis genes required for long-term survival in mouse lungs. As the input, we used high-throughput M. tuberculosis mutant library screen data, mycobacterial global transcriptional profiles in mice and macrophages, and functional interaction networks. We selected 57 unique, genetically defined mutants (18 previously tested and 39 untested) to assess the predictive power of this approach in the murine model of TB infection. We observed a 6-fold enrichment in the predicted set of M. tuberculosis genes required for persistence in mouse lungs relative to randomly selected mutant pools. Our results also allowed us to reclassify several genes as required for M. tuberculosis persistence in vivo. Finally, the new results implicated additional high-priority candidate genes for testing. Experimental validation of computational predictions demonstrates the power of this systems biology approach for elucidating M. tuberculosis persistence genes. Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), has a genetic repertoire that permits it to persist in the face of host immune responses. Identification of such persistence genes could reveal novel drug targets and elucidate mechanisms by which the organism eludes the immune system and resists drugs. Genetic screens have identified a total of 31 persistence genes, but to date only 15% of the ~4,000 M. tuberculosis genes have been tested experimentally. In this paper, as an alternative to brute force experimental screens, we describe computational methods that predict new persistence genes by combining known examples with growing databases of biological networks. Experimental testing demonstrated that these predictions are highly accurate, validating the computational approach and providing new information

  6. Multi-walled carbon nanotube-induced gene signatures in the mouse lung: potential predictive value for human lung cancer risk and prognosis

    PubMed Central

    Guo, Nancy L; Wan, Ying-Wooi; Denvir, James; Porter, Dale W; Pacurari, Maricica; Wolfarth, Michael G; Castranova, Vincent; Qian, Yong

    2012-01-01

    Concerns over the potential for multi-walled carbon nanotubes (MWCNT) to induce lung carcinogenesis have emerged. This study sought to (1) identify gene expression signatures in the mouse lungs following pharyngeal aspiration of well-dispersed MWCNT and (2) determine if these genes were associated with human lung cancer risk and progression. Genome-wide mRNA expression profiles were analyzed in mouse lungs (n=160) exposed to 0, 10, 20, 40, or 80 µg of MWCNT by pharyngeal aspiration at 1, 7, 28, and 56 days post-exposure. By using pairwise-Statistical Analysis of Microarray (SAM) and linear modeling, 24 genes were selected, which have significant changes in at least two time points, have a more than 1.5 fold change at all doses, and are significant in the linear model for the dose or the interaction of time and dose. Additionally, a 38-gene set was identified as related to cancer from 330 genes differentially expressed at day 56 post-exposure in functional pathway analysis. Using the expression profiles of the cancer-related gene set in 8 mice at day 56 post-exposure to 10 µg of MWCNT, a nearest centroid classification accurately predicts human lung cancer survival with a significant hazard ratio in training set (n=256) and test set (n=186). Furthermore, both gene signatures were associated with human lung cancer risk (n=164) with significant odds ratios. These results may lead to development of a surveillance approach for early detection of lung cancer and prognosis associated with MWCNT in the workplace. PMID:22891886

  7. Nonmucoid Pseudomonas aeruginosa Expresses Alginate in the Lungs of Patients with Cystic Fibrosis and in a Mouse Model

    PubMed Central

    Bragonzi, Alessandra; Worlitzsch, Dieter; Pier, Gerald B.; Timpert, Petra; Ulrich, Martina; Hentzer, Morten; Andersen, Jens Bo; Givskov, Michael; Conese, Massimo; Döring, Gerd

    2005-01-01

    Background. In patients with cystic fibrosis (CF), lung infection with mucoid Pseudomonas aeruginosa strains overexpressing the exopolysaccaride alginate is preceded by colonization with nonmucoid strains. We investigated the kinetics, impact of environmental signals, and genetics of P. aeruginosa alginate expression in a mouse model and in patients with CF. Methods. Using indirect immunofluorescence, microarray technology and real-time reverse-transcriptionpolymerase chain reaction, we assessed alginate gene expression during aerobic and anaerobic growth of the nonmucoid strain PAO1 in vitro, in a mouse lung-infection model and in sputum specimens from patients with CF infected with nonmucoid or mucoid P. aeruginosa strains. Results. Anaerobic conditions increased the transcription of alginate genes in vitro and in murine lungs within 24 h. Alginate production by PAO1 in murine lungs and by nonmucoid P. aeruginosa strains in patients with CF was reversible after in vitro culture under aerobic conditions. A subpopulation of P. aeruginosa clones revealing stable alginate production was detected in murine lungs 2 weeks after infection. Conclusions. Anaerobiosis and lung infection rapidly induce alginate production by gene regulation in nonmucoid P. aeruginosa. This trait may contribute to early persistence, leading to chronic P. aeruginosa infection once stable mucoid strains are generated. PMID:15995954

  8. Nonmucoid Pseudomonas aeruginosa expresses alginate in the lungs of patients with cystic fibrosis and in a mouse model.

    PubMed

    Bragonzi, Alessandra; Worlitzsch, Dieter; Pier, Gerald B; Timpert, Petra; Ulrich, Martina; Hentzer, Morten; Andersen, Jens Bo; Givskov, Michael; Conese, Massimo; Doring, Gerd

    2005-08-01

    In patients with cystic fibrosis (CF), lung infection with mucoid Pseudomonas aeruginosa strains overexpressing the exopolysaccaride alginate is preceded by colonization with nonmucoid strains. We investigated the kinetics, impact of environmental signals, and genetics of P. aeruginosa alginate expression in a mouse model and in patients with CF. Using indirect immunofluorescence, microarray technology and real-time reverse-transcription polymerase chain reaction, we assessed alginate gene expression during aerobic and anaerobic growth of the nonmucoid strain PAO1 in vitro, in a mouse lung-infection model and in sputum specimens from patients with CF infected with nonmucoid or mucoid P. aeruginosa strains. Anaerobic conditions increased the transcription of alginate genes in vitro and in murine lungs within 24 h. Alginate production by PAO1 in murine lungs and by nonmucoid P. aeruginosa strains in patients with CF was reversible after in vitro culture under aerobic conditions. A subpopulation of P. aeruginosa clones revealing stable alginate production was detected in murine lungs 2 weeks after infection. Anaerobiosis and lung infection rapidly induce alginate production by gene regulation in nonmucoid P. aeruginosa. This trait may contribute to early persistence, leading to chronic P. aeruginosa infection once stable mucoid strains are generated.

  9. Differential role of FGF9 on epithelium and mesenchyme in mouse embryonic lung.

    PubMed

    del Moral, Pierre-Marie; De Langhe, Stijn P; Sala, Frédéric G; Veltmaat, Jacqueline M; Tefft, Denise; Wang, Kasper; Warburton, David; Bellusci, Savério

    2006-05-01

    Mesothelial Fibroblast Growth Factor 9 (Fgf9) has been demonstrated by inactivation studies in mouse to be critical for the proliferation of the mesenchyme. We now show that Fgf9 is also expressed at significant levels in the distal epithelium from the mid-pseudoglandular stages. Using mesenchymal-free lung endoderm culture, we show that FGF9 triggers the proliferation of the distal epithelium leading to the formation of a cyst-like structure. On embryonic Fgfr2b-/- lungs, FGF9 induces proliferation of the mesenchyme but fails to trigger a similar effect on the epithelium, therefore involving the FGFR2b receptor in the proliferative response of the epithelium to FGF9. While FGF9 inhibits the differentiation of the mesenchyme, the epithelium appears to differentiate normally. At the molecular level, FGF9 up-regulates Fgf10 expression in the mesenchyme likely via increased expression of Tbx4 and 5 and controls the transcription of Hedgehog targets Ptc and Gli-1 in a Hedgehog-independent manner. We also show that FGF9 inhibits the activation of the canonical Wnt pathway in the epithelium by increasing Dkk1 expression, a canonical Wnt antagonist. Our work shows for the first time that FGF9 acts on the epithelium involving FGFR2b to control its proliferation but not its differentiation and contributes to the regulation of canonical Wnt signaling in the epithelium.

  10. Proteoglycans maintain lung stability in an elastase-treated mouse model of emphysema.

    PubMed

    Takahashi, Ayuko; Majumdar, Arnab; Parameswaran, Harikrishnan; Bartolák-Suki, Erzsébet; Suki, Béla

    2014-07-01

    Extracellular matrix remodeling and tissue rupture contribute to the progression of emphysema. Lung tissue elasticity is governed by the tensile stiffness of fibers and the compressive stiffness of proteoglycans. It is not known how proteoglycan remodeling affects tissue stability and destruction in emphysema. The objective of this study was to characterize the role of remodeled proteoglycans in alveolar stability and tissue destruction in emphysema. At 30 days after treatment with porcine pancreatic elastase, mouse lung tissue stiffness and alveolar deformation were evaluated under varying tonicity conditions that affect the stiffness of proteoglycans. Proteoglycans were stained and measured in the alveolar walls. Computational models of alveolar stability and rupture incorporating the mechanical properties of fibers and proteoglycans were developed. Although absolute tissue stiffness was only 24% of normal, changes in relative stiffness and alveolar shape distortion due to changes in tonicity were increased in emphysema (P < 0.01 and P < 0.001). Glycosaminoglycan amount per unit alveolar wall length, which is responsible for proteoglycan stiffness, was higher in emphysema (P < 0.001). Versican expression increased in the tissue, but decorin decreased. Our network model predicted that the rate of tissue deterioration locally governed by mechanical forces was reduced when proteoglycan stiffness was increased. Consequently, this general network model explains why increasing proteoglycan deposition protects the alveolar walls from rupture in emphysema. Our results suggest that the loss of proteoglycans observed in human emphysema contributes to disease progression, whereas treatments that promote proteoglycan deposition in the extracellular matrix should slow the progression of emphysema.

  11. PCR detection of retinoblastoma gene deletions in radiation-induced mouse lung adenocarcinomas

    SciTech Connect

    Churchill, M.E.; Gemmell, M.A.; Woloschak, G.E.

    1994-05-01

    From 1971--1986, Argonne National Laboratory conducted a series of large-scale studies of tumor incidence in 40,000 BCF{sub 1} mice irradiated with {sup 60}Co {gamma}-rays or JANUS fission-spectrum neutrons. Polymerase chain reaction (PCR) technique was used to detect deletions in the mouse retinoblastoma (mRb) gene. Six mRb gene exon fragments were amplified in a 40-cycle, 3-temperature PCR protocol. Absence of any of these fragments on a Southern blot indicated a deletion of that portion of the mRb gene. Tumors chosen for analysis were lung adenocarcinomas that were judged to be the cause of death in post-mortem analyses. Spontaneous tumors as well as those from irradiated mice were analyzed for mRb deletions. In all normal mouse tissues studies all six mRb exon fragments were present on Southern blots. Tumors in six neutron-irradiated mice also had no mRb deletions. However, 1 of 6 tumors from {gamma}-irradiated mice and 6 of 18 spontaneous tumors from unirradiated mice showed a deletion in one or both mRb alleles. All deletions detected were in the 5{prime} region of the mRb gene.

  12. Comparison of two methods used to prepare smears of mouse lung tissue for detection of Pneumocystis carinii.

    PubMed Central

    Thomson, R B; Smith, T F; Wilson, W R

    1982-01-01

    The laboratory diagnosis of Pneumocystis carinii pneumonia in humans includes the identification of cysts in stained lung tissue impression smears. By using a mouse model, we compared the number of cysts in lung tissue impression smears with those contained in a concentrate of homogenized lung tissue. Eleven C3H/HEN mice developed P. carinii infection after corticosteroid injections, a low protein (8%) diet, and tetracycline administered in drinking water. Impression smears were prepared with freshly bisected lung tissue. Smears of concentrates were prepared with sediment from centrifuged lung tissue homogenates. All smears were made in duplicate, stained with toluidine blue O or methenamine silver, coded, randomized, and examined. The concentrate preparations contained more cysts per microscopic field than the impression preparations (P less than 0.01). Concentrates prepared by grinding with a mortar and pestle contained more cysts than concentrates prepared by blending with a Stomacher (P less than 0.05). Cysts were detected equally well with either the toluidine blue O or silver stain (not significant). Lung tissue concentrates were superior to lung tissue impressions for detecting P. carinii cysts in mice. Use of lung tissue concentrates should be considered for the diagnosis of human P. carinii infection. PMID:6181088

  13. Phenotypical and ultrastructural features of Oct4-positive cells in the adult mouse lung

    PubMed Central

    Galiger, Celimene; Kostin, Sawa; Golec, Anita; Ahlbrecht, Katrin; Becker, Sven; Gherghiceanu, Mihaela; Popescu, Laurentiu M; Morty, Rory E; Seeger, Werner; Voswinckel, Robert

    2014-01-01

    Octamer binding trascription factor 4 (Oct4) is a transcription factor of POU family specifically expressed in embryonic stem cells (ESCs). A role for maintaining pluripotency and self-renewal of ESCs is assigned to Oct4 as a pluripotency marker. Oct4 can also be detected in adult stem cells such as bone marrow-derived mesenchymal stem cells. Several studies suggest a role for Oct4 in sustaining self-renewal capacity of adult stem cells. However, Oct4 gene ablation in adult stem cells revealed no abnormalities in tissue turnover or regenerative capacity. In the present study we have conspicuously found pulmonary Oct4-positive cells closely resembling the morphology of telocytes (TCs). These cells were found in the perivascular and peribronchial areas and their presence and location were confirmed by electron microscopy. Moreover, we have used Oct4-GFP transgenic mice which revealed a similar localization of the Oct4-GFP signal. We also found that Oct4 co-localized with several described TC markers such as vimentin, Sca-1, platelet-derived growth factor receptor-beta C-kit and VEGF. By flow cytometry analyses carried out with Oct4-GFP reporter mice, we described a population of EpCAMneg/CD45neg/Oct4-GFPpos that in culture displayed TC features. These results were supported by qRT-PCR with mRNA isolated from lungs by using laser capture microdissection. In addition, Oct4-positive cells were found to express Nanog and Klf4 mRNA. It is concluded for the first time that TCs in adult lung mouse tissue comprise Oct4-positive cells, which express pluripotency-related genes and represent therefore a population of adult stem cells which might contribute to lung regeneration. PMID:24889158

  14. The composition of cigarette smoke determines inflammatory cell recruitment to the lung in COPD mouse models.

    PubMed

    John, Gerrit; Kohse, Katrin; Orasche, Jürgen; Reda, Ahmed; Schnelle-Kreis, Jürgen; Zimmermann, Ralf; Schmid, Otmar; Eickelberg, Oliver; Yildirim, Ali Önder

    2014-02-01

    COPD (chronic obstructive pulmonary disease) is caused by exposure to toxic gases and particles, most often CS (cigarette smoke), leading to emphysema, chronic bronchitis, mucus production and a subsequent decline in lung function. The disease pathogenesis is related to an abnormal CS-induced inflammatory response of the lungs. Similar to active (mainstream) smoking, second hand (sidestream) smoke exposure severely affects respiratory health. These processes can be studied in vivo in models of CS exposure of mice. We compared the acute inflammatory response of female C57BL/6 mice exposed to two concentrations [250 and 500 mg/m3 TPM (total particulate matter)] of sidestream and mainstream CS for 3 days and interpreted the biological effects based on physico-chemical differences in the gas and particulate phase composition of CS. BAL (bronchoalveolar lavage fluid) was obtained to perform differential cell counts and to measure cytokine release. Lung tissue was used to determine mRNA and protein expression of proinflammatory genes and to assess tissue inflammation. A strong acute inflammatory response characterized by neutrophilic influx, increased cytokine secretion [KC (keratinocyte chemoattractant), TNF-α (tumour necrosis factor α), MIP-2 (macrophage inflammatory protein 2), MIP-1α and MCP-1 (monocyte chemoattractant protein-1)], pro-inflammatory gene expression [KC, MIP-2 and MMP12 (matrix metalloproteinase 12)] and up-regulated GM-CSF (granulocyte macrophage colony-stimulating factor) production was observed in the mainstream model. After sidestream exposure there was a dampened inflammatory reaction consisting only of macrophages and diminished GM-CSF levels, most likely caused by elevated CO concentrations. These results demonstrate that the composition of CS determines the dynamics of inflammatory cell recruitment in COPD mouse models. Different initial inflammatory processes might contribute to COPD pathogenesis in significantly varying ways, thereby

  15. The composition of cigarette smoke determines inflammatory cell recruitment to the lung in COPD mouse models

    PubMed Central

    John, Gerrit; Kohse, Katrin; Orasche, Jürgen; Reda, Ahmed; Schnelle-Kreis, Jürgen; Zimmermann, Ralf; Schmid, Otmar; Eickelberg, Oliver; Yildirim, Ali Önder

    2013-01-01

    COPD (chronic obstructive pulmonary disease) is caused by exposure to toxic gases and particles, most often CS (cigarette smoke), leading to emphysema, chronic bronchitis, mucus production and a subsequent decline in lung function. The disease pathogenesis is related to an abnormal CS-induced inflammatory response of the lungs. Similar to active (mainstream) smoking, second hand (sidestream) smoke exposure severely affects respiratory health. These processes can be studied in vivo in models of CS exposure of mice. We compared the acute inflammatory response of female C57BL/6 mice exposed to two concentrations [250 and 500 mg/m3 TPM (total particulate matter)] of sidestream and mainstream CS for 3 days and interpreted the biological effects based on physico-chemical differences in the gas and particulate phase composition of CS. BAL (bronchoalveolar lavage fluid) was obtained to perform differential cell counts and to measure cytokine release. Lung tissue was used to determine mRNA and protein expression of proinflammatory genes and to assess tissue inflammation. A strong acute inflammatory response characterized by neutrophilic influx, increased cytokine secretion [KC (keratinocyte chemoattractant), TNF-α (tumour necrosis factor α), MIP-2 (macrophage inflammatory protein 2), MIP-1α and MCP-1 (monocyte chemoattractant protein-1)], pro-inflammatory gene expression [KC, MIP-2 and MMP12 (matrix metalloproteinase 12)] and up-regulated GM-CSF (granulocyte macrophage colony-stimulating factor) production was observed in the mainstream model. After sidestream exposure there was a dampened inflammatory reaction consisting only of macrophages and diminished GM-CSF levels, most likely caused by elevated CO concentrations. These results demonstrate that the composition of CS determines the dynamics of inflammatory cell recruitment in COPD mouse models. Different initial inflammatory processes might contribute to COPD pathogenesis in significantly varying ways, thereby

  16. Interactions between the otitis media gene, Fbxo11, and p53 in the mouse embryonic lung.

    PubMed

    Tateossian, Hilda; Morse, Susan; Simon, Michelle M; Dean, Charlotte H; Brown, Steve D M

    2015-12-01

    Otitis media with effusion (OME) is the most common cause of hearing loss in children, and tympanostomy (ear tube insertion) to alleviate the condition remains the commonest surgical intervention in children in the developed world. Chronic and recurrent forms of otitis media (OM) are known to have a very substantial genetic component; however, until recently, little was known of the underlying genes involved. The Jeff mouse mutant carries a mutation in the Fbxo11 gene, a member of the F-box family, and develops deafness due to a chronic proliferative OM. We previously reported that Fbxo11 is involved in the regulation of transforming growth factor beta (TGF-β) signalling by regulating the levels of phospho-Smad2 in the epithelial cells of palatal shelves, eyelids and airways of the lungs. It has been proposed that FBXO11 regulates the cell's response to TGF-β through the ubiquitination of CDT2. Additional substrates for FBXO11 have been identified, including p53. Here, we have studied both the genetic and biochemical interactions between FBXO11 and p53 in order to better understand the function of FBXO11 in epithelial development and its potential role in OM. In mice, we show that p53 (also known as Tp53) homozygous mutants and double heterozygous mutants (Jf/+ p53/+) exhibit similar epithelial developmental defects to Fbxo11 homozygotes. FBXO11 and p53 interact in the embryonic lung, and mutation in Fbxo11 prevents the interaction with p53. Both p53 and double mutants show raised levels of pSMAD2, recapitulating that seen in Fbxo11 homozygotes. Overall, our results support the conclusion that FBXO11 regulates the TGF-β pathway in the embryonic lung via cross-talk with p53.

  17. Curcumin inhibits paraquat induced lung inflammation and fibrosis by extracellular matrix modifications in mouse model.

    PubMed

    Tyagi, Namitosh; Dash, D; Singh, Rashmi

    2016-12-01

    Paraquat (PQ), a potent herbicide can cause severe toxicity. We report here that fibroproliferation phase of acute lung injury (ALI) is initiated much earlier (within 48 h) after PQ intoxication than previously reported (after 2 weeks) and we aimed to study the protective effects of intranasal curcumin as new therapeutic strategy in mouse model. Mice (Park's strain) were divided into five experimental groups (I) control, received only saline (0.9 % NaCl) (II) PQ, mice intoxicated with PQ (50 mg/kg, i.p., single dose); (III) curcumin, treated with curcumin (5 mg/kg, i.n) an hour before PQ administration; (IV)Veh, DMSO (equal volume to curcumin) given an hour before PQ exposure; (V) DEXA, mice treated with dexamethasone (1 mg/kg, i.p) before an hour of PQ intoxication. After 48 h of the PQ exposure, all mice were sacrificed and samples were analyzed. Pretreatment with intranasal curcumin (5 mg/kg) could modify the PQ-intoxication (50 mg/kg, i.p) induced structural remodeling of lung parenchyma at an early phase of acute lung injury. Significant increase in inflammatory cell count, reactive oxygen species and hydroxyproline levels were decreased after curcumin pretreatment (all p < 0.05). Histological examination and zymography results were also found consistent. Our results show that curcumin pretreatment decreased the expression of alpha smooth muscle actin (α-SMA), matrix metalloproteinases-9 (MMP-9) and changed the expression of tissue inhibitors of metalloproteinase (TIMP-1) after PQ intoxication. Single toxic dose of PQ has initiated fibroproliferation within 48 h and intranasal curcumin may prove as new therapeutic strategy for PQ induced ALI and fibroproliferation.

  18. The sickle cell mouse lung: pro-inflammatory and primed for allergic inflammation

    PubMed Central

    Andemariam, Biree; Adami, Alexander J.; McNamara, Jeffrey T.; Secor, Eric R.; Guernsey, Linda A.; Thrall, Roger S.

    2015-01-01

    Co-morbid asthma in sickle cell disease (SCD) confers higher rates of vaso-occlusive pain and mortality, yet the physiological link between these two distinct diseases remains puzzling. We utilized a mouse model of SCD to study pulmonary immunology and physiology before and after the induction of allergic airway disease (AAD). SCD mice were sensitized with ovalbumin (OVA) and aluminum hydroxide by the intraperitoneal (IP) route followed by daily, nose-only OVA-aerosol challenge to induce AAD. The lungs of naive SCD mice showed signs of inflammatory and immune processes: (1) histologic and cytochemical evidence of airway inflammation as compared to naïve wildtype mice; (2) bronchoalveolar lavage fluid (BAL) contained increased total lymphocytes, %CD8+ T cells, G-CSF, IL-5, IL-7, and CXCL1, and (3) lung tissue and hilar lymph node (HLN) had increased CD4+, CD8+, and regulatory T cells (Tregs). Further, SCD mice at AAD demonstrated significant changes compared to the naïve state: (1) BAL with increased %CD4+ T cells and Tregs, lower %CD8+ T cells, and decreased IFNγ, CXCL10, CCL2, and IL-17, (2) serum with increased OVA-specific IgE, IL-6, and IL-13, and decreased IL-1α and CXCL10, (3) no increase in Tregs in the lung tissue or HLN, and (4) hypo-responsiveness to methacholine challenge. In conclusion, SCD mice have an altered immunologic pulmonary milieu and physiologic responsiveness. These findings suggest that the clinical phenotype of AAD in SCD mice differs from that of wildtype mice and suggests that individuals with SCD may also have a unique, divergent phenotype perhaps amenable to a different therapeutic approach. PMID:25843670

  19. Interactions between the otitis media gene, Fbxo11, and p53 in the mouse embryonic lung

    PubMed Central

    Tateossian, Hilda; Morse, Susan; Simon, Michelle M.; Dean, Charlotte H.; Brown, Steve D. M.

    2015-01-01

    ABSTRACT Otitis media with effusion (OME) is the most common cause of hearing loss in children, and tympanostomy (ear tube insertion) to alleviate the condition remains the commonest surgical intervention in children in the developed world. Chronic and recurrent forms of otitis media (OM) are known to have a very substantial genetic component; however, until recently, little was known of the underlying genes involved. The Jeff mouse mutant carries a mutation in the Fbxo11 gene, a member of the F-box family, and develops deafness due to a chronic proliferative OM. We previously reported that Fbxo11 is involved in the regulation of transforming growth factor beta (TGF-β) signalling by regulating the levels of phospho-Smad2 in the epithelial cells of palatal shelves, eyelids and airways of the lungs. It has been proposed that FBXO11 regulates the cell's response to TGF-β through the ubiquitination of CDT2. Additional substrates for FBXO11 have been identified, including p53. Here, we have studied both the genetic and biochemical interactions between FBXO11 and p53 in order to better understand the function of FBXO11 in epithelial development and its potential role in OM. In mice, we show that p53 (also known as Tp53) homozygous mutants and double heterozygous mutants (Jf/+ p53/+) exhibit similar epithelial developmental defects to Fbxo11 homozygotes. FBXO11 and p53 interact in the embryonic lung, and mutation in Fbxo11 prevents the interaction with p53. Both p53 and double mutants show raised levels of pSMAD2, recapitulating that seen in Fbxo11 homozygotes. Overall, our results support the conclusion that FBXO11 regulates the TGF-β pathway in the embryonic lung via cross-talk with p53. PMID:26471094

  20. Genetically determined heterogeneity of lung disease in a mouse model of airway mucus obstruction.

    PubMed

    Livraghi-Butrico, Alessandra; Grubb, Barbara R; Kelly, Elizabeth J; Wilkinson, Kristen J; Yang, Huifang; Geiser, Marianne; Randell, Scott H; Boucher, Richard C; O'Neal, Wanda K

    2012-04-15

    Mucus clearance is an important airway innate defense mechanism. Airway-targeted overexpression of the epithelial Na(+) channel β-subunit [encoded by sodium channel nonvoltage gated 1, beta subunit (Scnn1b)] in mice [Scnn1b-transgenic (Tg) mice] increases transepithelial Na(+) absorption and dehydrates the airway surface, which produces key features of human obstructive lung diseases, including mucus obstruction, inflammation, and air-space enlargement. Because the first Scnn1b-Tg mice were generated on a mixed background, the impact of genetic background on disease phenotype in Scnn1b-Tg mice is unknown. To explore this issue, congenic Scnn1b-Tg mice strains were generated on C57BL/6N, C3H/HeN, BALB/cJ, and FVB/NJ backgrounds. All strains exhibited a two- to threefold increase in tracheal epithelial Na(+) absorption, and all developed airway mucus obstruction, inflammation, and air-space enlargement. However, there were striking differences in neonatal survival, ranging from 5 to 80% (FVB/NJlung atelectasis. The spontaneous occurrence of a high surviving BALB/cJ line, which exhibited delayed onset of Na(+) hyperabsorption, provided evidence that: 1) air-space enlargement and postnatal death were only present when Na(+) hyperabsorption occurred early, and 2) inflammation and mucus obstruction developed whenever Na(+) hyperabsorption was expressed. In summary, the genetic context and timing of airway innate immune dysfunction critically determines lung disease phenotype. These mouse strains may be useful to identify key modifier genes and pathways.

  1. PCR detection of retinoblastoma gene deletions in radiation-induced mouse lung adenocarcinomas

    SciTech Connect

    Churchill, M.E.; Gemmell, M.A.; Woloschak, G.E.

    1993-04-01

    From 1971 to 1986, Argonne National Laboratory conducted a series of large-scale studies of tumor incidence in 40,000 BCF{sub 1} mice irradiated with {sup 60}Co {gamma} rays or JANUS fission-spectrum neutrons; normal and tumor tissues from mice in these studies were preserved in paraffin blocks. A polymerase chain reaction (PCR) technique has been developed to detect deletions in the mouse retinoblastoma (mRb) gene in the paraffin-embedded tissues. Microtomed sections were used as the DNA source in PCR reaction mixtures. Six mRb gene exon fragments were amplified in a 40-cycle, 3-temperature PCR protocol. The absence of any of these fragments (relative to control PCR products) on a Southern blot indicated a deletion of that portion of the mRb gene. The tumors chosen for analysis were lung adenocarcinomas that were judged to be the cause of death in post-mortem analyses. Spontaneous tumors as well as those from irradiated mice (569 cGy of {sup 60}Co {gamma} rays or 60 cGy of JANUS neutrons, doses that have been found to have approximately equal biological effectiveness in the BCF, mouse) were analyzed for mRb deletions. In all normal mouse tissues studies, all six mRb exon fragments were present on Southem blots. Tumors in six neutron-irradiated mice also had no mRb deletions. However, I of 6 tumors from {gamma}-irradiated mice and 6 of 18 spontaneous tumors from unirradiated mice had a deletion in one or both mRb alleles. All deletions detected were in the 5{prime} region of the mRb gene.

  2. PCR detection of retinoblastoma gene deletions in radiation-induced mouse lung adenocarcinomas

    SciTech Connect

    Churchill, M.E.; Gemmell, M.A.; Woloschak, G.E.

    1993-01-01

    From 1971 to 1986, Argonne National Laboratory conducted a series of large-scale studies of tumor incidence in 40,000 BCF[sub 1] mice irradiated with [sup 60]Co [gamma] rays or JANUS fission-spectrum neutrons; normal and tumor tissues from mice in these studies were preserved in paraffin blocks. A polymerase chain reaction (PCR) technique has been developed to detect deletions in the mouse retinoblastoma (mRb) gene in the paraffin-embedded tissues. Microtomed sections were used as the DNA source in PCR reaction mixtures. Six mRb gene exon fragments were amplified in a 40-cycle, 3-temperature PCR protocol. The absence of any of these fragments (relative to control PCR products) on a Southern blot indicated a deletion of that portion of the mRb gene. The tumors chosen for analysis were lung adenocarcinomas that were judged to be the cause of death in post-mortem analyses. Spontaneous tumors as well as those from irradiated mice (569 cGy of [sup 60]Co [gamma] rays or 60 cGy of JANUS neutrons, doses that have been found to have approximately equal biological effectiveness in the BCF, mouse) were analyzed for mRb deletions. In all normal mouse tissues studies, all six mRb exon fragments were present on Southem blots. Tumors in six neutron-irradiated mice also had no mRb deletions. However, I of 6 tumors from [gamma]-irradiated mice and 6 of 18 spontaneous tumors from unirradiated mice had a deletion in one or both mRb alleles. All deletions detected were in the 5[prime] region of the mRb gene.

  3. In vitro radiation sensitivity of mouse lung fibroblasts isolated by flow cytometry

    SciTech Connect

    Keng, P.C.; Phipps, R.; Penney, D.P.

    1995-02-01

    Recently, we have isolated two major fibroblast cells (Thy-1{sup +}, Thy-1{sup {minus}}) from mouse LAF1 lung tissue using the anti-Thy-1 antibody expression and fluorescence activated cell sorter. To examine the possibility that x- or {gamma}-ray-induced pulmonary fibrosis at the late stage of injury could arise from radioresistant cell subpopulations, the radiation sensitivities of Thy-1{sup +} and Thy-1{sup {minus}} cells were evaluated by the colony forming assay. Cell survival curves, repair of potentially lethal damage (PLD) and sublethal damage (SLD), and cell-age response curves were obtained after Cs-137 {gamma}-ray irradiation. The cell survival curves measured after 0-10 Gy {gamma}-ray showed that Thy-1{sup +} cells were slightly more radioresistant than Thy-1{sup {minus}} cells. The D{sub 0}, n, alpha, and beta values measured from the survival curves also confirmed this observation. After a single dose of 10 Gy, a small amount of PLD repair was observed in Thy-1{sup {minus}} cells, while no PLD repair was found in Thy-1{sup +} cells. Although the initial cell survival level of Thy-1{sup {minus}} cells was lower, the final survival levels of Thy-1{sup +} and Thy-1{sup {minus}} cells became identical at 8 h after irradiation due to the PLD repair. After split-dose irradiation of 4 Gy followed by 4 Gy, a similar extent and rate of SLD repair was found in Thy-1{sup +} and Thy-1{sup {minus}} cells. Cell-age response curves were obtained from irradiated G{sub 0}/G{sub 1}, S, and G{sub 2}/M cells separated by centrifugal elutriation and irradiated with 8 Gy gamma-ray. The results indicated that Thy-1{sup +} and Thy-1{sup {minus}} cells had a similar S resistant, and G{sub 1}, G{sub 2}M-sensitive radiation cell-age response curve. This study suggests that the selection of radioresistant lung fibroblast may not be responsible for the development of lung fibrosis in irradiated LAF{sub 1} mouse. 16 refs., 4 figs., 1 tab.

  4. Metabolism of the anti-tuberculosis drug ethionamide by mouse and human FMO1, FMO2 and FMO3 and mouse and human lung microsomes

    SciTech Connect

    Henderson, Marilyn C.; Siddens, Lisbeth K.; Morre, Jeffrey T.; Krueger, Sharon K.; Williams, David E.

    2008-12-15

    Tuberculosis (TB) results from infection with Mycobacterium tuberculosis and remains endemic throughout the world with one-third of the world's population infected. The prevalence of multi-drug resistant strains necessitates the use of more toxic second-line drugs such as ethionamide (ETA), a pro-drug requiring bioactivation to exert toxicity. M. tuberculosis possesses a flavin monooxygenase (EtaA) that oxygenates ETA first to the sulfoxide and then to 2-ethyl-4-amidopyridine, presumably through a second oxygenation involving sulfinic acid. ETA is also a substrate for mammalian flavin-containing monooxygenases (FMOs). We examined activity of expressed human and mouse FMOs toward ETA, as well as liver and lung microsomes. All FMOs converted ETA to the S-oxide (ETASO), the first step in bioactivation. Compared to M. tuberculosis, the second S-oxygenation to the sulfinic acid is slow. Mouse liver and lung microsomes, as well as human lung microsomes from an individual expressing active FMO, oxygenated ETA in the same manner as expressed FMOs, confirming this reaction functions in the major target organs for therapeutics (lung) and toxicity (liver). Inhibition by thiourea, and lack of inhibition by SKF-525A, confirm ETASO formation is primarily via FMO, particularly in lung. ETASO production was attenuated in a concentration-dependent manner by glutathione. FMO3 in human liver may contribute to the toxicity and/or affect efficacy of ETA administration. Additionally, there may be therapeutic implications of efficacy and toxicity in human lung based on the FMO2 genetic polymorphism, though further studies are needed to confirm that suggestion.

  5. A comprehensive analysis of oxidative stress in the ozone-induced lung inflammation mouse model.

    PubMed

    Wiegman, Coen H; Li, Feng; Clarke, Colin J; Jazrawi, Elen; Kirkham, Paul; Barnes, Peter J; Adcock, Ian M; Chung, Kian F

    2014-03-01

    Ozone is an oxidizing environmental pollutant that contributes significantly to respiratory health. Exposure to increased levels of ozone has been associated with worsening of symptoms of patients with asthma and COPD (chronic obstructive pulmonary disease). In the present study, we investigated the acute and chronic effects of ozone exposure-induced oxidative stress-related inflammation mechanics in mouse lung. In particular, we investigated the oxidative stress-induced effects on HDAC2 (histone deacetylase 2) modification and activation of the Nrf2 (nuclear factor erythroid-related factor 2) and HIF-1α (hypoxia-inducible factor-1α) signalling pathways. Male C57BL/6 mice were exposed to ozone (3 p.p.m.) for 3 h a day, twice a week for a period of 1, 3 or 6 weeks. Control mice were exposed to normal air. After the last exposure, mice were killed for BAL (bronchoalveolar lavage) fluid and lung tissue collection. BAL total cell counts were elevated at all of the time points studied. This was associated with increased levels of chemokines and cytokines in all ozone-exposed groups, indicating the presence of a persistent inflammatory environment in the lung. Increased inflammation and Lm (mean linear intercept) scores were observed in chronic exposed mice, indicating emphysematous changes were present in lungs of chronic exposed mice. The antioxidative stress response was active (indicated by increased Nrf2 activity and protein) after 1 week of ozone exposure, but this ability was lost after 3 and 6 weeks of ozone exposure. The transcription factor HIF-1α was elevated in 3- and 6-week ozone-exposed mice and this was associated with increased gene expression levels of several HIF-1α target genes including Hdac2 (histone deacetylase 2), Vegf (vascular endothelial growth factor), Keap1 (kelch-like ECH-associated protein 1) and Mif (macrophage migration inhibitory factor). HDAC2 protein was found to be phosphorylated and carbonylated in nuclear and cytoplasm fractions

  6. Metabolite signatures in hydrophilic extracts of mouse lungs exposed to cigarette smoke revealed by 1H NMR metabolomics investigation

    DOE PAGES

    Hu, Jian Z.; Wang, Xuan; Feng, Ju; ...

    2015-05-12

    Herein, 1H-NMR metabolomics are carried out to evaluate the changes of metabolites in lungs of mice exposed to cigarette smoke. It is found that the concentrations of adenosine derivatives (i.e. ATP, ADP and AMP), inosine and uridine are significantly fluctuated in the lungs of mice exposed to cigarette smoke compared with those of controls regardless the mouse is obese or regular weight. The decreased ATP, ADP, AMP and elevated inosine predict that the deaminases in charge of adenosine derivatives to inosine derivatives conversion are altered in lungs of mice exposed to cigarette smoke. Transcriptional analysis reveals that the concentrations ofmore » adenosine monophosphate deaminase and adenosine deaminase are different in the lungs of mice exposed to cigarette smoke, confirming the prediction from metabolomics studies. We also found, for the first time, that the ratio of glycerophosphocholine (GPC) to phosphocholine (PC) is significantly increased in the lungs of obese mice compared with regular weight mice. The ratio of GPC/PC is further elevated in the lungs of obese group by cigarette smoke exposure. Since GPC/PC ratio is a known biomarker for cancer, these results may suggest that obese group is more susceptible to lung cancer when exposed to cigarette smoke.« less

  7. Bioluminescent Orthotopic Mouse Models of Human Localized Non-Small Cell Lung Cancer: Feasibility and Identification of Circulating Tumour Cells

    PubMed Central

    Lahon, Benoit; Castier, Yves; Lesèche, Guy; Soria, Jean-Charles; Vozenin, Marie-Catherine; Decraene, Charles; Deutsch, Eric

    2011-01-01

    Background Preclinical models of non-small cell lung cancer (NSCLC) require better clinical relevance to study disease mechanisms and innovative therapeutics. We sought to compare and refine bioluminescent orthotopic mouse models of human localized NSCLC. Methods Athymic nude mice underwent subcutaneous injection (group 1-SC, n = 15, control), percutaneous orthotopic injection (group 2-POI, n = 30), surgical orthotopic implantation of subcutaneously grown tumours (group 3-SOI, n = 25), or transpleural orthotopic injection (group 4-TOI, n = 30) of A549-luciferase cells. Bioluminescent in vivo imaging was then performed weekly. Circulating tumour cells (CTCs) were searched using Cellsearch® system in SC and TOI models. Results Group 2-POI was associated with unexpected direct pleural spreading of the cellular solution in 53% of the cases, forbidding further evaluation of any localized lung tumour. Group 3-SOI was characterized by high perioperative mortality, initially localized lung tumours, and local evolution. Group 4-TOI was associated with low perioperative mortality, initially localized lung tumours, loco regional extension, and distant metastasis. CTCs were detected in 83% of nude mice bearing subcutaneous or orthotopic NSCLC tumours. Conclusions Transpleural orthotopic injection of A549-luc cells in nude mouse lung induces localized tumour, followed by lymphatic extension and specific mortality, and allowed the first time identification of CTCs in a NSCLC mice model. PMID:22022511

  8. The glucocorticoid-glucocorticoid receptor signal transduction pathway, transforming growth factor-beta, and embryonic mouse lung development in vivo.

    PubMed

    Jaskoll, T; Choy, H A; Melnick, M

    1996-05-01

    Lung morphogenesis has been shown to be regulated by glucocorticoids (CORT). Because CORT has been primarily thought to affect fetal lung development, previous studies have focused on the role of CORT receptor (GR)-mediated regulation of fetal lung development. Although endogenous CORT increases during embryonic and fetal stages and exogenous CORT treatment in vivo and in vitro clearly accelerates embryonic lung development, little is known about the morphoregulatory role of the embryonic CORT-GR signal transduction pathway during lung development. In this study, we characterize the embryonic mouse CORT-GR pathway and demonstrate: stage-specific in situ patterns of GR immunolocalization; similarity in GR relative mobility with progressive (E13 --> E17) development; that embryonic GR can be activated to bind a GR response element (GRE); significantly increasing levels of functional GR with increasing lung maturation; and the presence of heat shock protein (hsp) 70 and hsp90 from early (E13) to late (E17) developmental stages. These results support the purported importance of the embryonic CORT-GR signal transduction pathway in progressive lung differentiation. To demonstrate that the embryonic CORT-GR directed pathway plays a role in lung development, early embryonic (E12) lungs were exposed to CORT in utero and surfactant-associated protein A (SP-A) expression was analyzed; CORT treatment up-regulates SP-A mRNA expression and spatiotemporal protein distribution. Finally, to determine whether CORT-GR-directed pulmonary morphogenesis in vivo involves the modulation of growth factors, we studied the effect of CORT on TGF-beta gene expression. Northern analysis of TGF-beta 1, TGF-beta 2, and TGF-beta 3 transcript levels in vivo indicates that CORT regulates the rate of lung morpho- and histodifferentiation by down-regulating TGF-beta 3 gene expression.

  9. Low oxygen tension enhances the generation of lung progenitor cells from mouse embryonic and induced pluripotent stem cells

    PubMed Central

    Garreta, Elena; Melo, Esther; Navajas, Daniel; Farré, Ramon

    2014-01-01

    Abstract Whole‐organ decellularization technology has emerged as a new alternative for the fabrication of bioartificial lungs. Embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) are potentially useful for recellularization since they can be directed to express phenotypic marker genes of lung epithelial cells. Normal pulmonary development takes place in a low oxygen environment ranging from 1 to 5%. By contrast, in vitro ESC and iPSC differentiation protocols are usually carried out at room‐air oxygen tension. Here, we sought to determine the role played by oxygen tension on the derivation of Nkx2.1+ lung/thyroid progenitor cells from mouse ESC and iPSC. A step‐wise differentiation protocol was used to generate Nkx2.1+ lung/thyroid progenitors under 20% and 5% oxygen tension. On day 12, gene expression analysis revealed that Nkx2.1 and Foxa2 (endodermal and early lung epithelial cell marker) were significantly upregulated at 5% oxygen tension in ESC and iPSC differentiated cultures compared to 20% oxygen conditions. In addition, quantification of Foxa2+Nkx2.1+Pax8‐ cells corresponding to the lung field, with exclusion of the potential thyroid fate identified by Pax8 expression, confirmed that the low physiologic oxygen tension exerted a significant positive effect on early pulmonary differentiation of ESC and iPSC. In conclusion, we found that 5% oxygen tension enhanced the derivation of lung progenitors from mouse ESC and iPSC compared to 20% room‐air oxygen tension. PMID:25347858

  10. [Effect of thalidomide in a mouse model of paraquat-induced acute lung injury and the underlying mechanisms].

    PubMed

    Zhao, Guangju; Cai, Xiaoxia; She, Xingrong; Li, Dong; Hong, Guangliang; Wu, Bin; Li, Mengfang; Lu, Zhongqiu

    2014-11-01

    To investigate the effects of thalidomide in a mouse model of paraquat-induced acute lung injury and the mechanisms underlying the properties of thalidomide. Male ICR mice were randomly allocated into four groups: nomal control group (n = 30), thalidomide control group (n = 30), paraquat poisioning group (n = 30) and thalidomide treatment group (n = 90). Mice were sacrificed at 1d, 3d and 7d after paraquat poisioning. The level of (MDA) malondialdehyde, Superoxidedi-smutase (SOD) and glutathione (GSH) in the lung tissue were measuerd by chemical colorimetry. The expression of Nrf2 mRNA was determined by RT-PCR; Nuclear protein Nrf2 was abserved by Western blotting; Pathological changes of lung tissue were observed under light microscope by HE stain; the lung apoptosis cells were detected by TUNEL. The levels of MDA, SOD and the expressions Nrf2 mRNA and protein Nrf2 in lung tissue were all markedly increased in mice of paraquat poisioning group than those in nomal group at 1 d, 3 d, 7 d. In contrast, the levels of GSH were decreaseel (P<0.05). Compared with paraquat poisioning group, the pulmonary SOD, Nrf2 mRNA and protein were increased and the lung wet dry ratio were all significantly decreased in mice of THD treatment group at 1 d, 3 d, 7 d (P<0.05). THD alleviated the pulmonary damage in the lightmicroscope at 3d after paraquat poisioning. The apoptosis index was markedly decreased in THD treatment groups comparing to paraquat piosioning group (P<0.05). Lipid peroxide damage was one of the mechanisms of paraquat poisioning, thalidomide could attenuate paraquat-induced acute lung injury and its mechanism may be activating the Nrf2-ARE signaling pathway to protect mouse from Lipid peroxide damage.

  11. Network Inference Algorithms Elucidate Nrf2 Regulation of Mouse Lung Oxidative Stress

    PubMed Central

    Singhal, Mudita; Malhotra, Deepti; Biswal, Shyam

    2008-01-01

    A variety of cardiovascular, neurological, and neoplastic conditions have been associated with oxidative stress, i.e., conditions under which levels of reactive oxygen species (ROS) are elevated over significant periods. Nuclear factor erythroid 2-related factor (Nrf2) regulates the transcription of several gene products involved in the protective response to oxidative stress. The transcriptional regulatory and signaling relationships linking gene products involved in the response to oxidative stress are, currently, only partially resolved. Microarray data constitute RNA abundance measures representing gene expression patterns. In some cases, these patterns can identify the molecular interactions of gene products. They can be, in effect, proxies for protein–protein and protein–DNA interactions. Traditional techniques used for clustering coregulated genes on high-throughput gene arrays are rarely capable of distinguishing between direct transcriptional regulatory interactions and indirect ones. In this study, newly developed information-theoretic algorithms that employ the concept of mutual information were used: the Algorithm for the Reconstruction of Accurate Cellular Networks (ARACNE), and Context Likelihood of Relatedness (CLR). These algorithms captured dependencies in the gene expression profiles of the mouse lung, allowing the regulatory effect of Nrf2 in response to oxidative stress to be determined more precisely. In addition, a characterization of promoter sequences of Nrf2 regulatory targets was conducted using a Support Vector Machine classification algorithm to corroborate ARACNE and CLR predictions. Inferred networks were analyzed, compared, and integrated using the Collective Analysis of Biological Interaction Networks (CABIN) plug-in of Cytoscape. Using the two network inference algorithms and one machine learning algorithm, a number of both previously known and novel targets of Nrf2 transcriptional activation were identified. Genes predicted as

  12. Proteoglycans Maintain Lung Stability in an Elastase-Treated Mouse Model of Emphysema

    PubMed Central

    Takahashi, Ayuko; Majumdar, Arnab; Parameswaran, Harikrishnan; Bartolák-Suki, Erzsébet

    2014-01-01

    Extracellular matrix remodeling and tissue rupture contribute to the progression of emphysema. Lung tissue elasticity is governed by the tensile stiffness of fibers and the compressive stiffness of proteoglycans. It is not known how proteoglycan remodeling affects tissue stability and destruction in emphysema. The objective of this study was to characterize the role of remodeled proteoglycans in alveolar stability and tissue destruction in emphysema. At 30 days after treatment with porcine pancreatic elastase, mouse lung tissue stiffness and alveolar deformation were evaluated under varying tonicity conditions that affect the stiffness of proteoglycans. Proteoglycans were stained and measured in the alveolar walls. Computational models of alveolar stability and rupture incorporating the mechanical properties of fibers and proteoglycans were developed. Although absolute tissue stiffness was only 24% of normal, changes in relative stiffness and alveolar shape distortion due to changes in tonicity were increased in emphysema (P < 0.01 and P < 0.001). Glycosaminoglycan amount per unit alveolar wall length, which is responsible for proteoglycan stiffness, was higher in emphysema (P < 0.001). Versican expression increased in the tissue, but decorin decreased. Our network model predicted that the rate of tissue deterioration locally governed by mechanical forces was reduced when proteoglycan stiffness was increased. Consequently, this general network model explains why increasing proteoglycan deposition protects the alveolar walls from rupture in emphysema. Our results suggest that the loss of proteoglycans observed in human emphysema contributes to disease progression, whereas treatments that promote proteoglycan deposition in the extracellular matrix should slow the progression of emphysema. PMID:24450478

  13. Acinetobacter baumannii Infection Inhibits Airway Eosinophilia and Lung Pathology in a Mouse Model of Allergic Asthma

    PubMed Central

    Qiu, Hongyu; KuoLee, Rhonda; Harris, Greg; Zhou, Hongyan; Miller, Harvey; Patel, Girishchandra B.; Chen, Wangxue

    2011-01-01

    Allergic asthma is a dysregulation of the immune system which leads to the development of Th2 responses to innocuous antigens (allergens). Some infections and microbial components can re-direct the immune response toward the Th1 response, or induce regulatory T cells to suppress the Th2 response, thereby inhibiting the development of allergic asthma. Since Acinetobacter baumannii infection can modulate lung cellular and cytokine responses, we studied the effect of A. baumannii in modulating airway eosinophilia in a mouse model of allergic asthma. Ovalbumin (OVA)-sensitized mice were treated with live A. baumannii or phosphate buffered saline (PBS), then intranasally challenged with OVA. Compared to PBS, A. baumannii treatment significantly reduced pulmonary Th2 cytokine and chemokine responses to OVA challenge. More importantly, the airway inflammation in A. baumannii-treated mice was strongly suppressed, as seen by the significant reduction of the proportion and the total number of eosinophils in the bronchoalveolar lavage fluid. In addition, A. baumannii-treated mice diminished lung mucus overproduction and pathology. However, A. baumannii treatment did not significantly alter systemic immune responses to OVA. Serum OVA-specific IgE, IgG1 and IgG2a levels were comparable between A. baumannii- and PBS-treated mice, and tracheobronchial lymph node cells from both treatment groups produced similar levels of Th1 and Th2 cytokines in response to in vitro OVA stimulation. Moreover, it appears that TLR-4 and IFN-γ were not directly involved in the A. baumannii-induced suppression of airway eosinophilia. Our results suggest that A. baumannii inhibits allergic airway inflammation by direct suppression of local pulmonary Th2 cytokine responses to the allergen. PMID:21789200

  14. api, A novel Medicago truncatula symbiotic mutant impaired in nodule primordium invasion.

    PubMed

    Teillet, Alice; Garcia, Joseph; de Billy, Françoise; Gherardi, Michèle; Huguet, Thierry; Barker, David G; de Carvalho-Niebel, Fernanda; Journet, Etienne-Pascal

    2008-05-01

    Genetic approaches have proved to be extremely useful in dissecting the complex nitrogen-fixing Rhizobium-legume endosymbiotic association. Here we describe a novel Medicago truncatula mutant called api, whose primary phenotype is the blockage of rhizobial infection just prior to nodule primordium invasion, leading to the formation of large infection pockets within the cortex of noninvaded root outgrowths. The mutant api originally was identified as a double symbiotic mutant associated with a new allele (nip-3) of the NIP/LATD gene, following the screening of an ethylmethane sulphonate-mutagenized population. Detailed characterization of the segregating single api mutant showed that rhizobial infection is also defective at the earlier stage of infection thread (IT) initiation in root hairs, as well as later during IT growth in the small percentage of nodules which overcome the primordium invasion block. Neither modulating ethylene biosynthesis (with L-alpha-(2-aminoethoxyvinylglycine or 1-aminocyclopropane-1-carboxylic acid) nor reducing ethylene sensitivity in a skl genetic background alters the basic api phenotype, suggesting that API function is not closely linked to ethylene metabolism or signaling. Genetic mapping places the API gene on the upper arm of the M. truncatula linkage group 4, and epistasis analyses show that API functions downstream of BIT1/ERN1 and LIN and upstream of NIP/LATD and the DNF genes.

  15. Reaction-diffusion finite element model of lateral line primordium migration to explore cell leadership.

    PubMed

    Allena, R; Maini, P K

    2014-12-01

    Collective cell migration plays a fundamental role in many biological phenomena such as immune response, embryogenesis and tumorigenesis. In the present work, we propose a reaction-diffusion finite element model of the lateral line primordium migration in zebrafish. The population is modelled as a continuum with embedded discrete motile cells, which are assumed to be viscoelastic and able to undergo large deformations. The Wnt/ß-catenin-FGF and cxcr4b-cxcr7b signalling pathways inside the cohort regulating the migration are described through coupled reaction-diffusion equations. The coupling between mechanics and the molecular scenario occurs in two ways. Firstly, the intensity of the protrusion-contraction movement of the cells depends on the cxcr4b concentration. Secondly, the intra-synchronization between the active deformations and the adhesion forces inside each cell is triggered by the cxcr4b-cxcr7b polarity. This influences the inter-synchronization between the cells and results in two main modes of migration: uncoordinated and coordinated. The main objectives of the work were (i) to validate our assumptions with respect to the experimental observations and (ii) to decipher the mechanical conditions leading to efficient migration of the primordium. To achieve the second goal, we will specifically focus on the role of the leader cells and their position inside the population.

  16. Stereological assessment of mouse lung parenchyma via nondestructive, multiscale micro-CT imaging validated by light microscopic histology.

    PubMed

    Vasilescu, Dragos M; Klinge, Christine; Knudsen, Lars; Yin, Leilei; Wang, Ge; Weibel, Ewald R; Ochs, Matthias; Hoffman, Eric A

    2013-03-15

    Quantitative assessment of the lung microstructure using standard stereological methods such as volume fractions of tissue, alveolar surface area, or number of alveoli, are essential for understanding the state of normal and diseased lung. These measures are traditionally obtained from histological sections of the lung tissue, a process that ultimately destroys the three-dimensional (3-D) anatomy of the tissue. In comparison, a novel X-ray-based imaging method that allows nondestructive sectioning and imaging of fixed lungs at multiple resolutions can overcome this limitation. Scanning of the whole lung at high resolution and subsequent regional sampling at ultrahigh resolution without physically dissecting the organ allows the application of design-based stereology for assessment of the whole lung structure. Here we validate multiple stereological estimates performed on micro-computed tomography (μCT) images by comparing them with those obtained via conventional histology on the same mouse lungs. We explore and discuss the potentials and limitations of the two approaches. Histological examination offers higher resolution and the qualitative differentiation of tissues by staining, but ultimately loses 3-D tissue relationships, whereas μCT allows for the integration of morphometric data with the spatial complexity of lung structure. However, μCT has limited resolution satisfactory for the sterological estimates presented in this study but not for differentiation of tissues. We conclude that introducing stereological methods in μCT studies adds value by providing quantitative information on internal structures while not curtailing more complex approaches to the study of lung architecture in the context of physiological or pathological studies.

  17. Stereological assessment of mouse lung parenchyma via nondestructive, multiscale micro-CT imaging validated by light microscopic histology

    PubMed Central

    Vasilescu, Dragoş M.; Klinge, Christine; Knudsen, Lars; Yin, Leilei; Wang, Ge; Weibel, Ewald R.; Ochs, Matthias

    2013-01-01

    Quantitative assessment of the lung microstructure using standard stereological methods such as volume fractions of tissue, alveolar surface area, or number of alveoli, are essential for understanding the state of normal and diseased lung. These measures are traditionally obtained from histological sections of the lung tissue, a process that ultimately destroys the three-dimensional (3-D) anatomy of the tissue. In comparison, a novel X-ray-based imaging method that allows nondestructive sectioning and imaging of fixed lungs at multiple resolutions can overcome this limitation. Scanning of the whole lung at high resolution and subsequent regional sampling at ultrahigh resolution without physically dissecting the organ allows the application of design-based stereology for assessment of the whole lung structure. Here we validate multiple stereological estimates performed on micro–computed tomography (μCT) images by comparing them with those obtained via conventional histology on the same mouse lungs. We explore and discuss the potentials and limitations of the two approaches. Histological examination offers higher resolution and the qualitative differentiation of tissues by staining, but ultimately loses 3-D tissue relationships, whereas μCT allows for the integration of morphometric data with the spatial complexity of lung structure. However, μCT has limited resolution satisfactory for the sterological estimates presented in this study but not for differentiation of tissues. We conclude that introducing stereological methods in μCT studies adds value by providing quantitative information on internal structures while not curtailing more complex approaches to the study of lung architecture in the context of physiological or pathological studies. PMID:23264542

  18. PR-Set7 is degraded in a conditional Cul4A transgenic mouse model of lung cancer

    DOE PAGES

    Wang, Yang; Xu, Zhidong; Mao, Jian -Hua; ...

    2015-06-01

    Background and objective. Maintenance of genomic integrity is essential to ensure normal organismal development and to prevent diseases such as cancer. PR-Set7 (also known as Set8) is a cell cycle regulated enzyme that catalyses monomethylation of histone 4 at Lys20 (H4K20me1) to promote chromosome condensation and prevent DNA damage. Recent studies show that CRL4CDT2-mediated ubiquitylation of PR-Set7 leads to its degradation during S phase and after DNA damage. This might occur to ensure appropriate changes in chromosome structure during the cell cycle or to preserve genome integrity after DNA damage. Methods. We developed a new model of lung tumor developmentmore » in mice harboring a conditionally expressed allele of Cul4A. We have therefore used a mouse model to demonstrate for the first time that Cul4A is oncogenic in vivo. With this model, staining of PR-Set7 in the preneoplastic and tumor lesions in AdenoCre-induced mouse lungs was performed. Meanwhile we identified higher protein level changes of γ-tubulin and pericentrin by IHC. Results. The level of PR-Set7 down-regulated in the preneoplastic and adenocarcinomous lesions following over-expression of Cul4A. We also identified higher levels of the proteins pericentrin and γ-tubulin in Cul4A mouse lungs induced by AdenoCre. Conclusion. PR-Set7 is a direct target of Cul4A for degradation and involved in the formation of lung tumors in the conditional Cul4A transgenic mouse model.« less

  19. Mouse lung slices: An ex vivo model for the evaluation of antiviral and anti-inflammatory agents against influenza viruses.

    PubMed

    Liu, Rui; An, Liwei; Liu, Ge; Li, Xiaoyu; Tang, Wei; Chen, Xulin

    2015-08-01

    The influenza A virus is notoriously known for its ability to cause recurrent epidemics and global pandemics. Antiviral therapy is effective when treatment is initiated within 48h of symptom onset, and delaying treatment beyond this time frame is associated with decreased efficacy. Research on anti-inflammatory therapy to ameliorate influenza-induced inflammation is currently underway and seems important to the impact on the clinical outcome. Both antiviral and anti-inflammatory drugs with novel mechanisms of action are urgently needed. Current methods for evaluating the efficacy of anti-influenza drugs rely mostly on transformed cells and animals. Transformed cell models are distantly related to physiological and pathological conditions. Although animals are the best choices for preclinical drug testing, they are not time- or cost-efficient. In this study, we established an ex vivo model using mouse lung slices to evaluate both antiviral and anti-inflammatory agents against influenza virus infection. Both influenza virus PR8 (H1N1) and A/Human/Hubei/3/2005 (H3N2) can replicate efficiently in mouse lung slices and trigger significant cytokine and chemokine responses. The induction of selected cytokines and chemokines were found to have a positive correlation between ex vivo and in vivo experiments, suggesting that the ex vivo cultured lung slices may closely resemble the lung functionally in an in vivo configuration when challenged by influenza virus. Furthermore, a set of agents with known antiviral and/or anti-inflammatory activities were tested to validate the ex vivo model. Our results suggested that mouse lung slices provide a robust, convenient and cost-efficient model for the assessment of both antiviral and anti-inflammatory agents against influenza virus infection in one assay. This ex vivo model may predict the efficacy of drug candidates' antiviral and anti-inflammatory activities in vivo.

  20. Ion-Current-Based Temporal Proteomic Profiling of Influenza-A-Virus-Infected Mouse Lungs Revealed Underlying Mechanisms of Altered Integrity of the Lung Microvascular Barrier.

    PubMed

    Shen, Shichen; Li, Jun; Hilchey, Shannon; Shen, Xiaomeng; Tu, Chengjian; Qiu, Xing; Ng, Andrew; Ghaemmaghami, Sina; Wu, Hulin; Zand, Martin S; Qu, Jun

    2016-02-05

    Investigation of influenza-A-virus (IAV)-infected lung proteomes will greatly promote our understanding on the virus-host crosstalk. Using a detergent-cocktail extraction and digestion procedure and a reproducible ion-current-based method, we performed the first comprehensive temporal analysis of mouse IAV infection. Mouse lung tissues at three time points post-inoculation were compared with controls (n = 4/group), and >1600 proteins were quantified without missing value in any animal. Significantly changed proteins were identified at 4 days (n = 144), 7 days (n = 695), and 10 days (n = 396) after infection, with low false altered protein rates (1.73-8.39%). Functional annotation revealed several key biological processes involved in the systemic host responses. Intriguingly, decreased levels of several cell junction proteins as well as increased levels of tissue metalloproteinase MMP9 were observed, reflecting the IAV-induced structural breakdown of lung epithelial barrier. Supporting evidence of MMP9 activation came from immunoassays examining the abundance and phosphorylation states of all MAPKs and several relevant molecules. Importantly, IAV-induced MMP gelatinase expression was suggested to be specific to MMP9, and p38 MAPK may contribute predominantly to MMP9 elevation. These findings help to resolve the long-lasting debate regarding the signaling pathways of IAV-induced MMP9 expression and shed light on the molecular mechanisms underlying pulmonary capillary-alveolar leak syndrome that can occur during influenza infection.

  1. The Shc 66 and 46 kD isoforms are differentially downregulated at parturition in the fetal mouse lung.

    PubMed

    Lee, M K; Zhao, J; Smith, S M; Tefft, J D; Bringas, P; Hwang, C; Warburton, D

    1998-12-01

    Many of the signaling pathways regulating fetal lung mesenchymal cell proliferation are mediated by the Shc intracellular signaling proteins. Shc is expressed as three isoforms: 52 kD and 46 kD proteins (Shc 52 and Shc 46, respectively) translated from the same mRNA, and a 66 kD form (Shc 66) translated from a separate mRNA. Shc 52 is an activator of Ras and mitogen-activated protein kinase, whereas Shc 66 antagonizes Ras activation. The function of Shc 46 is unclear. We hypothesized that the Shc isoforms are differentially regulated during fetal mouse lung morphogenesis. Relative Shc 66 and Shc 46 protein expression are high until parturition (term = 18.5 d), when a dramatic decrease begins; by postconceptual d 20, relative Shc 66 and Shc 46 expression have fallen by 75 and 69%, respectively. A similar pattern of decreasing Shc 66 mRNA expression in the peripartum period was detected by reverse transcription and competitive polymerase chain reaction during the same period. By isoform-specific immunohistochemistry, Shc 66 is widely distributed in the embryonic lung but becomes restricted to the bronchial smooth muscle and overlying epithelia, periarterial smooth muscle, and the interlobar pleura late in gestation. After parturition, Shc 66 is virtually absent from the lung. All three Shc isoforms are phosphorylated by epidermal growth factor stimulation in fetal lung mesenchymal cells, indicating that Shc 66 is functional in these cells. These data indicate that Shc isoforms are differentially regulated during lung development.

  2. In vivo tomographic imaging of lung colonization of tumour in mouse with simultaneous fluorescence and X-ray CT.

    PubMed

    Zhang, Bin; Gao, Fuping; Wang, Mengjiao; Cao, Xu; Liu, Fei; Wang, Xin; Luo, Jianwen; Wang, Guangzhi; Bai, Jing

    2014-01-01

    Non-invasive in vivo imaging of diffuse and wide-spread colonization within the lungs, rather than distinct solid primary tumors, is still a challenging work. In this work, a lung colonization mouse model bearing A549 human lung tumor was simultaneously scanned by a dual-modality fluorescence molecular tomography (FMT) and X-ray computed tomography (CT) system in vivo. A two steps method which incorporates CT structural information into the FMT reconstruction procedure is employed to provide concurrent anatomical and functional information. By using the target-specific fluorescence agent, the fluorescence tomographic results show elevated fluorescence intensity deep within the lungs which is colonized with diffuse and wide-spread tumors. The results were confirmed with ex vivo fluorescence reflectance imaging and histological examination of the lung tissues. With FMT reconstruction combined with the CT information, the dual-modality FMT/micro-CT system is expected to offer sensitive and noninvasive imaging of diffuse tumor colonization within the lungs in vivo. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. DNA vaccine elicits an efficient antitumor response by targeting the mutant Kras in a transgenic mouse lung cancer model.

    PubMed

    Weng, T-Y; Yen, M-C; Huang, C-T; Hung, J-J; Chen, Y-L; Chen, W-C; Wang, C-Y; Chang, J-Y; Lai, M-D

    2014-10-01

    Mutant Kras (V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) is observed in more than 20% of non-small-cell lung cancers; however, no effective Kras target therapy is available at present. The Kras DNA vaccine may represent as a novel immunotherapeutic agent in lung cancer. In this study, we investigated the antitumor efficacy of the Kras DNA vaccine in a genetically engineered inducible mouse lung tumor model driven by Kras(G12D). Lung tumors were induced by doxycycline, and the therapeutic effects of Kras DNA vaccine were evaluated with delivery of Kras(G12D) plasmids. Mutant Kras(G12D) DNA vaccine significantly decreased the tumor nodules. A dominant-negative mutant Kras(G12D)N17, devoid of oncogenic activity, achieved similar therapeutic effects. The T-helper 1 immune response was enhanced in mice treated with Kras DNA vaccine. Splenocytes from mice receiving Kras DNA vaccine presented an antigen-specific response by treatment with peptides of Kras but not Hras or OVA. The number of tumor-infiltrating CD8(+) T cells increased after Kras vaccination. In contrast, Kras DNA vaccine was not effective in the lung tumor in transgenic mice, which was induced by mutant L858R epidermal growth factor receptor. Overall, these results indicate that Kras DNA vaccine produces an effective antitumor response in transgenic mice, and may be useful in treating lung cancer-carrying Ras mutation.

  4. Cationic amphiphiles with fatty acyl chain asymmetry of coconut oil deliver genes selectively to mouse lung.

    PubMed

    Chandrashekhar, Voshavar; Srujan, Marepally; Prabhakar, Rairala; Reddy, Rakesh C; Sreedhar, Bojja; Rentam, Kiran K R; Kanjilal, Sanjit; Chaudhuri, Arabinda

    2011-03-16

    Recent structure-activity studies have revealed a dramatic influence of hydrophobic chain asymmetry in enhancing gene delivery efficacies of synthetic cationic amphiphiles (Nantz, M. H. et al. Mol. Pharmaceutics2010, 7, 786-794; Koynova, R. et al. Mol. Pharmaceutics2009, 6, 951-958). The present findings demonstrate for the first time that such a transfection enhancing influence of asymmetric hydrocarbon chains observed in pure synthetic cationic amphiphiles also works for cationic amphiphiles designed with natural, asymmetric fatty acyl chains of a food-grade oil. Herein, we demonstrate that cationic amphiphiles designed with the natural fatty acyl chain asymmetry of food-grade coconut oil are less cytotoxic and deliver genes selectively to mouse lung. Despite lauroyl chains being the major fatty acyl chains of coconut oil, both the in vitro and In vivo gene transfer efficiencies of such cationic amphiphiles were found to be remarkably superior (>4-fold) to those of their pure dilauroyl analogue. Mechanistic studies involving the technique of fluorescence resonance energy transfer (FRET) revealed higher biomembrane fusibility of the cationic liposomes of the coconut amphiphiles than that of the symmetric dilauroyl analogue. AFM study revealed pronounced fusogenic nonlamellar structures of the liposomes of coconut amphiphiles. Findings in the FRET and cellular uptake study, taken together, support the notion that the higher cellular uptake resulting from the more fusogenic nature of the liposomes of coconut amphiphiles 1 are likely to play a dominant role in making the coconut amphiphiles transfection competent.

  5. Arsenic trioxide inhibits lung metastasis of mouse colon cancer via reducing the infiltration of regulatory T cells.

    PubMed

    Wang, Lei; Hu, Xiang; Xu, Yingxin; Liu, Zhong

    2016-11-01

    The purpose of this study was to investigate the effects of arsenic trioxide (As2O3) on the infiltration of regulatory T cells (Tregs) in the local lung metastasis of mouse colon cancer in vivo and the regulation of Tregs in cytokine-induced killer cells (CIKs) in vitro. A high Tregs infiltration mouse colon cancer lung metastasis model was established by intravenous injection of CT26 murine colon carcinoma cells. Tumor-bearing mice were randomly divided into three groups: control group, low-dose As2O3 group, and high-dose As2O3 group. For in vitro studies, CIKs were treated with vehicle control or 0.1, 1, or 5 μM As2O3. The level of Tregs was detected via flow cytometry, Foxp3 expression was assessed by immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR), the level of interferon gamma (IFN-γ) was evaluated by enzyme-linked immunoassay (ELISA), and the cytotoxic activity of As2O3-treated CIKs was assessed through a lactate dehydrogenase (LDH) release assay. Obvious lung metastasis was observed 3 days after CT26 murine colon carcinoma cell injection. The numbers of Tregs in the lungs and spleens of tumor-bearing mice were significantly higher than those of the normal group (p < 0.01). As2O3 treatment increased the mouse weight as well as reduced the number of metastatic lung nodules and the lung/body weight ratio (p < 0.01). Moreover, As2O3 treatment significantly reduced the Tregs proportion and the Foxp3 messenger RNA (mRNA) levels in metastatic lung tissues (p < 0.01). In vitro, As2O3 significantly reduced the Tregs proportion and the Foxp3 mRNA levels (p < 0.01) and significantly increased the cytotoxic activity of CIKs and the IFN-γ levels in the supernatant of cultured CIKs (p < 0.01). As2O3 might inhibit lung metastasis of colon cancer by reducing the local infiltration of Tregs and increase the cytotoxic activity of CIKs by suppressing Tregs.

  6. Airway segmentation and analysis for the study of mouse models of lung disease using micro-CT

    NASA Astrophysics Data System (ADS)

    Artaechevarria, X.; Pérez-Martín, D.; Ceresa, M.; de Biurrun, G.; Blanco, D.; Montuenga, L. M.; van Ginneken, B.; Ortiz-de-Solorzano, C.; Muñoz-Barrutia, A.

    2009-11-01

    Animal models of lung disease are gaining importance in understanding the underlying mechanisms of diseases such as emphysema and lung cancer. Micro-CT allows in vivo imaging of these models, thus permitting the study of the progression of the disease or the effect of therapeutic drugs in longitudinal studies. Automated analysis of micro-CT images can be helpful to understand the physiology of diseased lungs, especially when combined with measurements of respiratory system input impedance. In this work, we present a fast and robust murine airway segmentation and reconstruction algorithm. The algorithm is based on a propagating fast marching wavefront that, as it grows, divides the tree into segments. We devised a number of specific rules to guarantee that the front propagates only inside the airways and to avoid leaking into the parenchyma. The algorithm was tested on normal mice, a mouse model of chronic inflammation and a mouse model of emphysema. A comparison with manual segmentations of two independent observers shows that the specificity and sensitivity values of our method are comparable to the inter-observer variability, and radius measurements of the mainstem bronchi reveal significant differences between healthy and diseased mice. Combining measurements of the automatically segmented airways with the parameters of the constant phase model provides extra information on how disease affects lung function.

  7. Immunohistochemical and immunocytochemical detection of SchS34 antigen in Stachybotrys chartarum spores and spore impacted mouse lungs.

    PubMed

    Rand, Thomas G; Miller, J David

    2008-02-01

    The purpose of this study was to evaluate the distribution of a 34 kD antigen isolated from S. chartarum sensu lato in spores and in the mouse lung 48 h after intra-tracheal instillation of spores by immuno-histochemistry. This antigen was localized in spore walls, primarily in the outer and inner wall layers and on the external wall surfaces with modest labelling observed in cytoplasm. Immuno-histochemistry revealed that in spore impacted mouse lung, antigen was again observed in spore walls, along the outside surface of the outer wall and in the intercellular space surrounding spores. In lung granulomas the labelled antigen formed a diffusate, some 2-3x the size of the long axis of spores, with highest concentrations nearest to spores. Collectively, these observations indicated that this protein not only displayed a high degree of specificity with respect to its location in spores and wall fragments, but also that it slowly diffuses into surrounding lungs.

  8. Kinase domain activation of FGFR2 yields high-grade lung adenocarcinoma sensitive to a Pan-FGFR inhibitor in a mouse model of NSCLC.

    PubMed

    Tchaicha, Jeremy H; Akbay, Esra A; Altabef, Abigail; Mikse, Oliver R; Kikuchi, Eiki; Rhee, Kevin; Liao, Rachel G; Bronson, Roderick T; Sholl, Lynette M; Meyerson, Matthew; Hammerman, Peter S; Wong, Kwok-Kin

    2014-09-01

    Somatic mutations in FGFR2 are present in 4% to 5% of patients diagnosed with non-small cell lung cancer (NSCLC). Amplification and mutations in FGFR genes have been identified in patients with NSCLCs, and clinical trials are testing the efficacy of anti-FGFR therapies. FGFR2 and other FGFR kinase family gene alterations have been found in both lung squamous cell carcinoma and lung adenocarcinoma, although mouse models of FGFR-driven lung cancers have not been reported. Here, we generated a genetically engineered mouse model (GEMM) of NSCLC driven by a kinase domain mutation in FGFR2. Combined with p53 ablation, primary grade 3/4 adenocarcinoma was induced in the lung epithelial compartment exhibiting locally invasive and pleiotropic tendencies largely made up of multinucleated cells. Tumors were acutely sensitive to pan-FGFR inhibition. This is the first FGFR2-driven lung cancer GEMM, which can be applied across different cancer indications in a preclinical setting.

  9. Radiation-induced lung fibrosis in a tumor-bearing mouse model is associated with enhanced Type-2 immunity.

    PubMed

    Chen, Jing; Wang, Yacheng; Mei, Zijie; Zhang, Shimin; Yang, Jie; Li, Xin; Yao, Ye; Xie, Conghua

    2016-03-01

    Lung fibrosis may be associated with Type-2 polarized inflammation. Herein, we aim to investigate whether radiation can initiate a Type-2 immune response and contribute to the progression of pulmonary fibrosis in tumor-bearing animals. We developed a tumor-bearing mouse model with Lewis lung cancer to receive either radiation therapy alone or radiation combined with Th1 immunomodulator unmethylated cytosine-phosphorothioate-guanine containing oligodeoxynucleotide (CpG-ODN). The Type-2 immune phenotype in tumors and the histological grade of lung fibrosis were evaluated in mice sacrificed three weeks after irradiation. Mouse lung tissues were analyzed for hydroxyproline and the expression of Type-1/Type-2 key transcription factors (T-bet/GATA-3). The concentration of Type-1/Type-2 cytokines in serum was measured by cytometric bead array. Lung fibrosis was observed to be more serious in tumor-bearing mice than in normal mice post-irradiation. The fibrosis score in irradiated tumor-bearing mice on Day 21 was 4.33 ± 0.82, which was higher than that of normal mice (2.00 ± 0.63; P < 0.05). Hydroxyproline and GATA-3 expression were increased in the lung tissues of tumor-bearing mice following irradiation. CpG-ODN attenuated fibrosis by markedly decreasing GATA-3 expression. Serum IL-13 and IL-5 were elevated, whereas INF-γ and IL-12 expression were decreased in irradiated tumor-bearing mice. These changes were reversed after CpG-ODN treatment. Thus, Type-2 immunity in tumors appeared to affect the outcome of radiation damage and might be of interest for future studies on developing approaches in which Type-1-related immunotherapy and radiotherapy are used in combination. © The Author 2015. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.

  10. A Novel Nontoxic Inhibitor of the Activation of NADPH Oxidase Reduces Reactive Oxygen Species Production in Mouse LungS⃞

    PubMed Central

    Lee, Intae; Dodia, Chandra; Chatterjee, Shampa; Zagorski, John; Mesaros, Clementina; Blair, Ian A.; Feinstein, Sheldon I.; Jain, Mahendra

    2013-01-01

    1-Hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33) is a fluorinated phospholipid analog that inhibits the phospholipase A2 (PLA2) activity of peroxiredoxin 6 (Prdx6). Prdx6 PLA2 activity is required for activation of NADPH oxidase 2 and subsequent generation of reactive oxygen species (ROS). In vitro, MJ33 inhibited agonist-stimulated production of ROS by the isolated perfused mouse lung, lung microvascular endothelial cells, and polymorphonuclear leukocytes. MJ33 (0.02–0.5 µmol MJ33/kg body weight) in mixed unilamellar liposomes was administered to C57BL/6 mice by either intratracheal (i.t.) or i.v. routes. Lung MJ33 content, measured by liquid chromatography/mass spectroscopy, showed uptake of 67–87% of the injected dose for i.t. and 23–42% for i.v. administration at 4 hours postinjection. PLA2 activity of lung homogenates was markedly inhibited (>85%) at 4 hours postadministration. Both MJ33 content and PLA2 activity gradually returned to near control levels over the subsequent 24–72 hours. Mice treated with MJ33 at 12.5–25 µmol/kg did not show changes (compared with control) in clinical symptomatology, body weight, hematocrit, and histology of lung, liver, and kidney during a 30- to 50-day observation period. Thus, the toxic dose of MJ33 was >25 µmol/kg, whereas the PLA2 inhibitory dose was approximately 0.02 µmol/kg, indicating a high margin of safety. MJ33 administered to mice prior to lung isolation markedly reduced ROS production and tissue lipid and protein oxidation during ischemia followed by reperfusion. Thus, MJ33 could be useful as a therapeutic agent to prevent ROS-mediated tissue injury associated with lung inflammation or in harvested lungs prior to transplantation. PMID:23475902

  11. Transalveolar osmotic and diffusional water permeability in intact mouse lung measured by a novel surface fluorescence method.

    PubMed

    Carter, E P; Matthay, M A; Farinas, J; Verkman, A S

    1996-09-01

    A surface fluorescence method was developed to measure transalveolar transport of water, protons, and solutes in intact perfused lungs. Lungs from c57 mice were removed and perfused via the pulmonary artery (approximately 2 ml/min). The airspace was filled via the trachea with physiological saline containing a membrane-impermeant fluorescent indicator (FITC-dextran or aminonapthalene trisulfonic acid, ANTS). Because fluorescence is detected only near the lung surface due to light absorption by lung tissue, the surface fluorescence signal is directly proportional to indicator concentration. Confocal microscopy confirmed that the fluorescence signal arises from fluorophores in alveoli just beneath the pleural surface. Osmotic water permeability (Pf) was measured from the time course of intraalveolar FITC-dextran fluorescence in response to changes in perfusate osmolality. Transalveolar Pf was 0.017 +/- 0.001 cm/s at 23 degrees C, independent of the solute used to induce osmosis (sucrose, NaCl, urea), independent of osmotic gradient size and direction, weakly temperature dependent (Arrhenius activation energy 5.3 kcal/mol) and inhibited by HgCl2. Pf was not affected by cAMP activation but was decreased by 43% in lung exposed to hyperoxia for 5 d. Diffusional water permeability (Pd) and Pf were measured in the same lung from intraalveolar ANTS fluorescence, which increased by 1.8-fold upon addition of 50% D2O to the perfusate, Pd was 1.3 x 10(-5) cm/s at 23 degrees C. Transalveolar proton transport was measured from FITC-dextran fluorescence upon switching perfusate pH between 7.4 and 5.6; alveolar pH half-equilibrated in 1.9 and 1.0 min without and with HCO3-, respectively. These results indicate high transalveolar water permeability in mouse lung, implicating the involvement of molecular water channels, and establish a quantitative surface fluorescence method to measure water and solute permeabilities in intact lung.

  12. Macrophages mediate lung inflammation in a mouse model of ischemic acute kidney injury

    PubMed Central

    Altmann, Christopher; Andres-Hernando, Ana; McMahan, Rachel H.; Ahuja, Nilesh; He, Zhibin; Rivard, Chris J.; Edelstein, Charles Louis; Barthel, Lea; Janssen, William J.

    2012-01-01

    Serum IL-6 is increased in acute kidney injury (AKI) and inhibition of IL-6 reduces AKI-mediated lung inflammation. We hypothesized that circulating monocytes produce IL-6 and that alveolar macrophages mediate lung inflammation after AKI via chemokine (CXCL1) production. To investigate systemic and alveolar macrophages in lung injury after AKI, sham operation or 22 min of renal pedicle clamping (AKI) was performed in three experimental settings: 1) systemic macrophage depletion via diphtheria toxin (DT) injection to CD11b-DTR transgenic mice, 2) DT injection to wild-type mice, and 3) alveolar macrophage depletion via intratracheal (IT) liposome-encapsulated clodronate (LEC) administration to wild-type mice. In mice with AKI and systemic macrophage depletion (CD11b-DTR transgenic administered DT) vs. vehicle-treated AKI, blood monocytes and lung interstitial macrophages were reduced, renal function was similar, serum IL-6 was increased, lung inflammation was improved, lung CXCL1 was reduced, and lung capillary leak was increased. In wild-type mice with AKI administered DT vs. vehicle, serum IL-6 was increased. In mice with AKI and alveolar macrophage depletion (IT-LEC) vs. AKI with normal alveolar macrophage content, blood monocytes and lung interstitial macrophages were similar, alveolar macrophages were reduced, renal function was similar, lung inflammation was improved, lung CXCL1 was reduced, and lung capillary leak was increased. In conclusion, administration of DT in AKI is proinflammatory, limiting the use of the DTR-transgenic model to study systemic effects of AKI. Mice with AKI and either systemic mononuclear phagocyte depletion or alveolar macrophage depletion had reduced lung inflammation and lung CXCL1, but increased lung capillary leak; thus, mononuclear phagocytes mediate lung inflammation, but they protect against lung capillary leak after ischemic AKI. Since macrophage activation and chemokine production are key events in the development of acute

  13. Molecular and functional characterization of a calcium-sensitive chloride channel from mouse lung.

    PubMed

    Gandhi, R; Elble, R C; Gruber, A D; Schreur, K D; Ji, H L; Fuller, C M; Pauli, B U

    1998-11-27

    A protein (mCLCA1) has been cloned from a mouse lung cDNA library that bears strong sequence homology with the recently described bovine tracheal, Ca2+-sensitive chloride channel protein (bCLCA1), bovine lung endothelial cell adhesion molecule-1 (Lu-ECAM-1), and the human intestinal Ca2+-sensitive chloride channel protein (hCLCA1). In vitro, its 3.1-kilobase message translates into a 100-kDa protein that can be glycosylated to an approximately 125-kDa product. SDS-polyacrylamide gel electrophoresis from lysates of mCLCA1 cDNA-transfected transformed human embryonic kidney cells (HEK293) reveals proteins of 130, 125, and 90 kDa as well as a protein triplet in the 32-38 kDa size range. Western analyses with antisera raised against Lu-ECAM-1 peptides show that the N-terminal region of the predicted open reading frame is present only in the larger size proteins (i.e. 130, 125, and 90 kDa), whereas the C-terminal region of the open reading frame is observed in the 32-38 kDa size proteins, suggesting a posttranslational, proteolytic processing of a precursor protein (125/130 kDa) into 90 kDa and 32-38 kDa components similar to that reported for Lu-ECAM-1. Hydrophobicity analyses predict four transmembrane domains for the 90-kDa protein. The mCLCA1 mRNA is readily detected by Northern analysis and by in situ hybridization in the respiratory epithelia of trachea and bronchi. Transient expression of mCLCA1 in HEK293 cells was associated with an increase in whole cell Cl- current that could be activated by Ca2+ and ionomycin and inhibited by 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid, dithiothreitol, and niflumic acid. The discovery of mCLCA1 opens the door for further investigating the possible contribution of a Ca2+-sensitive chloride conductance to the pathogenesis of cystic fibrosis.

  14. Analysis of the pathological lesions of the lung in a mouse model of cutaneous infection with Streptococcus pyogenes.

    PubMed

    Minami, Masaaki; Sobue, Sayaka; Ichihara, Masatoshi; Hasegawa, Tadao

    2012-02-01

    Invasive diseases such as toxic shock syndrome caused by Streptococcus pyogenes (S. pyogenes) are re-emerging infectious diseases. The mechanism of pathogenesis is not completely understood although the virulence of this organism has been analyzed using animal model systems, particularly using mice. The analysis of the progression of infection, however, is difficult. Computed tomography (CT) scanning is an extremely powerful technique that we applied to the mouse model of cutaneous infection with S. pyogenes. Two or three days after subcutaneous administration of bacteria, high density reticular areas were detected in the lung by CT. Histopathological examination of the lung was performed to examine the results of CT. Increased numbers of cytokeratin-positive epithelial cells, probably alveolar type II epithelial cells, were detected but no remarkable increase of inflammatory cell infiltrates was observed. Our results show that the pathological lesions of the lung in this model, wherein relatively few numbers of neutrophils were in the alveoli, are well correlated with the lung of a part of streptococcal toxic shock syndrome patients. Therefore, CT may be useful in assessing the progression of S. pyogenes infection, particularly in the pathological lesions of the lung in this model. © 2011 The Authors. Pathology International © 2011 Japanese Society of Pathology and Blackwell Publishing Asia Pty Ltd.

  15. The lateral root initiation index: an integrative measure of primordium formation

    PubMed Central

    Dubrovsky, J. G.; Soukup, A.; Napsucialy-Mendivil, S.; Jeknić, Z.; Ivanchenko, M. G.

    2009-01-01

    Background and Aims Lateral root initiation is an essential and continuous process in the formation of root systems; therefore, its quantitative analysis is indispensable. In this study a new measure of lateral root initiation is proposed and analysed, namely the lateral root initiation index (ILRI), which defines how many lateral roots and/or primordia are formed along a parent-root portion corresponding to 100 cortical cells in a file. Methods For data collection, a commonly used root clearing procedure was employed, and a new simple root clearing procedure is also proposed. The ILRI was determined as 100dl, where d is the density of lateral root initiation events (number mm−1) and l is the average fully elongated cortical cell length (mm). Key Results Analyses of different Arabidopsis thaliana genotypes and of a crop plant, tomato (Solanum lycopersicum), showed that ILRI is a more precise parameter than others commonly used as it normalizes root growth for variations in cell length. Lateral root primordium density varied in the A. thaliana accessions Col, Ler, Ws, and C24; however, in all accessions except Ws, ILRI was similar under the same growth conditions. The nitrogen/carbon ratio in the growth medium did not change the lateral root primordium density but did affect ILRI. The ILRI was also modified in a number of auxin-related mutants, revealing new root branching phenotypes in some of these mutants. The rate of lateral root initiation increased with Arabidopsis seedling age; however, ILRI was not changed in plants between 8 and 14 d post-germination. Conclusions The ILRI allows for a more precise comparison of lateral root initiation under different growth conditions, treatments, genotypes and plant species than other comparable methods. PMID:19151042

  16. Initial Binding and Recellularization of Decellularized Mouse Lung Scaffolds with Bone Marrow-Derived Mesenchymal Stromal Cells

    PubMed Central

    Daly, Amanda B.; Wallis, John M.; Borg, Zachary D.; Bonvillain, Ryan W.; Deng, Bin; Ballif, Bryan A.; Jaworski, Diane M.; Allen, Gilman B.

    2012-01-01

    Recellularization of whole decellularized lung scaffolds provides a novel approach for generating functional lung tissue ex vivo for subsequent clinical transplantation. To explore the potential utility of stem and progenitor cells in this model, we investigated recellularization of decellularized whole mouse lungs after intratracheal inoculation of bone marrow-derived mesenchymal stromal cells (MSCs). The decellularized lungs maintained structural features of native lungs, including intact vasculature, ability to undergo ventilation, and an extracellular matrix (ECM) scaffold consisting primarily of collagens I and IV, laminin, and fibronectin. However, even in the absence of intact cells or nuclei, a number of cell-associated (non-ECM) proteins were detected using mass spectroscopy, western blots, and immunohistochemistry. MSCs initially homed and engrafted to regions enriched in types I and IV collagen, laminin, and fibronectin, and subsequently proliferated and migrated toward regions enriched in types I and IV collagen and laminin but not provisional matrix (fibronectin). MSCs cultured for up to 1 month in either basal MSC medium or in a small airways growth media (SAGM) localized in both parenchymal and airway regions and demonstrated several different morphologies. However, while MSCs cultured in basal medium increased in number, MSCs cultured in SAGM decreased in number over 1 month. Under both media conditions, the MSCs predominantly expressed genes consistent with mesenchymal and osteoblast phenotype. Despite a transient expression of the lung precursor TTF-1, no other airway or alveolar genes or vascular genes were expressed. These studies highlight the power of whole decellularized lung scaffolds to study functional recellularization with MSCs and other cells. PMID:21756220

  17. Toxicogenomics analysis of mouse lung responses following exposure to titanium dioxide nanomaterials reveal their disease potential at high doses

    PubMed Central

    Rahman, Luna; Wu, Dongmei; Johnston, Michael; William, Andrew; Halappanavar, Sabina

    2017-01-01

    Titanium dioxide nanoparticles (TiO2NPs) induce lung inflammation in experimental animals. In this study, we conducted a comprehensive toxicogenomic analysis of lung responses in mice exposed to six individual TiO2NPs exhibiting different sizes (8, 20 and 300nm), crystalline structure (anatase, rutile or anatase/rutile) and surface modifications (hydrophobic or hydrophilic) to investigate whether the mechanisms leading to TiO2NP-induced lung inflammation are property specific. A detailed histopathological analysis was conducted to investigate the long-term disease implications of acute exposure to TiO2NPs. C57BL/6 mice were exposed to 18, 54, 162 or 486 µg of TiO2NPs/mouse via single intratracheal instillation. Controls were exposed to dispersion medium only. Bronchoalveolar lavage fluid (BALF) and lung tissue were sampled on 1, 28 and 90 days post-exposure. Although all TiO2NPs induced lung inflammation as measured by the neutrophil influx in BALF, rutile-type TiO2NPs induced higher inflammation with the hydrophilic rutile TiO2NP showing the maximum increase. Accordingly, the rutile TiO2NPs induced higher number of differentially expressed genes. Histopathological analysis of lung sections on Day 90 post-exposure showed increased collagen staining and fibrosis-like changes following exposure to the rutile TiO2NPs at the highest dose tested. Among the anatase, the smallest TiO2NP of 8nm showed the maximum response. The anatase TiO2NP of 300nm was the least responsive of all. The results suggest that the severity of lung inflammation is property specific; however, the underlying mechanisms (genes and pathways perturbed) leading to inflammation were the same for all particle types. While the particle size clearly influenced the overall acute lung responses, a combination of small size, crystalline structure and hydrophilic surface contributed to the long-term pathological effects observed at the highest dose (486 µg/mouse). Although the dose at which the

  18. Flaxseed Mitigates Acute Oxidative Lung Damage in a Mouse Model of Repeated Radiation and Hyperoxia Exposure Associated with Space Exploration.

    PubMed

    Pietrofesa, Ralph A; Solomides, Charalambos C; Christofidou-Solomidou, Melpo

    Spaceflight missions may require crewmembers to conduct extravehicular activities (EVA). Pre-breathe protocols in preparation for an EVA entail 100% hyperoxia exposure that may last for a few hours and be repeated 2-3 times weekly. Each EVA is associated with additional challenges such as low levels of total body cosmic/galactic radiation exposure that may present a threat to crewmember health. We have developed a mouse model of total body radiation and hyperoxia exposure and identified acute damage of lung tissues. In the current study we evaluated the usefulness of dietary flaxseed (FS) as a countermeasure agent for such double-hit exposures. We evaluated lung tissue changes 2 weeks post-initiation of exposure challenges. Mouse cohorts (n=5/group) were pre-fed diets containing either 0% FS or 10% FS for 3 weeks and exposed to: a) normoxia (Untreated); b) >95% O2 (O2); c) 0.25Gy single fraction gamma radiation (IR); or d) a combination of O2 and IR (O2+IR) 3 times per week for 2 consecutive weeks, where 8-hour hyperoxia treatments were spanned by normoxic intervals. At 2 weeks post challenge, while control-diet fed mice developed significant lung injury and inflammation across all challenges, FS protected lung tissues by decreasing bronchoalveolar lavage fluid (BALF) neutrophils (p<0.003) and protein levels, oxidative tissue damage, as determined by levels of malondialdehyde (MDA) (p<0.008) and nitrosative stress as determined by nitrite levels. Lung hydroxyproline levels, a measure of lung fibrosis, were significantly elevated in mice fed 0% FS (p<0.01) and exposed to hyperoxia/radiation or the combination treatment, but not in FS-fed mice. FS also decreased levels of a pro-inflammatory, pro-fibrogenic cytokine (TGF-β1) gene expression levels in lung. Flaxseed mitigated adverse effects in lung of repeat exposures to radiation/hyperoxia. This data will provide useful information in the design of countermeasures to early tissue oxidative damage associated with

  19. Flaxseed Mitigates Acute Oxidative Lung Damage in a Mouse Model of Repeated Radiation and Hyperoxia Exposure Associated with Space Exploration

    PubMed Central

    Pietrofesa, Ralph A.; Solomides, Charalambos C.; Christofidou-Solomidou, Melpo

    2015-01-01

    Background Spaceflight missions may require crewmembers to conduct extravehicular activities (EVA). Pre-breathe protocols in preparation for an EVA entail 100% hyperoxia exposure that may last for a few hours and be repeated 2-3 times weekly. Each EVA is associated with additional challenges such as low levels of total body cosmic/galactic radiation exposure that may present a threat to crewmember health. We have developed a mouse model of total body radiation and hyperoxia exposure and identified acute damage of lung tissues. In the current study we evaluated the usefulness of dietary flaxseed (FS) as a countermeasure agent for such double-hit exposures. Methods We evaluated lung tissue changes 2 weeks post-initiation of exposure challenges. Mouse cohorts (n=5/group) were pre-fed diets containing either 0% FS or 10% FS for 3 weeks and exposed to: a) normoxia (Untreated); b) >95% O2 (O2); c) 0.25Gy single fraction gamma radiation (IR); or d) a combination of O2 and IR (O2+IR) 3 times per week for 2 consecutive weeks, where 8-hour hyperoxia treatments were spanned by normoxic intervals. Results At 2 weeks post challenge, while control-diet fed mice developed significant lung injury and inflammation across all challenges, FS protected lung tissues by decreasing bronchoalveolar lavage fluid (BALF) neutrophils (p<0.003) and protein levels, oxidative tissue damage, as determined by levels of malondialdehyde (MDA) (p<0.008) and nitrosative stress as determined by nitrite levels. Lung hydroxyproline levels, a measure of lung fibrosis, were significantly elevated in mice fed 0% FS (p<0.01) and exposed to hyperoxia/radiation or the combination treatment, but not in FS-fed mice. FS also decreased levels of a pro-inflammatory, pro-fibrogenic cytokine (TGF-β1) gene expression levels in lung. Conclusion Flaxseed mitigated adverse effects in lung of repeat exposures to radiation/hyperoxia. This data will provide useful information in the design of countermeasures to early

  20. Phosphatidylinositol 3-Kinase Mediates Bronchioalveolar Stem Cell Expansion in Mouse Models of Oncogenic K-ras-Induced Lung Cancer

    PubMed Central

    Yang, Yanan; Iwanaga, Kentaro; Raso, Maria Gabriela; Wislez, Marie; Hanna, Amy E.; Wieder, Eric D.; Molldrem, Jeffrey J.; Wistuba, Ignacio I.; Powis, Garth; Demayo, Francesco J.; Kim, Carla F.; Kurie, Jonathan M.

    2008-01-01

    Background Non-small cell lung cancer (NSCLC) is the most common cause of cancer-related death in Western countries. Developing more effective NSCLC therapeutics will require the elucidation of the genetic and biochemical bases for this disease. Bronchioalveolar stem cells (BASCs) are a putative cancer stem cell population in mouse models of oncogenic K-ras-induced lung adenocarcinoma, an histologic subtype of NSCLC. The signals activated by oncogenic K-ras that mediate BASC expansion have not been fully defined. Methodology/Principal Findings We used genetic and pharmacologic approaches to modulate the activity of phosphatidylinositol 3-kinase (PI3K), a key mediator of oncogenic K-ras, in two genetic mouse models of lung adenocarcinoma. Oncogenic K-ras-induced BASC accumulation and tumor growth were blocked by treatment with a small molecule PI3K inhibitor and enhanced by inactivation of phosphatase and tensin homologue deleted from chromosome 10, a negative regulator of PI3K. Conclusions/Significance We conclude that PI3K is a critical regulator of BASC expansion, supporting treatment strategies to target PI3K in NSCLC patients. PMID:18493606

  1. TH-E-BRF-07: Raman Spectroscopy for Radiation Treatment Response Assessment in a Lung Metastases Mouse Model

    SciTech Connect

    Devpura, S; Barton, K; Brown, S; Siddiqui, F; Chetty, I; Sethi, S; Klein, M

    2014-06-15

    Purpose: Raman spectroscopy is an optical spectroscopic method used to probe chemical information about a target tissue. Our goal was to investigate whether Raman spectroscopy is able to distinguish lung tumors from normal lung tissue and whether this technique can identify the molecular changes induced by radiation. Methods: 4T1 mouse breast cancer cells were implanted subcutaneously into the flanks of 6 Balb/C female mice. Four additional mice were used as “normal lung” controls. After 14 days, 3 mice bearing tumors received 6Gy to the left lung with 6MV photons and the other three were treated as “unirradiated tumor” controls. At a 24-hour time point, lungs were excised and the specimens were sectioned using a cryostat; alternating sections were either stained with hematoxylin and eosin (H and E) for evaluation by a pathologist or unstained for Raman measurements. 240 total Raman spectra were collected; 84 from normal lung controls; 63 from unirradiated tumors and 64 from tumors irradiated with 6Gy in a single fraction. Raman spectra were also collected from normal lung tissues of mice with unirradiated tumors. Principal component analysis (PCA) and discriminant function analysis (DFA) were performed to analyze the data. Results: Raman bands assignable to DNA/RNA showed prominent contributions in tumor tissues while Raman bands associated with hemoglobin showed strong contributions in normal lung tissue. PCA/DFA analysis identified normal lung tissue and tumor with 100% and 98.4% accuracy, respectively, relative to pathologic scoring. Additionally, normal lung tissues from unirradiated mice bearing tumors were classified as normal with 100% accuracy. In a model consisting of unirradiated and irradiated tumors identification accuracy was 79.4% and 93.8% respectively, relative to pathologic assessment. Conclusion: Initial results demonstrate the promise for Raman spectroscopy in the diagnosis normal vs. lung metastases as well as the assessment of

  2. Age-Dependent Decline in Mouse Lung Regeneration with Loss of Lung Fibroblast Clonogenicity and Increased Myofibroblastic Differentiation

    PubMed Central

    Paxson, Julia A.; Gruntman, Alisha; Parkin, Christopher D.; Mazan, Melissa R.; Davis, Airiel; Ingenito, Edward P.; Hoffman, Andrew M.

    2011-01-01

    While aging leads to a reduction in the capacity for regeneration after pneumonectomy (PNX) in most mammals, this biological phenomenon has not been characterized over the lifetime of mice. We measured the age-specific (3, 9, 24 month) effects of PNX on physiology, morphometry, cell proliferation and apoptosis, global gene expression, and lung fibroblast phenotype and clonogenicity in female C57BL6 mice. The data show that only 3 month old mice were fully capable of restoring lung volumes by day 7 and total alveolar surface area by 21 days. By 9 months, the rate of regeneration was slower (with incomplete regeneration by 21 days), and by 24 months there was no regrowth 21 days post-PNX. The early decline in regeneration rate was not associated with changes in alveolar epithelial cell type II (AECII) proliferation or apoptosis rate. However, significant apoptosis and lack of cell proliferation was evident after PNX in both total cells and AECII cells in 24 mo mice. Analysis of gene expression at several time points (1, 3 and 7 days) post-PNX in 9 versus 3 month mice was consistent with a myofibroblast signature (increased Tnc, Lox1, Col3A1, Eln and Tnfrsf12a) and more alpha smooth muscle actin (αSMA) positive myofibroblasts were present after PNX in 9 month than 3 month mice. Isolated lung fibroblasts showed a significant age-dependent loss of clonogenicity. Moreover, lung fibroblasts isolated from 9 and 17 month mice exhibited higher αSMA, Col3A1, Fn1 and S100A expression, and lower expression of the survival gene Mdk consistent with terminal differentiation. These data show that concomitant loss of clonogenicity and progressive myofibroblastic differentiation contributes to the age-dependent decline in the rate of lung regeneration. PMID:21912590

  3. The effects of exogenous surfactant administration on ventilation-induced inflammation in mouse models of lung injury

    PubMed Central

    2013-01-01

    Background Mechanical ventilation (MV) is an essential supportive therapy for acute lung injury (ALI); however it can also contribute to systemic inflammation. Since pulmonary surfactant has anti-inflammatory properties, the aim of the study was to investigate the effect of exogenous surfactant administration on ventilation-induced systemic inflammation. Methods Mice were randomized to receive an intra-tracheal instillation of a natural exogenous surfactant preparation (bLES, 50 mg/kg) or no treatment as a control. MV was then performed using the isolated and perfused mouse lung (IPML) set up. This model allowed for lung perfusion during MV. In experiment 1, mice were exposed to mechanical ventilation only (tidal volume =20 mL/kg, 2 hours). In experiment 2, hydrochloric acid or air was instilled intra-tracheally four hours before applying exogenous surfactant and ventilation (tidal volume =5 mL/kg, 2 hours). Results For both experiments, exogenous surfactant administration led to increased total and functional surfactant in the treated groups compared to the controls. Exogenous surfactant administration in mice exposed to MV only did not affect peak inspiratory pressure (PIP), lung IL-6 levels and the development of perfusate inflammation compared to non-treated controls. Acid injured mice exposed to conventional MV showed elevated PIP, lung IL-6 and protein levels and greater perfusate inflammation compared to air instilled controls. Instillation of exogenous surfactant did not influence the development of lung injury. Moreover, exogenous surfactant was not effective in reducing the concentration of inflammatory cytokines in the perfusate. Conclusions The data indicates that exogenous surfactant did not mitigate ventilation-induced systemic inflammation in our models. Future studies will focus on altering surfactant composition to improve its immuno-modulating activity. PMID:24256698

  4. Tissue optical clearing, three-dimensional imaging, and computer morphometry in whole mouse lungs and human airways.

    PubMed

    Scott, Gregory D; Blum, Emily D; Fryer, Allison D; Jacoby, David B

    2014-07-01

    In whole adult mouse lung, full identification of airway nerves (or other cellular/subcellular objects) has not been possible due to patchy distribution and micron-scale size. Here we describe a method using tissue clearing to acquire the first complete image of three-dimensional (3D) innervation in the lung. We then created a method to pair analysis of nerve (or any other colabeled epitope) images with identification of 3D tissue compartments and airway morphometry by using fluorescent casting and morphometry software (which we designed and are making available as open-source). We then tested our method to quantify a sparse heterogeneous nerve population by examining visceral pleural nerves. Finally, we demonstrate the utility of our method in human tissue to image full thickness innervation in irregular 3D tissue compartments and to quantify sparse objects (intrinsic airway ganglia). Overall, this method can uniquely pair the advantages of whole tissue imaging and cellular/subcellular fluorescence microscopy.

  5. The genetic basis of strain-dependent differences in the early phase of radiation injury in mouse lung

    SciTech Connect

    Franko, A.J.; Sharplin, J.; Ward, W.F.; Hinz, J.M. )

    1991-06-01

    Substantial differences between mouse strains have been reported in the lesions present in the lung during the early phase of radiation injury. Some strains show only classical pneumonitis, while other strains develop substantial fibrosis and hyaline membranes which contribute appreciably to respiratory insufficiency, in addition to pneumonitis. Other strains are intermediate between these extremes. These differences correlate with intrinsic differences in activities of lung plasminogen activator and angiotensin converting enzyme. The genetic basis of these differences was assessed by examining histologically the early reaction in lungs of seven murine hybrids available commercially after whole-thorax irradiation. Crosses between fibrosing and nonfibrosing parents were uniformly nonfibrosing, and crosses between fibrosing and intermediate parents were uniformly intermediate. No evidence of sex linkage was seen. Thus the phenotype in which fibrosis is found is controlled by autosomal recessive determinants. Strains prone to radiation-induced pulmonary fibrosis and hyaline membranes exhibited intrinsically lower activities of lung plasminogen activator and angiotensin converting enzyme than either the nonfibrosing strains or the nonfibrosing hybrid crosses. The median time of death of the hybrids was genetically determined primarily by the longest-lived parent regardless of the types of lesions expressed.

  6. Azithromycin Attenuates Lung Inflammation in a Mouse Model of Ventilator-Associated Pneumonia by Multidrug-Resistant Acinetobacter baumannii

    PubMed Central

    Yamada, Koichi; Kaku, Norihito; Harada, Yosuke; Migiyama, Yohei; Nagaoka, Kentaro; Morinaga, Yoshitomo; Nakamura, Shigeki; Imamura, Yoshifumi; Miyazaki, Taiga; Izumikawa, Koichi; Kakeya, Hiroshi; Hasegawa, Hiroo; Mikamo, Hiroshige; Kohno, Shigeru

    2013-01-01

    Acinetobacter baumannii is one of the main pathogens that cause ventilator-associated pneumonia (VAP) and is associated with a high rate of mortality. Little is known about the efficacy of macrolides against A. baumannii. In order to confirm the efficacy of azithromycin (AZM) against VAP caused by multidrug-resistant A. baumannii (MDRAB), we used a mouse model that mimics VAP by placement of a plastic tube in the bronchus. AZM (10 and 100 mg/kg of body weight) was administered subcutaneously every 24 h beginning at 3 h after inoculation. Phosphate-buffered saline was administered as the control. Survival was evaluated over 7 days. At 48 h postinfection, mice were sacrificed and the numbers of viable bacteria in lungs and bronchoalveolar lavage fluid were compared. Histopathological analysis of lung specimens was also performed. The treatment groups displayed significantly longer survival than the control group (P < 0.05). AZM did not have an antimicrobial effect. Histopathological examination of lung specimens indicated that the progression of lung inflammation was prevented in the AZM-treated groups. Furthermore, total cell and neutrophil counts, as well as cytokine levels, in bronchoalveolar lavage fluid were significantly decreased (P < 0.05) in the AZM-treated groups. AZM may have a role for the treatment of VAP with MDRAB because of its anti-inflammatory effects. PMID:23733468

  7. Anti-tumor activity of fenretinide complexed with human serum albumin in lung cancer xenograft mouse model.

    PubMed

    Durante, Sandra; Orienti, Isabella; Teti, Gabriella; Salvatore, Viviana; Focaroli, Stefano; Tesei, Anna; Pignatta, Sara; Falconi, Mirella

    2014-07-15

    Sufficient knowledge regarding cellular and molecular basis of lung cancer progression and metastasis would help in the development of novel and effective strategies for the treatment of lung cancer. 4HPR is a synthetic retinoid with potential anti-tumor activity but is still limited because of its poor bioavailability. The use of albumin as a complexing agent for a hydrophobic drug is expected to improve the water solubility and consequently their bioavailability.This study investigated the antitumor activity of a novel complex between albumin and 4-HPR in a mouse model of human lung cancer and focuses on role and mechanism of Cav-1 mainly involved in regulating cancer and ACSVL3 mainly connected with tumor growth. Their expressions were assayed by immunohistochemistry and qRT-PCR, to demonstrate the reduction of the tumor growth following the drug treatment. Our results showed a high antitumor activity of 4HPR-HSA by reduction of the volume of tumor mass and the presence of a high level of apoptotic cell by TUNEL assay. The downregulation of Cav-1 and ACSVL3 suggested a reduction of tumor growth. In conclusion, we demonstrated the great potential of 4HPR-HSA in the treatment of lung cancer. More data about the mechanism of drug delivery the 4HPR-HSA are necessary.

  8. Feasibility Assessment of a MALDI FTICR Imaging Approach for the 3D Reconstruction of a Mouse Lung

    NASA Astrophysics Data System (ADS)

    Jones, E. Ellen; Quiason, Cristine; Dale, Stephanie; Shahidi-Latham, Sheerin K.

    2017-08-01

    Matrix assisted laser desorption ionization imaging mass spectrometry (MALDI IMS) has proven to be a quick, robust, and label-free tool to produce two-dimensional (2D) ion-density maps representing the distribution of a variety of analytes across a tissue section of interest. In addition, three-dimensional (3D) imaging mass spectrometry workflows have been developed that are capable of visualizing these same analytes throughout an entire volume of a tissue rather than a single cross-section. Until recently, the use of Fourier transform ion cyclotron resonance (FTICR) mass spectrometers for 3D volume reconstruction has been impractical due to software limitations, such as inadequate capacity to manipulate the extremely large data files produced during an imaging experiment. Fortunately with recent software and hardware advancements, 3D reconstruction from MALDI FTICR IMS datasets is now feasible. Here we describe the first proof of principle study for a 3D volume reconstruction of an entire mouse lung using data collected on a FTICR mass spectrometer. Each lung tissue section was analyzed with high mass resolution and mass accuracy, and considered as an independent dataset. Each subsequent lung section image, or lung dataset, was then co-registered to its adjacent section to reconstruct a 3D volume. Volumes representing various endogenous lipid species were constructed, including sphingolipids and phosphatidylcholines (PC), and species confirmation was performed with on-tissue collision induced dissociation (CID).

  9. A novel mouse model of conditional IRAK-M deficiency in myeloid cells: application in lung Pseudomonas aeruginosa infection.

    PubMed

    Jiang, Di; Matsuda, Jennifer; Berman, Reena; Schaefer, Niccolette; Stevenson, Connor; Gross, James; Zhang, Bicheng; Sanchez, Amelia; Li, Liwu; Chu, Hong Wei

    2017-02-01

    Myeloid cells such as macrophages are critical to innate defense against infection. IL-1 receptor-associated kinase M (IRAK-M) is a negative regulator of TLR signaling during bacterial infection, but the role of myeloid cell IRAK-M in bacterial infection is unclear. Our goal was to generate a novel conditional knockout mouse model to define the role of myeloid cell IRAK-M during bacterial infection. Myeloid cell-specific IRAK-M knockout mice were generated by crossing IRAK-M floxed mice with LysM-Cre knock-in mice. The resulting LysM-Cre(+)/IRAK-M(fl/wt) and control (LysM-Cre(-)/IRAK-M(fl/wt)) mice were intranasally infected with Pseudomonas aeruginosa (PA). IRAK-M deletion, inflammation, myeloperoxidase (MPO) activity and PA load were measured in leukocytes, bronchoalveolar lavage (BAL) fluid and lungs. PA killing assay with BAL fluid was performed to determine mechanisms of IRAK-M-mediated host defense. IRAK-M mRNA and protein levels in alveolar and lung macrophages were significantly reduced in LysM-Cre(+)/IRAK-M(fl/wt) mice compared with control mice. Following PA infection, LysM-Cre(+)/IRAK-M(fl/wt) mice have enhanced lung neutrophilic inflammation, including MPO activity, but reduced PA load. The increased lung MPO activity in LysM-Cre(+)/IRAK-M(fl/wt) mouse BAL fluid reduced PA load. Generation of IRAK-M conditional knockout mice will enable investigators to determine precisely the function of IRAK-M in myeloid cells and other types of cells during infection and inflammation.

  10. Gene Profiles in a Smoke-Induced COPD Mouse Lung Model Following Treatment with Mesenchymal Stem Cells.

    PubMed

    Kim, You-Sun; Kokturk, Nurdan; Kim, Ji-Young; Lee, Sei Won; Lim, Jaeyun; Choi, Soo Jin; Oh, Wonil; Oh, Yeon-Mok

    2016-10-01

    Mesenchymal stem cells (MSCs) effectively reduce airway inflammation and regenerate the alveolus in cigarette- and elastase-induced chronic obstructive pulmonary disease (COPD) animal models. The effects of stem cells are thought to be paracrine and immune-modulatory because very few stem cells remain in the lung one day after their systemic injection, which has been demonstrated previously. In this report, we analyzed the gene expression profiles to compare mouse lungs with chronic exposure to cigarette smoke with non-exposed lungs. Gene expression profiling was also conducted in a mouse lung tissue with chronic exposure to cigarette smoke following the systemic injection of human cord blood-derived mesenchymal stem cells (hCB-MSCs). Globally, 834 genes were differentially expressed after systemic injection of hCB-MSCs. Seven and 21 genes, respectively, were up-and downregulated on days 1, 4, and 14 after HCB-MSC injection. The Hbb and Hba, genes with oxygen transport and antioxidant functions, were increased on days 1 and 14. A serine protease inhibitor was also increased at a similar time point after injection of hCB-MSCs. Gene Ontology analysis indicated that the levels of genes related to immune responses, metabolic processes, and blood vessel development were altered, indicating host responses after hCB-MSC injection. These gene expression changes suggest that MSCs induce a regeneration mechanism against COPD induced by cigarette smoke. These analyses provide basic data for understanding the regeneration mechanisms promoted by hCB-MSCs in cigarette smoke-induced COPD.

  11. Gene Profiles in a Smoke-Induced COPD Mouse Lung Model Following Treatment with Mesenchymal Stem Cells

    PubMed Central

    Kim, You-Sun; Kokturk, Nurdan; Kim, Ji-Young; Lee, Sei Won; Lim, Jaeyun; Choi, Soo Jin; Oh, Wonil; Oh, Yeon-Mok

    2016-01-01

    Mesenchymal stem cells (MSCs) effectively reduce airway inflammation and regenerate the alveolus in cigarette- and elastase-induced chronic obstructive pulmonary disease (COPD) animal models. The effects of stem cells are thought to be paracrine and immune-modulatory because very few stem cells remain in the lung one day after their systemic injection, which has been demonstrated previously. In this report, we analyzed the gene expression profiles to compare mouse lungs with chronic exposure to cigarette smoke with non-exposed lungs. Gene expression profiling was also conducted in a mouse lung tissue with chronic exposure to cigarette smoke following the systemic injection of human cord blood-derived mesenchymal stem cells (hCB-MSCs). Globally, 834 genes were differentially expressed after systemic injection of hCB-MSCs. Seven and 21 genes, respectively, were up-and downregulated on days 1, 4, and 14 after HCB-MSC injection. The Hbb and Hba, genes with oxygen transport and antioxidant functions, were increased on days 1 and 14. A serine protease inhibitor was also increased at a similar time point after injection of hCB-MSCs. Gene Ontology analysis indicated that the levels of genes related to immune responses, metabolic processes, and blood vessel development were altered, indicating host responses after hCB-MSC injection. These gene expression changes suggest that MSCs induce a regeneration mechanism against COPD induced by cigarette smoke. These analyses provide basic data for understanding the regeneration mechanisms promoted by hCB-MSCs in cigarette smoke-induced COPD. PMID:27802588

  12. Understanding Lung Deposition of Alpha-1 Antitrypsin in Acute Experimental Mouse Lung Injury Model Using Fluorescence Microscopy

    PubMed Central

    Zhan, Yutian; Chen, Jianqing; Rong, Haojing; O'Neil, Shawn P.; Ghosh, Brahma; Nguyen, Vuong; Li, Xianfeng

    2016-01-01

    Human plasma-derived α1-antitrypsin (AAT) delivered by intravenous infusion is used as augmentation therapy in patients with emphysema who have a genetic mutation resulting in deficiency of AAT. Inhalation is an alternative route of administration that can potentially increase the efficacy and convenience of treatment. This study was conducted to determine whether delivery to the lungs, initially via the intratracheal (IT) route of administration, would deliver efficacious levels of a recombinant AAT (rAAT) to the site of action in the lungs in mice. 125I-radiolabeled rAAT, fluorophore-conjugated rAAT (rAAT-Alexa488), and NE680 (neutrophil elastase 680, a silent fluorescent substrate of neutrophil elastase which fluoresces in the near-infrared range upon activation by neutrophil elastase) were used to characterize the pharmacokinetics and tissue distribution profile, distribution of rAAT within the lung, and efficacy of rAAT to inhibit neutrophil elastase at the site of action, respectively. The study has demonstrated that rAAT was able to gain access to locations where neutrophil elastase was localized. The histochemical quantification of rAAT activity relative to dose at the site of action provided here will improve confidence in predicting the human dose via the inhalation route. PMID:28050284

  13. Inhomogeneity of local stiffness in the extracellular matrix scaffold of fibrotic mouse lungs.

    PubMed

    Melo, Esther; Cárdenes, Nayra; Garreta, Elena; Luque, Tomas; Rojas, Mauricio; Navajas, Daniel; Farré, Ramon

    2014-09-01

    Lung disease models are useful to study how cell engraftment, proliferation and differentiation are modulated in lung bioengineering. The aim of this work was to characterize the local stiffness of decellularized lungs in aged and fibrotic mice. Mice (2- and 24-month old; 14 of each) with lung fibrosis (N=20) and healthy controls (N=8) were euthanized after 11 days of intratracheal bleomycin (fibrosis) or saline (controls) infusion. The lungs were excised, decellularized by a conventional detergent-based (sodium-dodecyl sulfate) procedure and slices of the acellular lungs were prepared to measure the local stiffness by means of atomic force microscopy. The local stiffness of the different sites in acellular fibrotic lungs was very inhomogeneous within the lung and increased according to the degree of the structural fibrotic lesion. Local stiffness of the acellular lungs did not show statistically significant differences caused by age. The group of mice most affected by fibrosis exhibited local stiffness that were ~2-fold higher than in the control mice: from 27.2±1.64 to 64.8±7.1kPa in the alveolar septa, from 56.6±4.6 to 99.9±11.7kPa in the visceral pleura, from 41.1±8.0 to 105.2±13.6kPa in the tunica adventitia, and from 79.3±7.2 to 146.6±28.8kPa in the tunica intima. Since acellular lungs from mice with bleomycin-induced fibrosis present considerable micromechanical inhomogeneity, this model can be a useful tool to better investigate how different degrees of extracellular matrix lesion modulate cell fate in the process of organ bioengineering from decellularized lungs. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. Characterizing dynamic regulatory programs in mouse lung development and their potential association with tumourigenesis via miRNA-TF-mRNA circuits.

    PubMed

    Liu, Juan; Ye, Xinghuo; Wu, Fang-Xiang

    2013-01-01

    In dynamic biological processes, genes, transcription factors(TF) and microRNAs(miRNAs) play vital regulation roles. Many researchers have focused on the transcription factors or miRNAs in transcriptional or post transcriptional stage, respectively. However, the transcriptional regulation and post transcriptional regulation is not isolated in the whole dynamic biological processes, there are few reserchers who have tried to consider the network composed by genes, miRNAs and TFs in this dynamic biological processes, especially in the mouse lung development. Moreover, it is widely acknowledged that cancer is a kind of developmental disorders, and some of pathways involved in tissue development might be also implicated in causing cancer. Although it has been found that many genes differentially expressed during mouse lung development are also differentially expressed in lung cancer, very little work has been reported to elucidate the combinational regulatory programs of such kind of associations. In order to investigate the association of transcriptional and post-transcriptional regulating activities in the mouse lung development, we define the significant triple relations among miRNAs, TFs and mRNAs as circuits. From the lung development time course data GSE21053, we mine 142610 circuit candidates including 96 TFs, 129 miRNAs and 13403 genes. After removing genes with little variation along different time points, we finally find 64760 circuit candidates, containing 8299 genes, 50 TFs, and 118 miRNAs in total. Further analysis on the circuits shows that the circuits vary in different stages of the lung development and play different roles. By investigating the circuits in the context of lung specific genes, we identify out the regulatory combinations for lung specific genes, as well as for those lung non-specific genes. Moreover, we show that the lung non-specific genes involved circuits are functionally related to the lung development. Noticing that some tissue

  15. Depletion of tumor-associated macrophages slows the growth of chemically induced mouse lung adenocarcinomas.

    PubMed

    Fritz, Jason M; Tennis, Meredith A; Orlicky, David J; Lin, Hao; Ju, Cynthia; Redente, Elizabeth F; Choo, Kevin S; Staab, Taylor A; Bouchard, Ronald J; Merrick, Daniel T; Malkinson, Alvin M; Dwyer-Nield, Lori D

    2014-01-01

    Chronic inflammation is a risk factor for lung cancer, and low-dose aspirin intake reduces lung cancer risk. However, the roles that specific inflammatory cells and their products play in lung carcinogenesis have yet to be fully elucidated. In mice, alveolar macrophage numbers increase as lung tumors progress, and pulmonary macrophage programing changes within 2 weeks of carcinogen exposure. To examine how macrophages specifically affect lung tumor progression, they were depleted in mice bearing urethane-induced lung tumors using clodronate-encapsulated liposomes. Alveolar macrophage populations decreased to ≤50% of control levels after 4-6 weeks of liposomal clodronate treatment. Tumor burden decreased by 50% compared to vehicle treated mice, and tumor cell proliferation, as measured by Ki67 staining, was also attenuated. Pulmonary fluid levels of insulin-like growth factor-I, CXCL1, IL-6, and CCL2 diminished with clodronate liposome treatment. Tumor-associated macrophages expressed markers of both M1 and M2 programing in vehicle and clodronate liposome-treated mice. Mice lacking CCR2 (the receptor for macrophage chemotactic factor CCL2) had comparable numbers of alveolar macrophages and showed no difference in tumor growth rates when compared to similarly treated wild-type mice suggesting that while CCL2 may recruit macrophages to lung tumor microenvironments, redundant pathways can compensate when CCL2/CCR2 signaling is inactivated. Depletion of pulmonary macrophages rather than inhibition of their recruitment may be an advantageous strategy for attenuating lung cancer progression.

  16. Nicotine does not enhance tumorigenesis in mutant K-ras-driven mouse models of lung cancer.

    PubMed

    Maier, Colleen R; Hollander, M Christine; Hobbs, Evthokia A; Dogan, Irem; Linnoila, R Ilona; Dennis, Phillip A

    2011-11-01

    Smoking is the leading cause of preventable cancer deaths in the United States. Nicotine replacement therapies (NRT) have been developed to aid in smoking cessation, which decreases lung cancer incidence. However, the safety of NRT is controversial because numerous preclinical studies have shown that nicotine enhances tumor cell growth in vitro and in vivo. We modeled NRT in mice to determine the effects of physiologic levels of nicotine on lung tumor formation, tumor growth, or metastasis. Nicotine administered in drinking water did not enhance lung tumorigenesis after treatment with the tobacco carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Tumors that develop in this model have mutations in K-ras, which is commonly observed in smoking-related, human lung adenocarcinomas. In a transgenic model of mutant K-ras-driven lung cancer, nicotine did not increase tumor number or size and did not affect overall survival. Likewise, in a syngeneic model using lung cancer cell lines derived from NNK-treated mice, oral nicotine did not enhance tumor growth or metastasis. These data show that nicotine does not enhance lung tumorigenesis when given to achieve levels comparable with those of NRT, suggesting that nicotine has a dose threshold, below which it has no appreciable effect. These studies are consistent with epidemiologic data showing that NRT does not enhance lung cancer risk in former smokers.

  17. Nicotine does not enhance tumorigenesis in mutant K-Ras-driven mouse models of lung cancer

    PubMed Central

    Maier, Colleen R.; Hollander, M. Christine; Hobbs, Evthokia A.; Dogan, Irem; Dennis, Phillip A.

    2011-01-01

    Smoking is the leading cause of preventable cancer deaths in the United States. Nicotine replacement therapies (NRT) have been developed to aid in smoking cessation, which decreases lung cancer incidence. However, the safety of NRT is controversial because numerous preclinical studies have shown that nicotine enhances tumor cell growth in vitro and in vivo. We modeled NRT in mice to determine the effects of physiological levels of nicotine on lung tumor formation, tumor growth or metastasis. Nicotine administered in drinking water did not enhance lung tumorigenesis after treatment with the tobacco carcinogen, NNK. Tumors that develop in this model have mutations in K-ras, which is a commonly observed in smoking-related, human lung adenocarcinomas. In a transgenic model of mutant K-ras-driven lung cancer, nicotine did not increase tumor number or size, and did not affect overall survival. Likewise, in a syngeneic model of lung cancer cell lines derived from NNK-treated mice, oral nicotine did not enhance tumor growth or metastasis. These data show that nicotine does not enhance lung tumorigenesis when given to achieve levels comparable to those of NRT, suggesting that nicotine has a dose threshold, below which it has no appreciable effect. These studies are consistent with epidemiological data showing that NRT does not enhance lung cancer risk in former smokers. PMID:22027685

  18. Pten Inactivation Accelerates Oncogenic K-ras-Initiated Tumorigenesis in a Mouse Model of Lung Cancer

    PubMed Central

    Iwanaga, Kentaro; Yang, Yanan; Raso, Maria Gabriela; Ma, Lijiang; Hanna, Amy E.; Thilaganathan, Nishan; Moghaddam, Seyed; Evans, Christopher M.; Li, Huaiguang; Cai, Wei-Wen; Sato, Mitsuo; Minna, John D.; Wu, Hong; Creighton, Chad J.; Demayo, Francesco J.; Wistuba, Ignacio I.; Kurie, Jonathan M.

    2009-01-01

    Phosphatase and tensin homologue deleted from chromosome 10 (Pten) is expressed aberrantly in non-small cell lung cancer cells, but the role of Pten in lung neoplasia has not been fully elucidated. In this study, we used a genetic approach to inactivate Pten in the bronchial epithelium of mice. Although, by itself, Pten inactivation had no discernible effect on bronchial epithelial histology, it accelerated lung tumorigenesis initiated by oncogenic K-ras, causing more rapid lethality than that induced by oncogenic K-ras alone (8 weeks versus 24 weeks of median duration of survival, respectively). Lung tumors arose in K-ras mutant, Pten-deficient mice that rapidly obstructed bronchial lumina and replaced alveolar spaces. Relative to K-ras mutant tumors, the K-ras mutant, Pten-deficient tumors exhibited more advanced histologic severity and more prominent inflammation and vascularity. Thus, Pten inactivation cooperated with oncogenic K-ras in promoting lung tumorigenesis. PMID:18281487

  19. Surfactant protein secreted by the maturing mouse fetal lung acts as a hormone that signals the initiation of parturition.

    PubMed

    Condon, Jennifer C; Jeyasuria, Pancharatnam; Faust, Julie M; Mendelson, Carole R

    2004-04-06

    Parturition is timed to begin only after the developing embryo is sufficiently mature to survive outside the womb. It has been postulated that the signal for the initiation of parturition arises from the fetus although the nature and source of this signal remain obscure. Herein, we provide evidence that this signal originates from the maturing fetal lung. In the mouse, secretion of the major lung surfactant protein, surfactant protein A (SP-A), was first detected in amniotic fluid (AF) at 17 days postcoitum, rising progressively to term (19 days postcoitum). Expression of IL-1beta in AF macrophages and activation of NF-kappaB in the maternal uterus increased with the gestational increase in SP-A. SP-A stimulated IL-1beta and NF-kappaB expression in cultured AF macrophages. Studies using Rosa 26 Lac-Z (B6;129S-Gt(rosa)26Sor) (Lac-Z) mice revealed that fetal AF macrophages migrate to the uterus with the gestational increase in AF SP-A. Intraamniotic (i.a.) injection of SP-A caused preterm delivery of fetuses within 6-24 h. By contrast, injection of an SP-A antibody or NF-kappaB inhibitor into AF delayed labor by >24 h. We propose that augmented production of SP-A by the fetal lung near term causes activation and migration of fetal AF macrophages to the maternal uterus, where increased production of IL-1beta activates NF-kappaB, leading to labor. We have revealed a response pathway that ties augmented surfactant production by the maturing fetal lung to the initiation of labor. We suggest that SP-A secreted by the fetal lung serves as a hormone of parturition.

  20. Characterization of a genetic mouse model of lung cancer: a promise to identify Non-Small Cell Lung Cancer therapeutic targets and biomarkers

    PubMed Central

    2014-01-01

    Background Non-small cell lung cancer (NSCLC) accounts for 81% of all cases of lung cancer and they are often fatal because 60% of the patients are diagnosed at an advanced stage. Besides the need for earlier diagnosis, there is a high need for additional effective therapies. In this work, we investigated the feasibility of a lung cancer progression mouse model, mimicking features of human aggressive NSCLC, as biological reservoir for potential therapeutic targets and biomarkers. Results We performed RNA-seq profiling on total RNA extracted from lungs of a 30 week-old K-rasLA1/p53R172HΔg and wild type (WT) mice to detect fusion genes and gene/exon-level differential expression associated to the increase of tumor mass. Fusion events were not detected in K-rasLA1/p53R172HΔg tumors. Differential expression at exon-level detected 33 genes with differential exon usage. Among them nine, i.e. those secreted or expressed on the plasma membrane, were used for a meta-analysis of more than 500 NSCLC RNA-seq transcriptomes. None of the genes showed a significant correlation between exon-level expression and disease prognosis. Differential expression at gene-level allowed the identification of 1513 genes with a significant increase in expression associated to tumor mass increase. 74 genes, i.e. those secreted or expressed on the plasma membrane, were used for a meta-analysis of two transcriptomics datasets of human NSCLC samples, encompassing more than 900 samples. SPP1 was the only molecule whose over-expression resulted statistically related to poor outcome regarding both survival and metastasis formation. Two other molecules showed over-expression associated to poor outcome due to metastasis formation: GM-CSF and ADORA3. GM-CSF is a secreted protein, and we confirmed its expression in the supernatant of a cell line derived by a K-rasLA1/p53R172HΔg mouse tumor. ADORA3 is instead involved in the induction of p53-mediated apoptosis in lung cancer cell lines. Since in our

  1. Aberrant lung remodeling in a mouse model of surfactant dysregulation induced by modulation of the Abca3 gene✩

    PubMed Central

    Beers, Michael F.; Knudsen, Lars; Tomer, Yaniv; Maronn, Julian; Zhao, Ming; Ochs, Matthias; Mulugeta, Surafel

    2017-01-01

    The lipid transporter, ATP binding cassette class A3 (ABCA3), plays a critical role in the biogenesis of alveolar type 2 (AT2) cell lamellar bodies (LBs). A relatively large number of mutations in the ABCA3 gene have been identified in association with diffuse parenchymal lung disease (DPLD), the most common of which is a missense mutation (valine substitution for lysine at residue 292 (ABCA3E292V)) that leads to functional impairment of the transporter in vitro. The consequences of ABCA3E292V gene expression in vivo are unknown. To address this question, we developed mouse models expressing ABCA3E292V knocked-in to the endogenous mouse locus. The parental (F1) mouse line (mAbca3E292V) that retained an intronic pgk-Neo selection cassette (inserted in reverse orientation) (mAbca3E292V–rNeo) demonstrated an allele dependent extracellular surfactant phospholipid (PL) deficiency. We hypothesize that this PL deficiency leads to aberrant parenchymal remodeling contributing to the pathophysiology of the DPLD phenotype. Compared to wild type littermates, baseline studies of mice homozygous for the pgk-Neo insert (mAbca3E292V–rNeo+/+) revealed nearly 50% reduction in bronchoalveolar lavage (BAL) PL content that was accompanied by quantitative reduction in AT2 LB size with a compensatory increase in LB number. The phenotypic alteration in surfactant lipid homeostasis resulted in an early macrophage predominant alveolitis which peaked at 8 weeks of age. This was followed by age-dependent development of histological DPLD characterized initially by peribronchial inflammatory cell infiltration and culminating in both an emphysema-like phenotype (which included stereologically quantifiable reductions in both alveolar septal surface area and volume of septal wall tissue) plus foci of trichrome-positive collagen deposition together with substantial proliferation of hyperplastic AT2 cells. In addition to spontaneous lung remodeling, mABCA3E292V–rNeo mice were rendered more

  2. Aberrant lung remodeling in a mouse model of surfactant dysregulation induced by modulation of the Abca3 gene.

    PubMed

    Beers, Michael F; Knudsen, Lars; Tomer, Yaniv; Maronn, Julian; Zhao, Ming; Ochs, Matthias; Mulugeta, Surafel

    2017-03-01

    The lipid transporter, ATP binding cassette class A3 (ABCA3), plays a critical role in the biogenesis of alveolar type 2 (AT2) cell lamellar bodies (LBs). A relatively large number of mutations in the ABCA3 gene have been identified in association with diffuse parenchymal lung disease (DPLD), the most common of which is a missense mutation (valine substitution for lysine at residue 292 (ABCA3(E292V))) that leads to functional impairment of the transporter in vitro. The consequences of ABCA3(E292)(V) gene expression in vivo are unknown. To address this question, we developed mouse models expressing ABCA3(E292V) knocked-in to the endogenous mouse locus. The parental (F1) mouse line (mAbca3(E292)(V)) that retained an intronic pgk-Neo selection cassette (inserted in reverse orientation) (mAbca3(E292)(V)-rNeo) demonstrated an allele dependent extracellular surfactant phospholipid (PL) deficiency. We hypothesize that this PL deficiency leads to aberrant parenchymal remodeling contributing to the pathophysiology of the DPLD phenotype. Compared to wild type littermates, baseline studies of mice homozygous for the pgk-Neo insert (mAbca3(E292)(V)-rNeo(+/)(+)) revealed nearly 50% reduction in bronchoalveolar lavage (BAL) PL content that was accompanied by quantitative reduction in AT2 LB size with a compensatory increase in LB number. The phenotypic alteration in surfactant lipid homeostasis resulted in an early macrophage predominant alveolitis which peaked at 8 weeks of age. This was followed by age-dependent development of histological DPLD characterized initially by peribronchial inflammatory cell infiltration and culminating in both an emphysema-like phenotype (which included stereologically quantifiable reductions in both alveolar septal surface area and volume of septal wall tissue) plus foci of trichrome-positive collagen deposition together with substantial proliferation of hyperplastic AT2 cells. In addition to spontaneous lung remodeling, mABCA3(E292V)-rNeo mice

  3. A data-driven integrative model of sepal primordium polarity in Arabidopsis.

    PubMed

    La Rota, Camilo; Chopard, Jérôme; Das, Pradeep; Paindavoine, Sandrine; Rozier, Frédérique; Farcot, Etienne; Godin, Christophe; Traas, Jan; Monéger, Françoise

    2011-12-01

    Flower patterning is determined by a complex molecular network but how this network functions remains to be elucidated. Here, we develop an integrative modeling approach that assembles heterogeneous data into a biologically coherent model to allow predictions to be made and inconsistencies among the data to be found. We use this approach to study the network underlying sepal development in the young flower of Arabidopsis thaliana. We constructed a digital atlas of gene expression and used it to build a dynamical molecular regulatory network model of sepal primordium development. This led to the construction of a coherent molecular network model for lateral organ polarity that fully recapitulates expression and interaction data. Our model predicts the existence of three novel pathways involving the HD-ZIP III genes and both cytokinin and ARGONAUTE family members. In addition, our model provides predictions on molecular interactions. In a broader context, this approach allows the extraction of biological knowledge from diverse types of data and can be used to study developmental processes in any multicellular organism.

  4. The Drosophila DOCK family protein Sponge is required for development of the air sac primordium.

    PubMed

    Morishita, Kazushge; Anh Suong, Dang Ngoc; Yoshida, Hideki; Yamaguchi, Masamitsu

    2017-05-15

    Dedicator of cytokinesis (DOCK) family genes are known as DOCK1-DOCK11 in mammals. DOCK family proteins mainly regulate actin filament polymerization and/or depolymerization and are GEF proteins, which contribute to cellular signaling events by activating small G proteins. Sponge (Spg) is a Drosophila counterpart to mammalian DOCK3/DOCK4, and plays a role in embryonic central nervous system development, R7 photoreceptor cell differentiation, and adult thorax development. In order to conduct further functional analyses on Spg in vivo, we examined its localization in third instar larval wing imaginal discs. Immunostaining with purified anti-Spg IgG revealed that Spg mainly localized in the air sac primordium (ASP) in wing imaginal discs. Spg is therefore predicted to play an important role in the ASP. The specific knockdown of Spg by the breathless-GAL4 driver in tracheal cells induced lethality accompanied with a defect in ASP development and the induction of apoptosis. The monitoring of ERK signaling activity in wing imaginal discs by immunostaining with anti-diphospho-ERK IgG revealed reductions in the ERK signal cascade in Spg knockdown clones. Furthermore, the overexpression of D-raf suppressed defects in survival and the proliferation of cells in the ASP induced by the knockdown of Spg. Collectively, these results indicate that Spg plays a critical role in ASP development and tracheal cell viability that is mediated by the ERK signaling pathway. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. A Data-Driven Integrative Model of Sepal Primordium Polarity in Arabidopsis[C][W

    PubMed Central

    La Rota, Camilo; Chopard, Jérôme; Das, Pradeep; Paindavoine, Sandrine; Rozier, Frédérique; Farcot, Etienne; Godin, Christophe; Traas, Jan; Monéger, Françoise

    2011-01-01

    Flower patterning is determined by a complex molecular network but how this network functions remains to be elucidated. Here, we develop an integrative modeling approach that assembles heterogeneous data into a biologically coherent model to allow predictions to be made and inconsistencies among the data to be found. We use this approach to study the network underlying sepal development in the young flower of Arabidopsis thaliana. We constructed a digital atlas of gene expression and used it to build a dynamical molecular regulatory network model of sepal primordium development. This led to the construction of a coherent molecular network model for lateral organ polarity that fully recapitulates expression and interaction data. Our model predicts the existence of three novel pathways involving the HD-ZIP III genes and both cytokinin and ARGONAUTE family members. In addition, our model provides predictions on molecular interactions. In a broader context, this approach allows the extraction of biological knowledge from diverse types of data and can be used to study developmental processes in any multicellular organism. PMID:22198150

  6. Inhaled resveratrol treatments slow ageing-related degenerative changes in mouse lung.

    PubMed

    Navarro, Sonia; Reddy, Raghava; Lee, Jooeun; Warburton, David; Driscoll, Barbara

    2017-05-01

    Lung ageing, a significant risk factor for chronic human lung diseases such as COPD and emphysema, is characterised by airspace enlargement and decreasing lung function. Likewise, in prematurely ageing telomerase null (terc-/-) mice, p53 stabilisation within diminishing numbers of alveolar epithelial type 2 cells (AEC2) accompanies reduced lung function. Resveratrol (RSL) is a plant phytoalexin that has previously showed efficacy in enhancing invertebrate longevity and supporting mammalian muscle metabolism when delivered orally. Here, we tested whether inhaled RSL could protect young, terc-/- mice from accelerated ageing of the lung. terc-/- mice aged 2 months inhaled 1 mg/kg RSL that was instilled intratracheally once per month for 3 months. One month after the last inhalation, whole lung function, structure and cellular DNA damage were evaluated and AEC2 survival was assessed by western blotting for survival pathway gene expression. RSL treatments delayed the loss of lung compliance (p<0.05), maintained lung structure (p<0.001) and blocked parenchymal cell DNA damage as measured by TdT Nick-End Labeling (TUNEL). RSL, a known agonist of deacetylase SIRT1, supported AEC2 survival by stimulating SIRT1 expression, promoting p53 destabilisation and decreasing Bax expression and by maintaining expression levels of Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), activated p-Akt and p-Mdm2 and inactivated Phospho-Phosphatase and tensin homolog (p-PTEN). RSL prophylaxis by inhalation is a potential approach for slowing ageing-related deterioration of lung function and structure by maintaining AEC2 integrity. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

  7. Influence of dietary selenium on mouse lung biochemical response and tolerance to ozone inhalation

    SciTech Connect

    Elsayed, N.M.

    1983-01-01

    This study examined whether altered selenium (Se) intake with or without ozone (O/sub 3/) stress would result in a possible 1) dose-response relationship between lung Se and glutathione peroxidase, 2) influence of Se on other lung parameters, 3) interrelationship between lung Se and vitamin E contents, and 4) alteration of lung sensitivity or tolerance to O/sub 3/. The results showed the following: 1) Omission of dietary Se resulted in a drastic decline in GP activity but did not affect the other enzyme activities studied. 2) A stimulation of the PPC and CAC activites with low-level O/sub 3/ exposure occurred only in Se-supplemented mice. The stimulation was greater in the lungs of mice fed 1.0 ppm Se compared to 0.15 ppm, i.e., the response was Se-dose dependent in this range. 3) Diminished GP activity possibly resulted in a decreased demand for NADPH produced via not only the PPC but also the CAC. 4) An inverse relationship was observed between Se and vitamin E contents in lung tissue, showing that a compensatory relationship existed between the two. 5) After each O/sub 3/ exposure Se content increased in lung tissue of both dietary groups, suggesting a possible mobilization of Se to the lung under O/sub 3/ stress. 6) Decreased GP activity due to Se deficiency and the ensuring lack of stimulated NADPH production in the lung did not alter the animal sensitivity to O/sub 3/, suggesting that GP reaction and NADPH production cycles were not crucial for animal tolerance.

  8. Pharmacological and genetic reappraisals of protease and oxidative stress pathways in a mouse model of obstructive lung diseases

    PubMed Central

    Shuto, Tsuyoshi; Kamei, Shunsuke; Nohara, Hirofumi; Fujikawa, Haruka; Tasaki, Yukihiro; Sugahara, Takuya; Ono, Tomomi; Matsumoto, Chizuru; Sakaguchi, Yuki; Maruta, Kasumi; Nakashima, Ryunosuke; Kawakami, Taisei; Suico, Mary Ann; Kondo, Yoshitaka; Ishigami, Akihito; Takeo, Toru; Tanaka, Ken-ichiro; Watanabe, Hiroshi; Nakagata, Naomi; Uchimura, Kohei; Kitamura, Kenichiro; Li, Jian-Dong; Kai, Hirofumi

    2016-01-01

    Protease-antiprotease imbalance and oxidative stress are considered to be major pathophysiological hallmarks of severe obstructive lung diseases including chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF), but limited information is available on their direct roles in the regulation of pulmonary phenotypes. Here, we utilized βENaC-transgenic (Tg) mice, the previously established mouse model of severe obstructive lung diseases, to produce lower-mortality but pathophysiologically highly useful mouse model by backcrossing the original line with C57/BL6J mice. C57/BL6J-βENaC-Tg mice showed higher survival rates and key pulmonary abnormalities of COPD/CF, including mucous hypersecretion, inflammatory and emphysematous phenotypes and pulmonary dysfunction. DNA microarray analysis confirmed that protease- and oxidative stress-dependent pathways are activated in the lung tissue of C57/BL6J-βENaC-Tg mice. Treatments of C57/BL6J-βENaC-Tg mice with a serine protease inhibitor ONO-3403, a derivative of camostat methylate (CM), but not CM, and with an anti-oxidant N-acetylcystein significantly improved pulmonary emphysema and dysfunction. Moreover, depletion of a murine endogenous antioxidant vitamin C (VC), by genetic disruption of VC-synthesizing enzyme SMP30 in C57/BL6J-βENaC-Tg mice, exaggerated pulmonary phenotypes. Thus, these assessments clarified that protease-antiprotease imbalance and oxidative stress are critical pathways that exacerbate the pulmonary phenotypes of C57/BL6J-βENaC-Tg mice, consistent with the characteristics of human COPD/CF. PMID:27982104

  9. Pharmacological and genetic reappraisals of protease and oxidative stress pathways in a mouse model of obstructive lung diseases.

    PubMed

    Shuto, Tsuyoshi; Kamei, Shunsuke; Nohara, Hirofumi; Fujikawa, Haruka; Tasaki, Yukihiro; Sugahara, Takuya; Ono, Tomomi; Matsumoto, Chizuru; Sakaguchi, Yuki; Maruta, Kasumi; Nakashima, Ryunosuke; Kawakami, Taisei; Suico, Mary Ann; Kondo, Yoshitaka; Ishigami, Akihito; Takeo, Toru; Tanaka, Ken-Ichiro; Watanabe, Hiroshi; Nakagata, Naomi; Uchimura, Kohei; Kitamura, Kenichiro; Li, Jian-Dong; Kai, Hirofumi

    2016-12-16

    Protease-antiprotease imbalance and oxidative stress are considered to be major pathophysiological hallmarks of severe obstructive lung diseases including chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF), but limited information is available on their direct roles in the regulation of pulmonary phenotypes. Here, we utilized βENaC-transgenic (Tg) mice, the previously established mouse model of severe obstructive lung diseases, to produce lower-mortality but pathophysiologically highly useful mouse model by backcrossing the original line with C57/BL6J mice. C57/BL6J-βENaC-Tg mice showed higher survival rates and key pulmonary abnormalities of COPD/CF, including mucous hypersecretion, inflammatory and emphysematous phenotypes and pulmonary dysfunction. DNA microarray analysis confirmed that protease- and oxidative stress-dependent pathways are activated in the lung tissue of C57/BL6J-βENaC-Tg mice. Treatments of C57/BL6J-βENaC-Tg mice with a serine protease inhibitor ONO-3403, a derivative of camostat methylate (CM), but not CM, and with an anti-oxidant N-acetylcystein significantly improved pulmonary emphysema and dysfunction. Moreover, depletion of a murine endogenous antioxidant vitamin C (VC), by genetic disruption of VC-synthesizing enzyme SMP30 in C57/BL6J-βENaC-Tg mice, exaggerated pulmonary phenotypes. Thus, these assessments clarified that protease-antiprotease imbalance and oxidative stress are critical pathways that exacerbate the pulmonary phenotypes of C57/BL6J-βENaC-Tg mice, consistent with the characteristics of human COPD/CF.

  10. Pharmacokinetics/Pharmacodynamics of Pulmonary Delivery of Colistin against Pseudomonas aeruginosa in a Mouse Lung Infection Model.

    PubMed

    Lin, Yu-Wei; Zhou, Qi Tony; Cheah, Soon-Ee; Zhao, Jinxin; Chen, Ke; Wang, Jiping; Chan, Hak-Kim; Li, Jian

    2017-03-01

    Colistin is often administered by inhalation and/or the parenteral route for the treatment of respiratory infections caused by multidrug-resistant (MDR) Pseudomonas aeruginosa However, limited pharmacokinetic (PK) and pharmacodynamic (PD) data are available to guide the optimization of dosage regimens of inhaled colistin. In the present study, PK of colistin in epithelial lining fluid (ELF) and plasma was determined following intratracheal delivery of a single dose of colistin solution in neutropenic lung-infected mice. The antimicrobial efficacy of intratracheal delivery of colistin against three P. aeruginosa strains (ATCC 27853, PAO1, and FADDI-PA022; MIC of 1 mg/liter for all strains) was examined in a neutropenic mouse lung infection model. Dose fractionation studies were conducted over 2.64 to 23.8 mg/kg of body weight/day. The inhibitory sigmoid model was employed to determine the PK/PD index that best described the antimicrobial efficacy of pulmonary delivery of colistin. In both ELF and plasma, the ratio of the area under the unbound concentration-time profile to MIC (fAUC/MIC) was the PK/PD index that best described the antimicrobial effect in mouse lung infection (R(2) = 0.60 to 0.84 for ELF and 0.64 to 0.83 for plasma). The fAUC/MIC targets required to achieve stasis against the three strains were 684 to 1,050 in ELF and 2.15 to 3.29 in plasma. The histopathological data showed that pulmonary delivery of colistin reduced infection-caused pulmonary inflammation and preserved the integrity of the lung epithelium, although colistin introduced mild pulmonary inflammation in healthy mice. This study showed pulmonary delivery of colistin provides antimicrobial effects against MDR P. aeruginosa lung infections superior to those of parenteral administrations. For the first time, our results provide important preclinical PK/PD information for optimization of inhaled colistin therapy. Copyright © 2017 American Society for Microbiology.

  11. Mycobacterium terrae isolated from indoor air of a moisture-damaged building induces sustained biphasic inflammatory response in mouse lungs.

    PubMed

    Jussila, Juha; Komulainen, Hannu; Huttunen, Kati; Roponen, Marjut; Iivanainen, Eila; Torkko, Pirjo; Kosma, Veli-Matti; Pelkonen, Jukka; Hirvonen, Maija-Riitta

    2002-11-01

    Occupants in moisture-damaged buildings suffer frequently from respiratory symptoms. This may be partly due to the presence of abnormal microbial growth or the altered microbial flora in the damaged buildings. However, the specific effects of the microbes on respiratory health and the way they provoke clinical manifestations are poorly understood. In the present study, we exposed mice via intratracheal instillation to a single dose of Mycobacterium terrae isolated from the indoor air of a moisture-damaged building (1 X 10(7), 5 X 10(7), or 1 X 10(8) microbes). Inflammation and toxicity in lungs were evaluated 2 hr later. The time course of the effects was assessed with the dose of 1 X 10(8) bacterial cells for up to 28 days. M. terrae caused a sustained biphasic inflammation in mouse lungs. The characteristic features for the first phase, which lasted from 6 hr to 3 days, were elevated proinflammatory cytokine [i.e., tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6)] levels in the bronchoalveolar lavage fluid (BALF). TNF-alpha was produced in the lungs more intensively than was IL-6. Neutrophils were the most abundant cells in the airways during the first phase, although their numbers in BALF remained elevated up to 21 days. The characteristics of the second phase, which lasted from 7 to 28 days, were elevated TNF-alpha levels in BALF, expression of inducible nitric oxide synthase in BAL cells, and recruitment of mononuclear cells such as lymphocytes and macrophages into the airways. Moreover, total protein, albumin, and lactate dehydrogenase concentrations were elevated in both phases in BALF. The bacteria were detected in lungs up to 28 days. In summary, these observations indicate that M. terrae is capable of provoking a sustained, biphasic inflammation in mouse lungs and can cause a moderate degree of cytotoxicity. Thus, M. terrae can be considered a species with potential to adversely affect the health of the occupants of moisture

  12. A natural protective function of invariant NKT cells in a mouse model of innate-cell-driven lung inflammation.

    PubMed

    Bourgeois, Elvire A; Levescot, Anaïs; Diem, Séverine; Chauvineau, Angélique; Bergès, Hortense; Milpied, Pierre; Lehuen, Agnès; Damotte, Diane; Gombert, Jean-Marc; Schneider, Elke; Girard, Jean-Philippe; Gourdy, Pierre; Herbelin, André

    2011-02-01

    Activation of invariant natural killer T (iNKT) cells by treatment with their α-galactosyl ceramide ligand provides therapeutic benefits in several immune inflammatory settings. Given the artificial nature of this stimulation, the natural regulatory functions of iNKT remain uncertain. Addressing this issue in a mouse model of innate-cell-driven lung inflammation induced by the cytokine/alarmin IL-33 that targets iNKT cells, we found that eosinophil and neutrophil recruitment was markedly increased in treated iNKT cell-deficient (Jα18 KO) mice, as was the local production of eotaxin and keratinocyte chemoattractant chemokines. By contrast, lung inflammation decreased after adoptive transfer of iNKT cells, which restored the WT inflammatory response in Jα18 KO mice. Finally, we established that this natural anti-inflammatory function of iNKT cells depends on their IFN-γ production and on endogenous IL-12. Our study provides the first evidence of a protective role of iNKT cells during lung inflammation that does not require pharmacological TCR engagement.

  13. A cell-impermeable cyclosporine A derivative reduces pathology in a mouse model of allergic lung inflammation.

    PubMed

    Balsley, Molly A; Malesevic, Miroslav; Stemmy, Erik J; Gigley, Jason; Jurjus, Rosalyn A; Herzog, Dallen; Bukrinsky, Michael I; Fischer, Gunter; Constant, Stephanie L

    2010-12-15

    Although the main regulators of leukocyte trafficking are chemokines, another family of chemotactic agents is cyclophilins. Intracellular cyclophilins function as peptidyl-prolyl cis-trans isomerases and are targets of the immunosuppressive drug cyclosporine A (CsA). Cyclophilins can also be secreted in response to stress factors, with elevated levels of extracellular cyclophilins detected in several inflammatory diseases. Extracellular cyclophilins are known to have potent chemotactic properties, suggesting that they might contribute to inflammatory responses by recruiting leukocytes into tissues. The objective of the present study was to determine the impact of blocking cyclophilin activity using a cell-impermeable derivative of CsA to specifically target extracellular pools of cyclophilins. In this study, we show that treatment with this compound in a mouse model of allergic lung inflammation demonstrates up to 80% reduction in inflammation, directly inhibits the recruitment of Ag-specific CD4(+) T cells, and works equally well when delivered at 100-fold lower doses directly to the airways. Our findings suggest that cell-impermeable analogs of CsA can effectively reduce inflammatory responses by targeting leukocyte recruitment mediated by extracellular cyclophilins. Specifically blocking the extracellular functions of cyclophilins may provide an approach for inhibiting the recruitment of one of the principal immune regulators of allergic lung inflammation, Ag-specific CD4(+) T cells, into inflamed airways and lungs.

  14. A Cell-Impermeable Cyclosporine A Derivative Reduces Pathology in a Mouse Model of Allergic Lung Inflammation

    PubMed Central

    Balsley, Molly A.; Malesevic, Miroslav; Stemmy, Erik J.; Gigley, Jason; Jurjus, Rosalyn A.; Herzog, Dallen; Bukrinsky, Michael I.; Fischer, Gunter; Constant, Stephanie L.

    2013-01-01

    Although the main regulators of leukocyte trafficking are chemokines, another family of chemotactic agents is cyclophilins. Intracellular cyclophilins function as peptidyl-protyl cis-trans isomerases and are targets of the immunosuppressive drug, cyclosporine A (CsA). Cyclophilins can also be secreted in response to stress factors, with elevated levels of extracellular cyclophilins detected in several inflammatory diseases. Extracellular cyclophilins are known to have potent chemotactic properties, suggesting they might contribute to inflammatory responses by recruiting leukocytes into tissues. The objective of the current study was to determine the impact of blocking cyclophilin activity using a cell-impermeable derivative of CsA, MM218, to specifically target extracellular pools of cyclophilins. We show that treatment with this compound in a mouse model of allergic lung inflammation: 1) demonstrates up to 80% reduction in inflammation, 2) directly inhibits the recruitment of antigen-specific CD4+ T cells, and 3) works equally well when delivered at 100-fold lower doses to the airways. Our findings suggest that cell-impermeable analogs of CsA can effectively reduce inflammatory responses by targeting leukocyte recruitment mediated by extracellular cyclophilins. Specifically blocking the extracellular function(s) of cyclophilins may provide a novel approach for inhibiting the recruitment of one of the principal immune regulators of allergic lung inflammation, antigen-specific CD4+ T cells, into inflamed airways and lungs. PMID:21057089

  15. Aerosolized bovine lactoferrin reduces neutrophils and pro-inflammatory cytokines in mouse models of Pseudomonas aeruginosa lung infections.

    PubMed

    Valenti, Piera; Frioni, Alessandra; Rossi, Alice; Ranucci, Serena; De Fino, Ida; Cutone, Antimo; Rosa, Luigi; Bragonzi, Alessandra; Berlutti, Francesca

    2017-02-01

    Lactoferrin (Lf), an iron-chelating glycoprotein of innate immunity, produced by exocrine glands and neutrophils in infection/inflammation sites, is one of the most abundant defence molecules in airway secretions. Lf, a pleiotropic molecule, exhibits antibacterial and anti-inflammatory functions. These properties may play a relevant role in airway infections characterized by exaggerated inflammatory response, as in Pseudomonas aeruginosa lung infection in cystic fibrosis (CF) subjects. To verify the Lf role in Pseudomonas aeruginosa lung infection, we evaluated the efficacy of aerosolized bovine Lf (bLf) in mouse models of P. aeruginosa acute and chronic lung infections. C57BL/6NCrl mice were challenged with 10(6) CFUs of P. aeruginosa PAO1 (acute infection) or MDR-RP73 strain (chronic infection) by intra-tracheal administration. In both acute and chronic infections, aerosolized bLf resulted in nonsignificant reduction of bacterial load but significant decrease of the neutrophil recruitment and pro-inflammatory cytokine levels. Moreover, in chronic infection the bLf-treated mice recovered body weight faster and to a higher extent than the control mice. These findings add new insights into the benefits of bLf as a mediator of general health and its potential therapeutic applications.

  16. Using gene expression profiling to evaluate cellular responses in mouse lungs exposed to V2O5 and a group of other mouse lung tumorigens and non-tumorigens.

    PubMed

    Black, Michael B; Dodd, Darol E; McMullen, Patrick D; Pendse, Salil; MacGregor, Judith A; Gollapudi, B Bhaskar; Andersen, Melvin E

    2015-10-01

    Many compounds test positive for lung tumors in two-year NTP carcinogenicity bioassays in B6C3F1 mice. V2O5 was identified as a lung carcinogen in this assay, leading to its IARC (International Agency for Research on Cancer) classification as group 2b or a "possible" human carcinogen. To assess potential tumorigenic mode of action of V2O5, we compared gene expression and gene ontology enrichment in lung tissue of female B6C3F1 mice exposed for 13 weeks to a V2O5 particulate aerosol at a tumorigenic level (2.0 mg/m(3)). Relative to 12 other compounds also tested for carcinogenicity in 2-year bioassays in mice, there were 1026 differentially expressed genes with V2O5, of which 483 were unique to V2O5. Ontology analysis of the 1026 V2O5 differentially expressed genes showed enrichment for hyaluronan and sphingolipid metabolism, adenylate cyclase functions, c-AMP signaling and PKA activation/signaling. Enrichment of lipids/lipoprotein metabolism and inflammatory pathways were consistent with previously reported clinical findings. Enrichment of c-AMP and PKA signaling pathways may arise due to inhibition of phosphatases, a known biological action of vanadate. We saw no enrichment for DNA-damage, oxidative stress, cell cycle, or apoptosis pathway signaling in mouse lungs exposed to V2O5 which is in contrast with past studies evaluating in vivo gene expression in target tissues of other carcinogens (arsenic, formaldehyde, naphthalene and chloroprene). Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Recruitment of cells into the Drosophila wing primordium by a feed-forward circuit of vestigial autoregulation.

    PubMed

    Zecca, Myriam; Struhl, Gary

    2007-08-01

    The Drosophila wing primordium is defined by expression of the selector gene vestigial (vg) in a discrete subpopulation of cells within the wing imaginal disc. Following the early segregation of the disc into dorsal (D) and ventral (V) compartments, vg expression is governed by signals generated along the boundary between the two compartments. Short-range DSL (Delta/Serrate/LAG-2)-Notch signaling between D and V cells drives vg expression in ;border' cells that flank the boundary. It also induces these same cells to secrete the long-range morphogen Wingless (Wg), which drives vg expression in surrounding cells up to 25-30 cell diameters away. Here, we show that Wg signaling is not sufficient to activate vg expression away from the D-V boundary. Instead, Wg must act in combination with a short-range signal produced by cells that already express vg. We present evidence that this vg-dependent, vg-inducing signal feeds forward from one cell to the next to entrain surrounding cells to join the growing wing primordium in response to Wg. We propose that Wg promotes the expansion of the wing primordium following the D-V segregation by fueling this non-autonomous autoregulatory mechanism.

  18. Low-dose nicotine does not promote lung tumors in mouse models

    Cancer.gov

    Experiments in mice show that low levels of exposure to nicotine, equivalent to those in humans who use nicotine replacement therapy (NRT) to help them quit smoking, did not promote lung tumor growth.

  19. Biosynthesis and degradation of collagen in X-irradiated mouse lung

    SciTech Connect

    Walklin, C.M.; Freedman, R.B.; Law, M.P.

    1987-11-01

    Fibrosis, characterized by accumulation of collagen, is a delayed result of radiation injury in many tissues, including lung. To investigate its development, synthesis and degradation of collagen were measured in lungs of mice after X irradiation of the whole thorax. The ratio of type I (coarse fibered) to type III (meshwork) collagen was also determined. Synthesis of procollagen, measured as the activities of prolyl-4-hydroxylase and protein disulfide isomerase in lung tissue, was increased at 2 months after X-ray doses of 5, 7.5, and 9 Gy. Maximal increases were observed 6 to 7 months after doses of 9 Gy and persisted up to 15 months after exposure. Increases after 5 and 7.5 Gy were more gradual, but by 1 year after irradiation they had reached levels similar to those after 9 Gy. X irradiation had no effect on the degradation of collagen as assessed by collagenase activity in lung. The ratio of type I to type III collagen, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of collagen-derived cyanogen bromide peptides, was the same in irradiated lungs as in age-matched controls. Therefore, increased synthesis of procollagen, rather than decreased degradation of collagen or changes in collagen type, is an important factor in the accumulation of collagen in irradiated lung.

  20. Mouse mammary tumor virus-like gene sequences are present in lung patient specimens.

    PubMed

    Trejo-Avila, Laura M; Zapata-Benavides, Pablo; Barrera-Rodríguez, Raúl; Badillo-Almaráz, Isaías; Saavedra-Alonso, Santiago; Zamora-Avila, Diana E; Morán-Santibañez, Karla; Garza-Sáenz, Jorge A; Tamez-Guerra, Reyes; Rodríguez-Padilla, Cristina

    2011-09-24

    Previous studies have reported on the presence of Murine Mammary Tumor Virus (MMTV)-like gene sequences in human cancer tissue specimens. Here, we search for MMTV-like gene sequences in lung diseases including carcinomas specimens from a Mexican population. This study was based on our previous study reporting that the INER51 lung cancer cell line, from a pleural effusion of a Mexican patient, contains MMTV-like env gene sequences. The MMTV-like env gene sequences have been detected in three out of 18 specimens studied, by PCR using a specific set of MMTV-like primers. The three identified MMTV-like gene sequences, which were assigned as INER6, HZ101, and HZ14, were 99%, 98%, and 97% homologous, respectively, as compared to GenBank sequence accession number AY161347. The INER6 and HZ-101 samples were isolated from lung cancer specimens, and the HZ-14 was isolated from an acute inflammatory lung infiltrate sample. Two of the env sequences exhibited disruption of the reading frame due to mutations. In summary, we identified the presence of MMTV-like gene sequences in 2 out of 11 (18%) of the lung carcinomas and 1 out of 7 (14%) of acute inflamatory lung infiltrate specimens studied of a Mexican Population.

  1. Mouse mammary tumor virus-like gene sequences are present in lung patient specimens

    PubMed Central

    2011-01-01

    Background Previous studies have reported on the presence of Murine Mammary Tumor Virus (MMTV)-like gene sequences in human cancer tissue specimens. Here, we search for MMTV-like gene sequences in lung diseases including carcinomas specimens from a Mexican population. This study was based on our previous study reporting that the INER51 lung cancer cell line, from a pleural effusion of a Mexican patient, contains MMTV-like env gene sequences. Results The MMTV-like env gene sequences have been detected in three out of 18 specimens studied, by PCR using a specific set of MMTV-like primers. The three identified MMTV-like gene sequences, which were assigned as INER6, HZ101, and HZ14, were 99%, 98%, and 97% homologous, respectively, as compared to GenBank sequence accession number AY161347. The INER6 and HZ-101 samples were isolated from lung cancer specimens, and the HZ-14 was isolated from an acute inflammatory lung infiltrate sample. Two of the env sequences exhibited disruption of the reading frame due to mutations. Conclusion In summary, we identified the presence of MMTV-like gene sequences in 2 out of 11 (18%) of the lung carcinomas and 1 out of 7 (14%) of acute inflamatory lung infiltrate specimens studied of a Mexican Population. PMID:21943279

  2. Mechanical properties of mouse lungs along organ decellularization by sodium dodecyl sulfate.

    PubMed

    Nonaka, Paula N; Uriarte, Juan J; Campillo, Noelia; Melo, Esther; Navajas, Daniel; Farré, Ramon; Oliveira, Luis V F

    2014-08-15

    Lung decellularization is based on the use of physical, chemical, or enzymatic methods to break down the integrity of the cells followed by a treatment to extract the cellular material from the lung scaffold. The aim of this study was to characterize the mechanical changes throughout the different steps of lung decellularization process. Four lungs from mice (C57BL/6) were decellularized by using a conventional protocol based on sodium dodecyl sulfate. Lungs resistance (R(L)) and elastance (E(L)) were measured along decellularization steps and were computed by linear regression fitting of tracheal pressure, flow, and volume during mechanical ventilation. Transients differences found were more distinct in an intermediate step after the lungs were rinsed with deionized water and treated with 1% SDS, whereupon the percentage of variation reached approximately 80% for resistance values and 30% for elastance values. In conclusion, although a variation in extracellular matrix stiffness was observed during the decellularization process, this variation can be considered negligible overall because the resistance and elastance returned to basal values at the final decellularization step.

  3. A cytokine signal inhibitor for rheumatoid arthritis enhances cancer metastasis via depletion of NK cells in an experimental lung metastasis mouse model of colon cancer.

    PubMed

    Shimaoka, Hideki; Takeno, Shinsuke; Maki, Kenji; Sasaki, Takahide; Hasegawa, Suguru; Yamashita, Yuichi

    2017-09-01

    Current therapy for rheumatoid arthritis (RA) relies on global suppression of the immune response or specific blockade of inflammatory cytokines. However, it is unclear how immunosuppressants affect patients with cancer. Therefore, in the present study, the effect of three biological agents, tofacitinib, anti-mouse IL-6 receptor antibody (MR16-1) and etanercept, which are used for the treatment of RA diseases, on a tumor-bearing mouse model was investigated. The effect of the three agents was examined using a mouse lung-metastasis model with the murine colon 26 cancer cell line. Lymphocyte subsets and natural killer (NK) cells in peripheral blood and spleen were analyzed using fluorescence-activated cell sorting, and the number of lung surface nodules was examined. In the continuous tofacitinib administration (15 mg/kg/day) group, the number of lung surface nodules was significantly increased compared with that of the vehicle-treated group (vehicle, 1.20±0.58; tofacitinib, 35.6±10.81; P<0.01). NK cell number in the blood and spleen of tofacitinib-treated mice was decreased 10-fold, and the percentage of cluster of differentiation (CD)11(+)CD27(-) NK cells was significantly reduced. MR16-1 [8 mg/mouse; once a week; intraperitoneal (i.p.)] or etanercept (1 mg/mouse; 3 times a week; i.p.) treatment did not affect the number of NK cells or lung metastasis. In the present study, immunosuppressants that target cytokines, including tofacitinib, were demonstrated to inhibit the proliferation and differentiation of NK cells, and exhibit the potential to promote cancer metastasis using a mouse model of lung metastasis.

  4. Comparison of efficacy and toxicity of traditional Chinese medicine (TCM) herbal mixture LQ and conventional chemotherapy on lung cancer metastasis and survival in mouse models.

    PubMed

    Zhang, Lei; Wu, Chengyu; Zhang, Yong; Liu, Fang; Wang, Xiaoen; Zhao, Ming; Hoffman, Robert M

    2014-01-01

    Unlike Western medicine that generally uses purified compounds and aims to target a single molecule or pathway, traditional Chinese medicine (TCM) compositions usually comprise multiple herbs and components that are necessary for efficacy. Despite the very long-time and wide-spread use of TCM, there are very few direct comparisons of TCM and standard cytotoxic chemotherapy. In the present report, we compared the efficacy of the TCM herbal mixture LQ against lung cancer in mouse models with doxorubicin (DOX) and cyclophosphamide (CTX). LQ inhibited tumor size and weight measured directly as well as by fluorescent-protein imaging in subcutaneous, orthotopic, spontaneous experimental metastasis and angiogenesis mouse models of lung cancer. LQ was efficacious against primary and metastatic lung cancer without weight loss and organ toxicity. In contrast, CTX and DOX, although efficacious in the lung cancer models caused significant weight loss, and organ toxicity. LQ also had anti-angiogenic activity as observed in lung tumors growing in nestin-driven green fluorescent protein (ND-GFP) transgenic nude mice, which selectively express GFP in nascent blood vessels. Survival of tumor-bearing mice was also prolonged by LQ, comparable to DOX. In vitro, lung cancer cells were killed by LQ as observed by time-lapse imaging, comparable to cisplatinum. LQ was more potent to induce cell death on cancer cell lines than normal cell lines unlike cytotoxic chemotherapy. The results indicate that LQ has non-toxic efficacy against metastatic lung cancer.

  5. Comparison of Efficacy and Toxicity of Traditional Chinese Medicine (TCM) Herbal Mixture LQ and Conventional Chemotherapy on Lung Cancer Metastasis and Survival in Mouse Models

    PubMed Central

    Zhang, Lei; Wu, Chengyu; Zhang, Yong; Liu, Fang; Wang, Xiaoen; Zhao, Ming; Hoffman, Robert M.

    2014-01-01

    Unlike Western medicine that generally uses purified compounds and aims to target a single molecule or pathway, traditional Chinese medicine (TCM) compositions usually comprise multiple herbs and components that are necessary for efficacy. Despite the very long-time and wide-spread use of TCM, there are very few direct comparisons of TCM and standard cytotoxic chemotherapy. In the present report, we compared the efficacy of the TCM herbal mixture LQ against lung cancer in mouse models with doxorubicin (DOX) and cyclophosphamide (CTX). LQ inhibited tumor size and weight measured directly as well as by fluorescent-protein imaging in subcutaneous, orthotopic, spontaneous experimental metastasis and angiogenesis mouse models of lung cancer. LQ was efficacious against primary and metastatic lung cancer without weight loss and organ toxicity. In contrast, CTX and DOX, although efficacious in the lung cancer models caused significant weight loss, and organ toxicity. LQ also had anti-angiogenic activity as observed in lung tumors growing in nestin-driven green fluorescent protein (ND-GFP) transgenic nude mice, which selectively express GFP in nascent blood vessels. Survival of tumor-bearing mice was also prolonged by LQ, comparable to DOX. In vitro, lung cancer cells were killed by LQ as observed by time-lapse imaging, comparable to cisplatinum. LQ was more potent to induce cell death on cancer cell lines than normal cell lines unlike cytotoxic chemotherapy. The results indicate that LQ has non-toxic efficacy against metastatic lung cancer. PMID:25286158

  6. Multi-Modal Imaging in a Mouse Model of Orthotopic Lung Cancer

    PubMed Central

    Patel, Priya; Kato, Tatsuya; Ujiie, Hideki; Wada, Hironobu; Lee, Daiyoon; Hu, Hsin-pei; Hirohashi, Kentaro; Ahn, Jin Young; Zheng, Jinzi; Yasufuku, Kazuhiro

    2016-01-01

    Background Investigation of CF800, a novel PEGylated nano-liposomal imaging agent containing indocyanine green (ICG) and iohexol, for real-time near infrared (NIR) fluorescence and computed tomography (CT) image-guided surgery in an orthotopic lung cancer model in nude mice. Methods CF800 was intravenously administered into 13 mice bearing the H460 orthotopic human lung cancer. At 48 h post-injection (peak imaging agent accumulation time point), ex vivo NIR and CT imaging was performed. A clinical NIR imaging system (SPY®, Novadaq) was used to measure fluorescence intensity of tumor and lung. Tumor-to-background-ratios (TBR) were calculated in inflated and deflated states. The mean Hounsfield unit (HU) of lung tumor was quantified using the CT data set and a semi-automated threshold-based method. Histological evaluation using H&E, the macrophage marker F4/80 and the endothelial cell marker CD31, was performed, and compared to the liposomal fluorescence signal obtained from adjacent tissue sections Results The fluorescence TBR measured when the lung is in the inflated state (2.0 ± 0.58) was significantly greater than in the deflated state (1.42 ± 0.380 (n = 7, p<0.003). Mean fluorescent signal in tumor was highly variable across samples, (49.0 ± 18.8 AU). CT image analysis revealed greater contrast enhancement in lung tumors (a mean increase of 110 ± 57 HU) when CF800 is administered compared to the no contrast enhanced tumors (p = 0.0002). Conclusion Preliminary data suggests that the high fluorescence TBR and CT tumor contrast enhancement provided by CF800 may have clinical utility in localization of lung cancer during CT and NIR image-guided surgery. PMID:27584018

  7. Difference in the toxicity mechanism between ion and nanoparticle forms of silver in the mouse lung and in macrophages.

    PubMed

    Arai, Yuta; Miyayama, Takamitsu; Hirano, Seishiro

    2015-02-03

    The health effects of silver nanoparticles (AgNPs) have not been well investigated, despite AgNPs now being widely used in consumer products. We investigated the metabolic behavior and toxicity of AgNPs in comparison to silver nitrate (AgNO3) both in vivo and in vitro. AgNPs (20 nm diameter) suspended in 1% albumin solution or AgNO3 solution was injected into the mouse lung. Less than 1% of the initial dose of AgNPs and more than 7% of the initial dose of AgNO3 was recovered in the liver 4h after administration, suggesting that the ionic form of silver was absorbed by the lung tissue and entered the systemic circulation more efficiently than AgNPs. The pro-inflammatory cytokine, IL-1β, and neutrophils in bronchoalveolar lavage fluid (BALF) increased following intratracheal instillation of AgNPs or AgNO3. AgNO3 recruited more neutrophils in the alveolar space than did AgNPs. In the in vitro study, AgNO3 was more cytotoxic than 20, 60, or 100 nm diameter AgNPs in a mouse macrophage cell line (J774.1). To investigate the intracellular distribution of Ag in detail, J774.1 cells were exposed to AgNO3 or 20 nm AgNPs and the distribution of Ag to cytosolic proteins was investigated using HPLC-inductively coupled plasma-mass spectrometry (HPLC-ICP-MS). Ag was mainly distributed to metallothioneins (MT) and to high molecular weight proteins in AgNO3- and AgNPs-exposed cells, respectively. Confocal laser microscopic examination of LysoTracker(®)-labeled cells indicated that AgNPs were colocalized with lysosomes in J774.1 cells. These results suggest that AgNPs were transported to lysosomes and only gradually dissolved in the macrophages, causing milder inflammatory stimulation in the mouse lung compared to AgNO3.

  8. Five-year update on the mouse model of orthotopic lung transplantation: Scientific uses, tricks of the trade, and tips for success

    PubMed Central

    Lin, Xue; Li, Wenjun; Lai, Jiaming; Okazaki, Mikio; Sugimoto, Seiichiro; Yamamoto, Sumiharu; Wang, Xingan; Gelman, Andrew E.; Kreisel, Daniel

    2012-01-01

    It has been 5 years since our team reported the first successful model of orthotopic single lung transplantation in the mouse. There has been great demand for this technique due to the obvious experimental advantages the mouse offers over other large and small animal models of lung transplantation. These include the availability of mouse-specific reagents as well as knockout and transgenic technology. Our laboratory has utilized this mouse model to study both immunological and non-immunological mechanisms of lung transplant physiology while others have focused on models of chronic rejection. It is surprising that despite our initial publication in 2007 only few other laboratories have published data using this model. This is likely due to the technical complexity of the surgical technique and perioperative complications, which can limit recipient survival. As two of the authors (XL and WL) have a combined experience of over 2500 left and right single lung transplants, this review will summarize their experience and delineate tips and tricks necessary for successful transplantation. We will also describe technical advances made since the original description of the model. PMID:22754663

  9. Inhaled Nitric Oxide Decreases Leukocyte Trafficking in the Neonatal Mouse Lung During Exposure to >95% Oxygen

    PubMed Central

    Rose, Melissa J.; Stenger, Michael R.; Joshi, Mandar S.; Welty, Stephen E.; Bauer, John Anthony; Nelin, Leif D.

    2010-01-01

    Chronic lung injury in the neonate is termed bronchopulmonary dysplasia (BPD). These patients generally require supplemental oxygen therapy, and hyperoxia has been implicated in the pathogenesis of BPD. The concomitant use of oxygen and inhaled nitric oxide (iNO) may result in the generation of reactive nitrogen species, or may have an anti-inflammatory effect in the neonatal lung. We tested the hypothesis that exposure to >95% O2 in neonatal mice would increase trafficking of leukocytes into the lung, and that the addition of iNO to >95% O2 would decrease this leukocyte trafficking. Hyperoxia resulted in fewer alveoli, increased presence of neutrophils and macrophages, and decreased number of mast cells within the lung parenchyma. Adding iNO to hyperoxia prevented the hyperoxia-induced changes and resulted in the numbers of alveoli, neutrophils, macrophages, and mast cells approximating those found in controls (room air exposure). Intercellular adhesion molecule (ICAM) and monocyte chemotactic protein-1 (MCP-1), two factors responsible for leukocyte recruitment, were upregulated by hyperoxic exposure, but the addition of iNO to the hyperoxic exposure prevented the hyperoxia-induced upregulation of ICAM and MCP-1. These data demonstrate that iNO alters the hyperoxia-induced recruitment of leukocytes into the lung. PMID:19915514

  10. Expanding signaling-molecule wavefront model of cell polarization in the Drosophila wing primordium.

    PubMed

    Wortman, Juliana C; Nahmad, Marcos; Zhang, Peng Cheng; Lander, Arthur D; Yu, Clare C

    2017-07-01

    In developing tissues, cell polarization and proliferation are regulated by morphogens and signaling pathways. Cells throughout the Drosophila wing primordium typically show subcellular localization of the unconventional myosin Dachs on the distal side of cells (nearest the center of the disc). Dachs localization depends on the spatial distribution of bonds between the protocadherins Fat (Ft) and Dachsous (Ds), which form heterodimers between adjacent cells; and the Golgi kinase Four-jointed (Fj), which affects the binding affinities of Ft and Ds. The Fj concentration forms a linear gradient while the Ds concentration is roughly uniform throughout most of the wing pouch with a steep transition region that propagates from the center to the edge of the pouch during the third larval instar. Although the Fj gradient is an important cue for polarization, it is unclear how the polarization is affected by cell division and the expanding Ds transition region, both of which can alter the distribution of Ft-Ds heterodimers around the cell periphery. We have developed a computational model to address these questions. In our model, the binding affinity of Ft and Ds depends on phosphorylation by Fj. We assume that the asymmetry of the Ft-Ds bond distribution around the cell periphery defines the polarization, with greater asymmetry promoting cell proliferation. Our model predicts that this asymmetry is greatest in the radially-expanding transition region that leaves polarized cells in its wake. These cells naturally retain their bond distribution asymmetry after division by rapidly replenishing Ft-Ds bonds at new cell-cell interfaces. Thus we predict that the distal localization of Dachs in cells throughout the pouch requires the movement of the Ds transition region and the simple presence, rather than any specific spatial pattern, of Fj.

  11. Leading and trailing cells cooperate in collective migration of the zebrafish posterior lateral line primordium.

    PubMed

    Dalle Nogare, Damian; Somers, Katherine; Rao, Swetha; Matsuda, Miho; Reichman-Fried, Michal; Raz, Erez; Chitnis, Ajay B

    2014-08-01

    Collective migration of cells in the zebrafish posterior lateral line primordium (PLLp) along a path defined by Cxcl12a expression depends on Cxcr4b receptors in leading cells and on Cxcr7b in trailing cells. Cxcr7b-mediated degradation of Cxcl12a by trailing cells generates a local gradient of Cxcl12a that guides PLLp migration. Agent-based computer models were built to explore how a polarized response to Cxcl12a, mediated by Cxcr4b in leading cells and prevented by Cxcr7b in trailing cells, determines unidirectional migration of the PLLp. These chemokine signaling-based models effectively recapitulate many behaviors of the PLLp and provide potential explanations for the characteristic behaviors that emerge when the PLLp is severed by laser to generate leading and trailing fragments. As predicted by our models, the bilateral stretching of the leading fragment is lost when chemokine signaling is blocked in the PLLp. However, movement of the trailing fragment toward the leading cells, which was also thought to be chemokine dependent, persists. This suggested that a chemokine-independent mechanism, not accounted for in our models, is responsible for this behavior. Further investigation of trailing cell behavior shows that their movement toward leading cells depends on FGF signaling and it can be re-oriented by exogenous FGF sources. Together, our observations reveal the simple yet elegant manner in which leading and trailing cells coordinate migration; while leading cells steer PLLp migration by following chemokine cues, cells further back play follow-the-leader as they migrate toward FGFs produced by leading cells.

  12. In toto imaging of the migrating Zebrafish lateral line primordium at single cell resolution.

    PubMed

    Nogare, Damian Dalle; Nikaido, Masataka; Somers, Katherine; Head, Jeffery; Piotrowski, Tatjana; Chitnis, Ajay B

    2017-02-01

    The zebrafish Posterior Lateral Line primordium (PLLp) has emerged as an important model system for studying many aspects of development, including cell migration, cell type specification and tissue morphogenesis. Despite this, basic aspects of PLLp biology remain incompletely understood. The PLLp is a group of approximately 140 cells which pioneers the formation of the Posterior Lateral Line (LL) system by migrating along the length of the embryo, periodically depositing clusters of epithelial cells, which will go on to form the mature sense organs of the lateral line, called neuromasts. The neuromasts are formed within the migrating PLLp as protoneuromasts: the first protoneuromast is formed close to the trailing end and additional protoneuromasts are formed sequentially, progressively closer to the leading edge of the migrating collective. We imaged the migration of PLL primordia and tracked every cell in the lateral line system over the course of migration. From this data set we unambiguously determined the lineage and fate of every cell deposited by the migrating PLLp. We show that, on average, proliferation across the entire PLLp is weakly patterned, with leading cells tending to divide more slowly than trailing cells. Neuromasts are formed sequentially by local expansion of existing cells along the length of the PLLp, not by self-renewing stem cell-like divisions of a restricted leading population that is highly proliferative. The fate of deposited cells, either within neuromasts or as interneuromast cells (in between deposited neuromasts) is not determined by any obvious stereotyped lineages. Instead, it is determined somewhat stochasitcailly, as a function of a cells distance from the center of a maturing protoneuromast. Together, our data provide a rigorous baseline for the behavior of the PLLp, which can be used to inform further study of this important model system. Published by Elsevier Inc.

  13. Leading and trailing cells cooperate in collective migration of the zebrafish posterior lateral line primordium

    PubMed Central

    Dalle Nogare, Damian; Somers, Katherine; Rao, Swetha; Matsuda, Miho; Reichman-Fried, Michal; Raz, Erez; Chitnis, Ajay B.

    2014-01-01

    Collective migration of cells in the zebrafish posterior lateral line primordium (PLLp) along a path defined by Cxcl12a expression depends on Cxcr4b receptors in leading cells and on Cxcr7b in trailing cells. Cxcr7b-mediated degradation of Cxcl12a by trailing cells generates a local gradient of Cxcl12a that guides PLLp migration. Agent-based computer models were built to explore how a polarized response to Cxcl12a, mediated by Cxcr4b in leading cells and prevented by Cxcr7b in trailing cells, determines unidirectional migration of the PLLp. These chemokine signaling-based models effectively recapitulate many behaviors of the PLLp and provide potential explanations for the characteristic behaviors that emerge when the PLLp is severed by laser to generate leading and trailing fragments. As predicted by our models, the bilateral stretching of the leading fragment is lost when chemokine signaling is blocked in the PLLp. However, movement of the trailing fragment toward the leading cells, which was also thought to be chemokine dependent, persists. This suggested that a chemokine-independent mechanism, not accounted for in our models, is responsible for this behavior. Further investigation of trailing cell behavior shows that their movement toward leading cells depends on FGF signaling and it can be re-oriented by exogenous FGF sources. Together, our observations reveal the simple yet elegant manner in which leading and trailing cells coordinate migration; while leading cells steer PLLp migration by following chemokine cues, cells further back play follow-the-leader as they migrate toward FGFs produced by leading cells. PMID:25063456

  14. Expanding signaling-molecule wavefront model of cell polarization in the Drosophila wing primordium

    PubMed Central

    Wortman, Juliana C.; Nahmad, Marcos; Zhang, Peng Cheng; Lander, Arthur D.; Yu, Clare C.

    2017-01-01

    In developing tissues, cell polarization and proliferation are regulated by morphogens and signaling pathways. Cells throughout the Drosophila wing primordium typically show subcellular localization of the unconventional myosin Dachs on the distal side of cells (nearest the center of the disc). Dachs localization depends on the spatial distribution of bonds between the protocadherins Fat (Ft) and Dachsous (Ds), which form heterodimers between adjacent cells; and the Golgi kinase Four-jointed (Fj), which affects the binding affinities of Ft and Ds. The Fj concentration forms a linear gradient while the Ds concentration is roughly uniform throughout most of the wing pouch with a steep transition region that propagates from the center to the edge of the pouch during the third larval instar. Although the Fj gradient is an important cue for polarization, it is unclear how the polarization is affected by cell division and the expanding Ds transition region, both of which can alter the distribution of Ft-Ds heterodimers around the cell periphery. We have developed a computational model to address these questions. In our model, the binding affinity of Ft and Ds depends on phosphorylation by Fj. We assume that the asymmetry of the Ft-Ds bond distribution around the cell periphery defines the polarization, with greater asymmetry promoting cell proliferation. Our model predicts that this asymmetry is greatest in the radially-expanding transition region that leaves polarized cells in its wake. These cells naturally retain their bond distribution asymmetry after division by rapidly replenishing Ft-Ds bonds at new cell-cell interfaces. Thus we predict that the distal localization of Dachs in cells throughout the pouch requires the movement of the Ds transition region and the simple presence, rather than any specific spatial pattern, of Fj. PMID:28671940

  15. Transcriptome Profiling of the Newborn Mouse Lung Response to Acute Ozone Exposure

    PubMed Central

    Loader, Joan E.; White, Carl W.; Dakhama, Azzeddine

    2014-01-01

    Ozone pollution is associated with adverse effects on respiratory health in adults and children but its effects on the neonatal lung remain unknown. This study was carried out to define the effect of acute ozone exposure on the neonatal lung and to profile the transcriptome response. Newborn mice were exposed to ozone or filtered air for 3h. Total RNA was isolated from lung tissues at 6 and 24h after exposure and was subjected to microarray gene expression analysis. Compared to filtered air-exposed littermates, ozone-exposed newborn mice developed a small but significant neutrophilic airway response associated with increased CXCL1 and CXCL5 expression in the lung. Transcriptome analysis indicated that 455 genes were down-regulated and 166 genes were up-regulated by at least 1.5-fold at 6h post-ozone exposure (t-test, p < .05). At 24h, 543 genes were down-regulated and 323 genes were up-regulated in the lungs of ozone-exposed, compared to filtered air-exposed, newborn mice (t-test, p < .05). After controlling for false discovery rate, 50 genes were identified as significantly down-regulated and only a few (RORC, GRP, VREB3, and CYP2B6) were up-regulated at 24h post-ozone exposure (q < .05). Gene ontology enrichment analysis revealed that cell cycle-associated functions including cell division/proliferation were the most impacted pathways, which were negatively regulated by ozone exposure, an adverse effect that was associated with reduced bromo-deoxyuridine incorporation. These results demonstrate that acute ozone exposure alters cell proliferation in the developing neonatal lung through a global suppression of cell cycle function. PMID:24336422

  16. High microvascular endothelial water permeability in mouse lung measured by a pleural surface fluorescence method.

    PubMed Central

    Carter, E P; Olveczky, B P; Matthay, M A; Verkman, A S

    1998-01-01

    Transport of water between the capillary and airspace compartments in lung encounters serial barriers: the alveolar epithelium, interstitium, and capillary endothelium. We previously reported a pleural surface fluorescence method to measure net capillary-to-airspace water transport. To measure the osmotic water permeability across the microvascular endothelial barrier in intact lung, the airspace was filled with a water-immiscible fluorocarbon. The capillaries were perfused via the pulmonary artery with solutions of specified osmolalites containing a high-molecular-weight fluorescent dextran. An increase in perfusate osmolality produced a prompt decrease in surface fluorescence due to dye dilution in the capillaries, followed by a slower return to initial fluorescence as capillary and lung interstitial osmolality equilibrate. A mathematical model was developed to determine the osmotic water permeability coefficient (Pf) of lung microvessels from the time course of pleural surface fluorescence. As predicted, the magnitude of the prompt change in surface fluorescence increased with decreased pulmonary artery perfusion rate and increased osmotic gradient size. With raffinose used to induce the osmotic gradient, Pf was 0.03 cm/s at 23 degrees C and was reduced 54% by 0.5 mM HgCl2. Temperature dependence measurements gave an Arrhenius activation energy (Ea) of 5.4 kcal/mol (12-37 degrees C). The apparent Pf induced by the smaller osmolytes mannitol and glycine was 0.021 and 0.011 cm/s (23 degrees C). Immunoblot analysis showed approximately 1.4 x 10(12) aquaporin-1 water channels/cm2 of capillary surface, which accounted quantitatively for the high Pf. These results establish a novel method for measuring osmotically driven water permeability across microvessels in intact lung. The high Pf, low Ea, and mercurial inhibition indicate the involvement of molecular water channels in water transport across the lung endothelium. PMID:9545071

  17. A humanized microbiota mouse model of ovalbumin-induced lung inflammation.

    PubMed

    Arrieta, Marie-Claire; Sadarangani, Manish; Brown, Eric M; Russell, Shannon L; Nimmo, Michael; Dean, John; Turvey, Stuart E; Chan, Edmond S; Finlay, B Brett

    2016-07-03

    There is increasing evidence for a role of early life gut microbiota in later development of asthma in children. In our recent study, children with reduced abundance of the bacterial genera Lachnospira, Veillonella, Faecalibacterium, and Rothia had an increased risk of development of asthma and addition of these bacteria in a humanized mouse model reduced airway inflammation. In this Addendum, we provide additional data on the use of a humanized gut microbiota mouse model to study the development of asthma in children, highlighting the differences in immune development between germ-free mice colonized with human microbes compared to those colonized with mouse gut microbiota. We also demonstrate that there is no association between the composition of the gut microbiota in older children and the diagnosis of asthma, further suggesting the importance of the gut microbiota-immune system axis in the first 3 months of life.

  18. Lung mechanics in the TIMP3 null mouse and its response to mechanical ventilation.

    PubMed

    Martin, Erica L; Truscott, Emily A; Bailey, Timothy C; Leco, Kevin J; McCaig, Lynda A; Lewis, James F; Veldhuizen, Ruud A W

    2007-03-01

    Tissue inhibitor of metalloproteinase-3 (TIMP3) null mice develop emphysema-like airspace enlargement due to an enzymatic imbalance. This study investigates how these abnormalities alter lung mechanics and the response to 2 different mechanical ventilation strategies. Phenotypically, TIMP3 null mice had increased compliance, and decreased resistance, tissue damping, and tissue elastance over wild-type controls. Decreased compliance and increased resistance were observed following the injurious ventilation strategy; however, the TIMP3 null response to both ventilation strategies was similar to wild-type mice. In conclusion, TIMP3 null mice have significant alterations in lung mechanics; however, this does not affect their response to ventilation.

  19. Effect of long-term simulated weightlessness on surfactant and water balance in mouse lungs.

    PubMed

    Bryndina, I G; Vasilieva, N N; Krivonogova, Yu A; Baranov, V M

    2013-07-01

    Weightlessness produces adaptive and maladaptive changes in the respiratory system. We assessed the effects of 30-day antiorthostatic hanging as a model of microgravity on the water balance in the lungs and surface activity and phospholipid composition of pulmonary surfactant in C57Bl/6 mice. Long-term antiorthostatic hanging increased water content in the lungs and reduced surface-active properties of the surfactant. This was accompanied by an increase in the content of alveolar phospholipids and changes in their fractional composition (increase in the relative content of lysophosphatidylcholine and phosphatidylethanolamine).

  20. Differences in gene expression profiles from asbestos-treated SPARC-null and wild type mouse lungs

    PubMed Central

    Pershouse, Mark A.; Smartt, Aubrey M.; Schwanke, Corbin; Putnam, Elizabeth A.

    2009-01-01

    The role of SPARC in the in vivo lung response to crocidolite asbestos was addressed by instillation of crocidolite asbestos in a series of wild type or SPARC -null mice. Animals were sacrificed at one week, one month, and three months post-instillation to assess the impact of SPARC on multiple stages in the development of fibrosis. RNA was harvested from 10 animals/time point, pooled, and used to probe a mouse array containing ∼10,000 probes. Gene expression data was analyzed for fold-change, and for broader functional group alterations. As expected, the one-week time point displayed alterations in genes involved in immune recognition, energy utilization, and growth factor production. Later time points showed expression alterations for genes involved in protein degradation, Wnt receptor signaling, membrane protein activity, and transport. Molecules in the Wnt pathway have been implicated in bone growth, mediation of fibroblast activity, and have been directly linked to SPARC regulation. PMID:19446018

  1. Delivery of siRNA to the Mouse Lung via a Functionalized Lipopolyamine

    PubMed Central

    Polach, Kevin J; Matar, Majed; Rice, Jennifer; Slobodkin, Gregory; Sparks, Jeff; Congo, Richard; Rea-Ramsey, Angela; McClure, Diane; Brunhoeber, Elaine; Krampert, Monika; Schuster, Andrea; Jahn-Hofmann, Kerstin; John, Matthias; Vornlocher, Hans-Peter; Fewell, Jason G; Anwer, Khursheed; Geick, Anke

    2012-01-01

    We have designed a series of versatile lipopolyamines which are amenable to chemical modification for in vivo delivery of small interfering RNA (siRNA). This report focuses on one such lipopolyamine (Staramine), its functionalized derivatives and the lipid nanocomplexes it forms with siRNA. Intravenous (i.v.) administration of Staramine/siRNA nanocomplexes modified with methoxypolyethylene glycol (mPEG) provides safe and effective delivery of siRNA and significant target gene knockdown in the lungs of normal mice, with much lower knockdown in liver, spleen, and kidney. Although siRNA delivered via Staramine is initially distributed across all these organs, the observed clearance rate from the lung tissue is considerably slower than in other tissues resulting in prolonged siRNA accumulation on the timescale of RNA interference (RNAi)-mediated transcript depletion. Complete blood count (CBC) analysis, serum chemistry analysis, and histopathology results are all consistent with minimal toxicity. An in vivo screen of mPEG modified Staramine nanocomplexes-containing siRNAs targeting lung cell-specific marker proteins reveal exclusive transfection of endothelial cells. Safe and effective delivery of siRNA to the lung with chemically versatile lipopolyamine systems provides opportunities for investigation of pulmonary cell function in vivo as well as potential treatments of pulmonary disease with RNAi-based therapeutics. PMID:21988874

  2. Macrophage depletion of mouse lung following inhalation of 239PuO2.

    PubMed

    Moores, S R; Talbot, R J; Evans, N; Lambert, B E

    1986-03-01

    Changes in the free-cell population of the lungs of two strains of mice (SAS/4 and CBA/H) were studied up to 4 months after inhalation exposure to a sized fraction of 239PuO2 particles (1.5 micron AMAD) to give initial alveolar depositions (IADs) ranging from 17 to 810 Bq. A sample of the free-cell population of the lung was recovered by bronchoalveolar lavage, and a radiometric method was used to estimate the total number of pulmonary alveolar macrophages (PAM) in the lung. The response of the lung to 239PuO2 was characterized by an initial, dose-dependent depression in the total number of PAM following an IAD as low as 50 Bq. At IADs greater than 150 Bq, the initial depression continued for longer, merging into a chronic phase in which the PAM were larger and were accompanied by a minor infiltration of leukocytes. These findings were confirmed by histology, which also revealed focal accumulations of Type II pneumocytes. The results indicate that inhaled alpha-emitting particles are effective at producing a depletion in the alveolar macrophage population at relatively low IADs and that chronic effects on the cells can be produced by higher concentrations.

  3. Macrophage depletion of mouse lung following inhalation of /sup 238/PuO/sub 2/

    SciTech Connect

    Moores, S.R.; Talbot, R.J.; Evans, N.; Lambert, B.E.

    1986-03-01

    Changes in the free-cell population of the lungs of two strains of mice (SAS/4 and CBA/H) were studied up to 4 months after inhalation exposure to a sized fraction of /sup 239/PuO/sub 2/ particles (1.5 micron AMAD) to give initial alveolar depositions (IADs) ranging from 17 to 810 Bq. A sample of the free-cell population of the lung was recovered by bronchoalveolar lavage, and a radiometric method was used to estimate the total number of pulmonary alveolar macrophages (PAM) in the lung. The response of the lung to /sup 239/PuO/sub 2/ was characterized by an initial, dose-dependent depression in the total number of PAM following an IAD as low as 50 Bq. At IADs greater than 150 Bq, the initial depression continued for longer, merging into a chronic phase in which the PAM were larger and were accompanied by a minor infiltration of leukocytes. These findings were confirmed by histology, which also revealed focal accumulations of Type II pneumocytes. The results indicate that inhaled alpha-emitting particles are effective at producing a depletion in the alveolar macrophage population at relatively low IADs and that chronic effects on the cells can be produced by higher concentrations.

  4. Efficacy and mechanism of action of yin lai tang (lung-stomach treatment) in dyspepsia mouse infected by FM1 virus.

    PubMed

    Liu, Tiegang; Yu, He; Zhang, Wang; Zhen, Jianhua; Li, Xiaofei; Lv, Guokai; Gu, Hong-Xiao; Murtaza, Ghulam

    2013-01-01

    The aim of this study was to assess the efficacy and elaborate the mechanism of action of Yin Lai Tang (Lung-Stomach Treatment) on dyspepsia mouse infected by FM1 virus. Ninety male, 4 week old Kunming mouse with 12-14 g weight, were randomly divided into 9 groups, i.e., normal, infected, dyspepsia, ribavirin, Shuanghuanglian, Children's indigestion tablet, YinLaiTang high dose, YinLaiTang middle dose and YinLaiTang low dose, and these groups had been treated by according drugs to get objectives. Compared with normal group, lung index significantly (p < 0.01) increased in all groups except ribavirin group where lung index obviously (p < 0.05) increased. There was non-significant (p > 0.05) difference in the values of lung homogenate virus titer between dyspepsia group and other groups. Compared to normal group, there was variable degree of inflammatory cell infiltrations in respiratory tract structures in the animals of other groups, and there was a significant (p < 0.01) increase in the level of serum IL-6, IL-10, and TNF-alpha in infected and dyspepsia group and significant (p < 0.01) decrease in the level of serum IFN-gamma was observed. Compared with single clearing stomach method and single clearing lung approach, lung-stomach treatment reduced the level of IL-6 with non-significant difference (p > 0.05) and increased the level of IL-10 obviously, and compared with the single clearing lung method, there was a significant difference (p < 0.05). Compared with the single clearing stomach method and the single clearing lung method, the lung-stomach treatment method had a better efficacy and showed effects on the expression of pro-inflammatory factor and anti-inflammatory factor.

  5. Deletion and differential expression of p16{sup INK4a} in mouse lung tumors

    SciTech Connect

    Belinsky, S.A.; Swafford, D.S.; Middleton, S.K.; Kennedy, C.H.; Tesfaigzi, J.

    1997-12-31

    Recent allelotyping of chemical-induced lung tumors in hybrid mice has detected loss of heterozygosity on chromosome 4 in a region involving the interferon-{alpha} (IFN-{alpha}) gene cluster that is syntenic to human chromosome 9p21-22, the location of the p16{sup INK4a}(p16) and (p15) tumor suppressor genes. The purpose of the current investigation was to characterize the expression of p16 and p15 in lung tumors and tumor-derived cell lines induced in Ad mice by exposure to the tobacco-specific nitrosamine, 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK). Expression of p16 and p15 was detected in all primary lung tumors; however, levels of expression of p16 differed by up to 15-fold between tumors. This is the first study to note a marked difference in the expression of the p16 gene in primary lung tumors. The apparent low levels of expression seen in approximately half of the tumors was not attributed to deletion, mutation or methylation of the p16 gene. Conversely, the high levels of p16 expression were not the result of effects on the retinoblastoma gene (Rb) or cyclin D1 proteins but most likely in response to a dysfunction elsewhere within this pathway. In contrast to the detection of p16 expression in primary tumors, this gene was deleted in all four cell lines. Three of four cell lines also showed loss of the p15 gene. Mapping of these homozygous deletions on chromosome 4 revealed that the p16 gene resides near the D4MIT77 marker, which is located approximately 12 cM proximal to the IFN-{alpha} gene cluster, thereby implicating the p16 gene as one of the targets within the allelic deletions detected previously in primary lung tumors from hybrid mice.

  6. Characterization of a nose-only inhaled phosgene acute lung injury mouse model

    PubMed Central

    Plahovinsak, Jennifer L.; Perry, Mark R.; Knostman, Katherine A.; Segal, Robert; Babin, Michael C.

    2016-01-01

    Context Phosgene’s primary mode of action is as a pulmonary irritant characterized by its early latent phase where life-threatening, non-cardiogenic pulmonary edema is typically observed 6–24 h post-exposure. Objective To develop an inhaled phosgene acute lung injury (ALI) model in C57BL/6 mice that can be used to screen potential medical countermeasures. Methods A Cannon style nose-only inhalation exposure tower was used to expose mice to phosgene (8 ppm) or air (sham). An inhalation lethality study was conducted to determine the 8 ppm median lethal exposure (LCt50) at 24 and 48 h post-exposure. The model was then developed at 1.2 times the 24 h LCt50. At predetermined serial sacrifice time points, survivors were euthanized, body and lung weights collected, and lung tissues processed for histopathology. Additionally, post-exposure clinical observations were used to assess quality of life. Results and discussion The 24-hour LCt50 was 226ppm*min (8 ppm for 28.2 min) and the 48-hour LCt50 was 215ppm*min (8 ppm for 26.9 min). The phosgene exposed animals had a distinct progression of clinical signs, histopathological changes and increased lung/body weight ratios. Early indicators of a 1.2 times the 24-hour LCt50 phosgene exposure were significant changes in the lung-to-body weight ratios by 4 h post-exposure. The progression of clinical signs and histopathological changes were important endpoints for characterizing phosgene-induced ALI for future countermeasure studies. Conclusion An 8 ppm phosgene exposure for 34 min (1.2 × LCt50) is the minimum challenge recommended for evaluating therapeutic interventions. The predicted higher mortality in the phosgene-only controls will help demonstrate efficacy of candidate treatments and increase the probability that a change in survival rate is statistically significant PMID:26671199

  7. [Pulmonary apoptosis and necrosis in hyperoxia-induced acute mouse lung injury].

    PubMed

    Zhang, Xiang-feng; Foda, Hussein D

    2004-07-01

    To investigate the pathways to cell death in hyperoxia-induced lung injury and the functional significance of apoptosis in vivo in response to hyperoxia. Seventy-two mice were exposed in sealed cages > 98% oxygen (for 24 - 72 h) or room air, and the severity of lung injury and epithelium sloughing was evaluated. The extent and location of apoptosis in injured lung tissues were studied by terminal transferase dUTP end labeling assay (TUNEL), reverse transcript-polymerase chain reaction (RT-PCR) and immunohistochemistry. Hyperoxia caused acute lung injury; the hyperoxic stress resulted in marked epithelium sloughing. TUNEL assay exhibited increased apoptosis index both in alveolar epithelial cells and bronchial epithelial cells in sections from mice after 48 h hyperoxia compared with their control group (0.51 +/- 0.10, 0.46 +/- 0.08 verse 0.04 +/- 0.02, 0.02 +/- 0.01). This was accompanied by increased expression of caspase-3 mRNA in lung tissues after 48 h hyperoxia compared with their control group (0.53 +/- 0.09 verse 0.34 +/- 0.07), the expression was higher at 72 h of hyperoxia (0.60 +/- 0.08). Immunohistochemistry study showed caspase-3 protein was located in cytoplasm and nuclei of airway epithelial cells, alveolar epithelial cells and macrophage in hyperoxia mice. The expression of caspase-3 protein in airway epithelium significantly increased at 24 h of hyperoxia compared with their control group (41.62 +/- 3.46 verse 15.86 +/- 1.84), the expression level was highest at 72 h of hyperoxia (55.24 +/- 6.80). Both apoptosis and necrosis contribute to cell death during hyperoxia. Apoptosis plays an important role in alveolar damage and cell death from hyperoxia.

  8. Increased cytosine DNA-methyltransferase activity in A/J mouse lung cells following carcinogen exposure and during tumor progression

    SciTech Connect

    Belinsky, S.A.; Issa, J.-P.J.; Baylin, S.B.

    1994-11-01

    Considerable evidence has accumulated that 5-methylcytosine modification of mammalian DNA, both in exons and CpG rich islands located in promoter regions, is important in gene regulation. For example, a decrease of 5-methylcytosine in 5{prime} flanking regions or exons of genes has been associated with increased gene transcription. In addition, hypermethylation at specific regions of chromosomes 17p and 3p have also been observed in lung and colon cancer. During colon cancer development, these hypermethylation changes precede allelic loss. In addition, the activity of the enzyme which maintains the methylation status at CpG dinucleotides, DNA methyltransferase (MT), has been shown to increase during colon cancer progression. These observations suggest changes in methylation patterns within specific genes could result in either inappropriate gene expression or gene deletion, both of which would contribute to the establishment of the malignant phenotype. The purpose of this investigation was to determine if DNA MT activity is elevated in target (alveolar type II), but not in nontarget (Clara, endothelial, macrophage) lung cells isolated from the A/J mouse following exposure to nitrosamine 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK). In addition, the activity of this enzyme during tumor progression was examined.

  9. Tissue Optical Clearing, Three-Dimensional Imaging, and Computer Morphometry in Whole Mouse Lungs and Human Airways

    PubMed Central

    Scott, Gregory D.; Blum, Emily D.; Fryer, Allison D.

    2014-01-01

    In whole adult mouse lung, full identification of airway nerves (or other cellular/subcellular objects) has not been possible due to patchy distribution and micron-scale size. Here we describe a method using tissue clearing to acquire the first complete image of three-dimensional (3D) innervation in the lung. We then created a method to pair analysis of nerve (or any other colabeled epitope) images with identification of 3D tissue compartments and airway morphometry by using fluorescent casting and morphometry software (which we designed and are making available as open-source). We then tested our method to quantify a sparse heterogeneous nerve population by examining visceral pleural nerves. Finally, we demonstrate the utility of our method in human tissue to image full thickness innervation in irregular 3D tissue compartments and to quantify sparse objects (intrinsic airway ganglia). Overall, this method can uniquely pair the advantages of whole tissue imaging and cellular/subcellular fluorescence microscopy. PMID:24471696

  10. Combination Effect of Regulatory T-Cell Depletion and Ionizing Radiation in Mouse Models of Lung and Colon Cancer

    SciTech Connect

    Son, Cheol-Hun; Bae, Jae-Ho; Shin, Dong-Yeok; Lee, Hong-Rae; Jo, Wol-Soon; Yang, Kwangmo; Park, You-Soo

    2015-06-01

    Purpose: To investigate the potential of low-dose cyclophosphamide (LD-CTX) and anti-CD25 antibody to prevent activation of regulatory T cells (Tregs) during radiation therapy. Methods and Materials: We used LD-CTX and anti-CD25 monoclonal antibody as a means to inhibit Tregs and improve the therapeutic effect of radiation in a mouse model of lung and colon cancer. Mice were irradiated on the tumor mass of the right leg and treated with LD-CTX and anti-CD25 antibody once per week for 3 weeks. Results: Combined treatment of LD-CTX or anti-CD25 antibody with radiation significantly decreased Tregs in the spleen and tumor compared with control and irradiation only in both lung and colon cancer. Combinatorial treatments resulted in a significant increase in the effector T cells, longer survival rate, and suppressed irradiated and distal nonirradiated tumor growth. Specifically, the combinatorial treatment of LD-CTX with radiation resulted in outstanding regression of local and distant tumors in colon cancer, and almost all mice in this group survived until the end of the study. Conclusions: Our results suggest that Treg depletion strategies may enhance radiation-mediated antitumor immunity and further improve outcomes after radiation therapy.

  11. Quantifying morphological parameters of the terminal branching units in a mouse lung by phase contrast synchrotron radiation computed tomography.

    PubMed

    Hwang, Jeongeun; Kim, Miju; Kim, Seunghwan; Lee, Jinwon

    2013-01-01

    An effective technique of phase contrast synchrotron radiation computed tomography was established for the quantitative analysis of the microstructures in the respiratory zone of a mouse lung. Heitzman's method was adopted for the whole-lung sample preparation, and Canny's edge detector was used for locating the air-tissue boundaries. This technique revealed detailed morphology of the respiratory zone components, including terminal bronchioles and alveolar sacs, with sufficiently high resolution of 1.74 µm isotropic voxel size. The technique enabled visual inspection of the respiratory zone components and comprehension of their relative positions in three dimensions. To check the method's feasibility for quantitative imaging, morphological parameters such as diameter, surface area and volume were measured and analyzed for sixteen randomly selected terminal branching units, each consisting of a terminal bronchiole and a pair of succeeding alveolar sacs. The four types of asymmetry ratios concerning alveolar sac mouth diameter, alveolar sac surface area, and alveolar sac volume are measured. This is the first ever finding of the asymmetry ratio for the terminal bronchioles and alveolar sacs, and it is noteworthy that an appreciable degree of branching asymmetry was observed among the alveolar sacs at the terminal end of the airway tree, despite the number of samples was small yet. The series of efficient techniques developed and confirmed in this study, from sample preparation to quantification, is expected to contribute to a wider and exacter application of phase contrast synchrotron radiation computed tomography to a variety of studies.

  12. Mechanics, nonlinearity, and failure strength of lung tissue in a mouse model of emphysema: possible role of collagen remodeling.

    PubMed

    Ito, Satoru; Ingenito, Edward P; Brewer, Kelly K; Black, Lauren D; Parameswaran, Harikrishnan; Lutchen, Kenneth R; Suki, Béla

    2005-02-01

    Enlargement of the respiratory air spaces is associated with the breakdown and reorganization of the connective tissue fiber network during the development of pulmonary emphysema. In this study, a mouse (C57BL/6) model of emphysema was developed by direct instillation of 1.2 IU of porcine pancreatic elastase (PPE) and compared with control mice treated with saline. The PPE treatment caused 95% alveolar enlargement (P = 0.001) associated with a 29% lower elastance along the quasi-static pressure-volume curves (P < 0.001). Respiratory mechanics were measured at several positive end-expiratory pressures in the closed-chest condition. The dynamic tissue elastance was 19% lower (P < 0.001), hysteresivity was 9% higher (P < 0.05), and harmonic distortion, a measure of collagen-related dynamic nonlinearity, was 33% higher in the PPE-treated group (P < 0.001). Whole lung hydroxyproline content, which represents the total collagen content, was 48% higher (P < 0.01), and alpha-elastin content was 13% lower (P = 0.16) in the PPE-treated group. There was no significant difference in airway resistance (P = 0.7). The failure stress at which isolated parenchymal tissues break during stretching was 40% lower in the PPE-treated mice (P = 0.002). These findings suggest that, after elastolytic injury, abnormal collagen remodeling may play a significant role in all aspects of lung functional changes and mechanical forces, leading to progressive emphysema.

  13. Exposure to arsenic at levels found inU.S. drinking water modifies expression in the mouse lung.

    PubMed

    Andrew, Angeline S; Bernardo, Viviane; Warnke, Linda A; Davey, Jennifer C; Hampton, Thomas; Mason, Rebecca A; Thorpe, Jessica E; Ihnat, Michael A; Hamilton, Joshua W

    2007-11-01

    The mechanisms of action of drinking water arsenic in the lung and the threshold for biologic effects remain controversial. Our study utilizes Affymetrix 22,690 transcript oligonucleotide microarrays to assess the long-term effects of increasing doses of drinking water arsenic on expression levels in the mouse lung. Mice were exposed at levels commonly found in contaminated drinking water wells in the United States (0, 0.1, 1 ppb), as well as the 50 ppb former maximum contaminant level, for 5 weeks. The expression profiles revealed modification of a number of important signaling pathways, many with corroborating evidence of arsenic responsiveness. We observed statistically significant expression changes for transcripts involved in angiogenesis, lipid metabolism, oxygen transport, apoptosis, cell cycle, and immune response. Validation by reverse transcription-PCR and immunoblot assays confirmed expression changes for a subset of transcripts. These data identify arsenic-modified signaling pathways that will help guide investigations into mechanisms of arsenic's health effects and clarify the threshold for biologic effects and potential disease risk.

  14. Genetic requirement for Mycl and efficacy of RNA Pol I inhibition in mouse models of small cell lung cancer

    PubMed Central

    Kim, Dong-Wook; Wu, Nan; Kim, Young-Chul; Cheng, Pei Feng; Basom, Ryan; Kim, Dongkyoon; Dunn, Colin T.; Lee, Anastasia Y.; Kim, Keebeom; Lee, Chang Sup; Singh, Andrew; Gazdar, Adi F.; Harris, Chris R.; Eisenman, Robert N.; Park, Kwon-Sik; MacPherson, David

    2016-01-01

    Small cell lung cancer (SCLC) is a devastating neuroendocrine carcinoma. MYCL (L-Myc) is frequently amplified in human SCLC, but its roles in SCLC progression are poorly understood. We isolated preneoplastic neuroendocrine cells from a mouse model of SCLC and found that ectopic expression of L-Myc, c-Myc, or N-Myc conferred tumor-forming capacity. We focused on L-Myc, which promoted pre-rRNA synthesis and transcriptional programs associated with ribosomal biogenesis. Deletion of Mycl in two genetically engineered models of SCLC resulted in strong suppression of SCLC. The high degree of suppression suggested that L-Myc may constitute a therapeutic target for a broad subset of SCLC. We then used an RNA polymerase I inhibitor to target rRNA synthesis in an autochthonous Rb/p53-deleted mouse SCLC model and found significant tumor inhibition. These data reveal that activation of RNA polymerase I by L-Myc and other MYC family proteins provides an axis of vulnerability for this recalcitrant cancer. PMID:27298335

  15. Vitamin D Repletion Reduces the Progression of Premalignant Squamous Lesions in the NTCU Lung Squamous Cell Carcinoma Mouse Model

    PubMed Central

    Mazzilli, Sarah A.; Hershberger, Pamela A.; Reid, Mary E.; Bogner, Paul N.; Atwood, Kristopher; Trump, Donald L.; Johnson, Candace S.

    2015-01-01

    The chemopreventive actions of vitamin D were examined in the N-nitroso-tris-chloroethylurea (NTCU) mouse model, a progressive model of lung squamous cell carcinoma (SCC). SWR/J mice were fed a deficient diet (D) containing no vitamin D3, a sufficient diet (S) containing 2000 IU/kg vitamin D3, or the same diets in combination with the active metabolite of vitamin D, calcitriol (C) (80 μg/kg, weekly). The percentage (%) of the mucosal surface of large airways occupied by dysplastic lesions was determined in mice after treatment with a total dose of 15 or 25 μmol NTCU (N). After treatment with 15 μmol NTCU, the % of the surface of large airways containing high-grade dysplastic (HGD) lesions were vitamin D-deficient +NTCU (DN), 22.7 % (p<0.05 compared to vitamin D-sufficient +NTCU (SN)); DN + C, 12.3%; SN, 8.7%; and SN + C, 6.6%. The extent of HGD increased with NTCU dose in the DN group. Proliferation, assessed by Ki-67 labeling, increased upon NTCU treatment. The highest Ki-67 labeling index was seen in the DN group. As compared to SN mice, DN mice exhibited a 3-fold increase (p <0.005) in circulating white blood cells (WBC), a 20% (p <0.05) increase in IL-6 levels, and a 4 -fold (p <0.005) increase in WBC in bronchial lavages. Thus, vitamin D repletion reduces the progression of premalignant lesions, proliferation, and inflammation, and may thereby suppress development of lung SCC. Further investigations of the chemopreventive effects of vitamin D in lung SCC are warranted. PMID:26276745

  16. Tracheal Dysplasia Precedes Bronchial Dysplasia in Mouse Model of N-Nitroso Trischloroethylurea Induced Squamous Cell Lung Cancer

    PubMed Central

    Ghosh, Moumita; Dwyer-Nield, Lori D.; Kwon, Jennifer B.; Barthel, Lea; Janssen, William J.; Merrick, Daniel T.; Keith, Robert L.

    2015-01-01

    Squamous cell lung cancer (SCC) is the second leading cause of lung cancer death in the US and has a 5-year survival rate of only 16%. Histological changes in the bronchial epithelium termed dysplasia are precursors to invasive SCC. However, the cellular mechanisms that cause dysplasia are unknown. To fill this knowledge gap, we used topical application of N-nitroso-tris chloroethylurea (NTCU) for 32 weeks to induce squamous dysplasia and SCC in mice. At 32 weeks the predominant cell type in the dysplastic airways was Keratin (K) 5 and K14 expressing basal cells. Notably, basal cells are extremely rare in the normal mouse bronchial epithelium but are abundant in the trachea. We therefore evaluated time-dependent changes in tracheal and bronchial histopathology after NTCU exposure (4, 8, 12, 16, 25 and 32 weeks). We show that tracheal dysplasia occurs significantly earlier than that of the bronchial epithelium (12 weeks vs. 25 weeks). This was associated with increased numbers of K5+/K14+ tracheal basal cells and a complete loss of secretory (Club cell secretory protein expressing CCSP+) and ciliated cells. TUNEL staining of NTCU treated tissues confirmed that the loss of CCSP+ and ciliated cells was not due to apoptosis. However, mitotic index (measured by bromodeoxyuridine incorporation) showed that NTCU treatment increased proliferation of K5+ basal cells in the trachea, and altered bronchial mitotic population from CCSP+ to K5+ basal cells. Thus, we demonstrate that NTCU-induced lung epithelial dysplasia starts in the tracheal epithelium, and is followed by basal cell metaplasia of the bronchial epithelium. This analysis extends our knowledge of the NTCU-SCC model by defining the early changes in epithelial cell phenotypes in distinct airway locations, and this may assist in identifying new targets for future chemoprevention studies. PMID:25860262

  17. Mouse ChemR23 is expressed in dendritic cell subsets and macrophages, and mediates an anti-inflammatory activity of chemerin in a lung disease model.

    PubMed

    Luangsay, Souphalone; Wittamer, Valérie; Bondue, Benjamin; De Henau, Olivier; Rouger, Laurie; Brait, Maryse; Franssen, Jean-Denis; de Nadai, Patricia; Huaux, François; Parmentier, Marc

    2009-11-15

    Chemerin is the ligand of the ChemR23 receptor and a chemoattractant factor for human immature dendritic cells (DCs), macrophages, and NK cells. In this study, we characterized the mouse chemerin/ChemR23 system in terms of pharmacology, structure-function, distribution, and in vivo biological properties. Mouse chemerin is synthesized as an inactive precursor (prochemerin) requiring, as in human, the precise processing of its C terminus for generating an agonist of ChemR23. Mouse ChemR23 is highly expressed in immature plasmacytoid DCs and at lower levels in myeloid DCs, macrophages, and NK cells. Mouse prochemerin is expressed in most epithelial cells acting as barriers for pathogens but not in leukocytes. Chemerin promotes calcium mobilization and chemotaxis on DCs and macrophages and these functional responses were abrogated in ChemR23 knockout mice. In a mouse model of acute lung inflammation induced by LPS, chemerin displayed potent anti-inflammatory properties, reducing neutrophil infiltration and inflammatory cytokine release in a ChemR23-dependent manner. ChemR23 knockout mice were unresponsive to chemerin and displayed an increased neutrophil infiltrate following LPS challenge. Altogether, the mouse chemerin/ChemR23 system is structurally and functionally conserved between human and mouse, and mouse can therefore be considered as a good model for studying the anti-inflammatory role of this system in the regulation of immune responses and inflammatory diseases.

  18. Iron homeostasis and its disruption in mouse lung in iron deficiency and overload.

    PubMed

    Giorgi, Gisela; D'Anna, María Cecilia; Roque, Marta Elena

    2015-10-01

    What is the central question of this study? The aim was to explore the role and hitherto unclear mechanisms of action of iron proteins in protecting the lung against the harmful effects of iron accumulation and the ability of pulmonary cells to mobilize iron in iron deficiency. What is the main finding and its importance? We show that pulmonary hepcidin appears not to modify cellular iron mobilization in the lung. We propose pathways for supplying iron to the lung in iron deficiency and for protecting the lung against iron excess in iron overload, mediated by the co-ordinated action of iron proteins, such as divalent metal transporter 1, ZRT-IRE-like-protein 14, transferrin receptor, ferritin, haemochromatosis-associated protein and ferroportin. Iron dyshomeostasis is associated with several forms of chronic lung disease, but its mechanisms of action remain to be elucidated. The aim of the present study was to determine the role of the lung in whole-animal models with iron deficiency and iron overload, studying the divalent metal transporter 1 (DMT1), ZRT-IRE-like protein 14 (ZIP14), transferrin receptor (TfR), haemochromatosis-associated protein (HFE), hepcidin, ferritin and ferroportin (FPN) expression. In each model, adult CF1 mice were divided into the following groups (six mice per group): (i) iron-overload model, iron saccharate i.p. and control group (iron adequate), 0.9% NaCl i.p.; and (ii) iron-deficiency model, induced by repeated bleeding, and control group (sham operated). Proteins were assessed by immunohistochemistry and Western blot. In control mice, DMT1 was localized in the cytoplasm of airway cells, and in iron deficiency and overload it was in the apical membrane. Divalent metal transporter 1 and TfR increased in iron deficiency, without changes in iron overload. ZRT-IRE-like protein 14 decreased in airway cells in iron deficiency and increased in iron overload. In iron deficiency, HFE and FPN were immunolocalized close to the apical membrane

  19. Gene Expression and Pathway Analysis of Effects of the CMAH Deactivation on Mouse Lung, Kidney and Heart

    PubMed Central

    Kwon, Deug-Nam; Chang, Byung-Soo; Kim, Jin-Hoi

    2014-01-01

    Background N-glycolylneuraminic acid (Neu5Gc) is generated by hydroxylation of CMP-Neu5Ac to CMP-Neu5Gc, catalyzed by CMP-Neu5Ac hydroxylase (CMAH). However, humans lack this common mammalian cell surface molecule, Neu5Gc, due to inactivation of the CMAH gene during evolution. CMAH is one of several human-specific genes whose function has been lost by disruption or deletion of the coding frame. It has been suggested that CMAH inactivation has resulted in biochemical or physiological characteristics that have resulted in human-specific diseases. Methodology/Principal Findings To identify differential gene expression profiles associated with the loss of Neu5Gc expression, we performed microarray analysis using Illumina MouseRef-8 v2 Expression BeadChip, using the main tissues (lung, kidney, and heart) from control mice and CMP-Neu5Ac hydroxylase (Cmah) gene knock-out mice, respectively. Out of a total of 25,697 genes, 204, 162, and 147 genes were found to be significantly modulated in the lung, kidney, and heart tissues of the Cmah null mouse, respectively. In this study, we examined the gene expression profiles, using three commercial pathway analysis software packages: Ingenuity Pathways Analysis, Kyoto Encyclopedia of Genes and Genomes analysis, and Pathway Studio. The gene ontology analysis revealed that the top 6 biological processes of these genes included protein metabolism and modification, signal transduction, lipid, fatty acid, and steroid metabolism, nucleoside, nucleotide and nucleic acid metabolism, immunity and defense, and carbohydrate metabolism. Gene interaction network analysis showed a common network that was common to the different tissues of the Cmah null mouse. However, the expression of most sialytransferase mRNAs of Hanganutziu-Deicher antigen, sialy-Tn antigen, Forssman antigen, and Tn antigen was significantly down-regulated in the liver tissue of Cmah null mice. Conclusions/Significance Mice bearing a human-like deletion of the Cmah gene

  20. Hormonal and extracellular matrix components act as mediators for mouse fetal lung development

    SciTech Connect

    Smith, C.I.

    1988-01-01

    The concentration of disaturated phosphatidylcholine (DPPC) in 16 day lung tissue was measured after 5 days in culture. When grown in the absence of serum and hormones, levels of DPPC, assayed by phosphorus content, increased over 17 day in vivo controls. Treated with thyroxine and dexamethasone, DPPC levels were comparable to 2 day postnatal controls. Levels of DPPC increased in cultures containing dexamethasone alone while thyroxine alone had significantly less effect. 16- and 19-day fetal lung tissues were labeled with {sup 35}S-sulfate and {sup 3}H-glucosamine. Each pool was analyzed by DEAE-sepharose chromatography and by digestion with nitrous acid and chondroitinase. GAG synthesis was inhibited using {beta}-xyloside. The {beta}-xyloside inhibition of GAG synthesis was examined morphologically by transmission and scanning electron microscopy and functionally by autoradiography, sequential extraction, chromatography, and digestion as above.

  1. Ureaplasma in lung. 1. Localization by in situ hybridization in a mouse model.

    PubMed

    Benstein, Barbara D; Crouse, Dennis T; Shanklin, D Radford; Ourth, Donald D

    2003-10-01

    Ureaplasma urealyticum is a common inhabitant of mucosal surfaces but is also associated with a higher incidence of pneumonia and bronchopulmonary dysplasia in preterm infants. Culture and polymerase chain reaction demonstrate high isolation rates of ureaplasma in clinical specimens documenting their presence but do not associate the organism directly with the diseased tissue. In this study, lung tissue samples from newborn mice inoculated intranasally with U. urealyticum were used to develop an in situ hybridization (ISH) test for the organism. In situ hybridization allows the localization of gene expression for visualization within the context of tissue morphology. New techniques which use biotinyl-tyramide based signal amplification have been able to greatly enhance the sensitivity of ISH. Using the Dako GenPoint Catalyzed Signal Amplification system to detect a biotinylated DNA probe specific for an internal nucleotide sequence within the urease gene of U. urealyticum, the organism was detected within the infected murine lung tissues. Electron microscopy was used to verify the presence of the organisms in the positive ISH areas. The ISH procedure developed in this study can be used to analyze the presence of ureaplasma in human neonatal lung tissue with the corresponding histopathology.

  2. Genetic variation in HTR4 and lung function: GWAS follow-up in mouse.

    PubMed

    House, John S; Li, Huiling; DeGraff, Laura M; Flake, Gordon; Zeldin, Darryl C; London, Stephanie J

    2015-01-01

    Human genome-wide association studies (GWASs) have identified numerous associations between single nucleotide polymorphisms (SNPs) and pulmonary function. Proving that there is a causal relationship between GWAS SNPs, many of which are noncoding and without known functional impact, and these traits has been elusive. Furthermore, noncoding GWAS-identified SNPs may exert trans-regulatory effects rather than impact the proximal gene. Noncoding variants in 5-hydroxytryptamine (serotonin) receptor 4 (HTR4) are associated with pulmonary function in human GWASs. To gain insight into whether this association is causal, we tested whether Htr4-null mice have altered pulmonary function. We found that HTR4-deficient mice have 12% higher baseline lung resistance and also increased methacholine-induced airway hyperresponsiveness (AHR) as measured by lung resistance (27%), tissue resistance (48%), and tissue elastance (30%). Furthermore, Htr4-null mice were more sensitive to serotonin-induced AHR. In models of exposure to bacterial lipopolysaccharide, bleomycin, and allergic airway inflammation induced by house dust mites, pulmonary function and cytokine profiles in Htr4-null mice differed little from their wild-type controls. The findings of altered baseline lung function and increased AHR in Htr4-null mice support a causal relationship between genetic variation in HTR4 and pulmonary function identified in human GWAS.

  3. Activation of latent metastases in the lung after resection of a metastatic lymph node in a lymph node metastasis mouse model.

    PubMed

    Shao, Lenan; Ouchi, Tomoki; Sakamoto, Maya; Mori, Shiro; Kodama, Tetsuya

    2015-05-08

    Iatrogenic induction of regional and distant cancer metastases is a risk associated with clinical resection of tumor-positive sentinel lymph nodes. However, there have been no studies of this risk in a mouse model of cancer metastasis. Here, we report that resection of a tumor-bearing subiliac lymph node (SiLN) enhanced lung metastasis in a mouse model of lymph node metastasis. Bioluminescence imaging revealed that metastatic tumor cells in the secondary lymph node continued to grow after resection of the SiLN, and that the probability of metastasis to the lungs was increased when the interval between SiLN inoculation and resection was reduced. Futhermore, histological analysis demonstrated that latents in the lung were stimulated to grow after resection of the SiLN. Fluorescence imaging indicated that the route of tumor cell dissemination from SiLN to the lung was the venous system located over the SiLN. We speculate that our mouse model will be useful for studying the mechanisms of tumor cell latency, with a view to improving the detection and treatment of latent metastases.

  4. A quantitative histological study of strain-dependent differences in the effects of irradiation on mouse lung during the intermediate and late phases

    SciTech Connect

    Sharplin, J.; Franko, A.J. )

    1989-07-01

    Strain differences in the intermediate and late phases of the radiation response of mouse lung were investigated histologically. The proportion of lung impairment in mice at 28 and 52 weeks postirradiation and in mice dying of respiratory insufficiency was assessed by scoring lung acini as nonfunctional due to lesions which obstructed airflow, or open and presumably functional. The nine strains tested were divided into three groups on the basis of the late fibrotic response. Group 1 mice, three C57 strains, developed extensive contracted fibrosis and usually showed enough damage to explain late deaths. Group 2, SWR, A, and BALB/c strains, developed foci of contracted fibrosis. Group 3, CBA and two C3H strains, did not form fibrotic scars. Mice in Groups 2 and 3 that died with no pleural effusions appeared to have insufficient late lung damage to account for respiratory distress. Problems with pulmonary blood flow were indicated by evidence of loss of fine vasculature and right ventricular hypertrophy. In nondistressed, late-stage mice in Groups 2 and 3, loss of capillary perfusion in lung parenchyma free of obvious lesions was demonstrated by infusion of colloidal carbon. In one strain, A, an estimate of the proportion of nonperfused lung was made on distressed late-stage mice. Almost 50% of lung acini were nonfunctional as a result of nonperfusion, and an additional 9% of acini were nonfunctional due to lesions obstructing ventilation. It is suggested that nonperfusion of apparently normal lung acini is a major factor in late-phase deaths in those mouse strains which show little or no fibrosis.

  5. Antitumor effect of para-toluenesulfonamide against lung cancer xenograft in a mouse model

    PubMed Central

    Gao, Yang; Gao, Yonghua; Guan, Weijie; Huang, Liyan; Xu, Xiaoming; Zhang, Chenting; Chen, Xiuqing; Wu, Yizhuang; Zeng, Guangqiao

    2013-01-01

    Background Conventional chemotherapy and radiation therapy against non-small cell lung cancer (NSCLC) are relatively insensitive and unsatisfactory. Para-toluenesulfonamide (PTS), a unique antitumor drug for local intratumoral injection, shows an efficacy of severely suppressing solid tumor growth with mild side effects in clinical trials. The aim of this study was to investigate the effect of PTS on lung cancer H460 cells in vivo in nude mice and its underlying mechanisms in vitro. Methods A lung cancer model for in vivo experiment was established in BALB/c nude mice using H460 cells to examine the effect of local injection of PTS on tumor suppression. We also assessed the injury to the normal tissue by subcutaneous injection of PTS. In vitro, PTS was diluted into different doses for study on its antitumor mechanisms. We evaluated the necrotic effect of PTS on H460 cells by PI and Hoechst 33342 staining. Cell viability and membrane permeability were also determined by using CCK-8 and LDH assays respectively. All these tests were conducted in comparison with traditional local injection of anhydrous ethanol. Results PTS was shown to significantly inhibit the growth of H460 tumor xenografts in nude mice by inducing necrosis of the tumor histologically. Its effect on tumor growth was significantly stronger than that of anhydrous ethanol. By contrast, the injured normal tissue by PTS injection was less than that by ethanol. In vitro, PTS still demonstrated excellent necrotizing effect on H460 cells when diluted to a lower concentration. Detailed analysis of PTS on H460 cells indicated that PTS had a better effect on attenuating the cell viability and increasing the cell membrane permeability than ethanol at the same level. Conclusions PTS exhibits excellent inhibition effect on the growth of lung cancer by necrotizing tumor in vivo and in vitro, reducing tumor cell viability and augmenting the membrane permeability in vitro, with only mild injury to normal tissue. The

  6. Characterization of a nose-only inhaled phosgene acute lung injury mouse model.

    PubMed

    Plahovinsak, Jennifer L; Perry, Mark R; Knostman, Katherine A; Segal, Robert; Babin, Michael C

    2015-01-01

    Phosgene's primary mode of action is as a pulmonary irritant characterized by its early latent phase where life-threatening, non-cardiogenic pulmonary edema is typically observed 6-24 h post-exposure. To develop an inhaled phosgene acute lung injury (ALI) model in C57BL/6 mice that can be used to screen potential medical countermeasures. A Cannon style nose-only inhalation exposure tower was used to expose mice to phosgene (8 ppm) or air (sham). An inhalation lethality study was conducted to determine the 8 ppm median lethal exposure (LCt50) at 24 and 48 h post-exposure. The model was then developed at 1.2 times the 24 h LCt50. At predetermined serial sacrifice time points, survivors were euthanized, body and lung weights collected, and lung tissues processed for histopathology. Additionally, post-exposure clinical observations were used to assess quality of life. The 24-hour LCt50 was 226 ppm*min (8 ppm for 28.2 min) and the 48-hour LCt50 was 215 ppm*min (8 ppm for 26.9 min). The phosgene exposed animals had a distinct progression of clinical signs, histopathological changes and increased lung/body weight ratios. Early indicators of a 1.2 times the 24-hour LCt50 phosgene exposure were significant changes in the lung-to-body weight ratios by 4 h post-exposure. The progression of clinical signs and histopathological changes were important endpoints for characterizing phosgene-induced ALI for future countermeasure studies. An 8 ppm phosgene exposure for 34 min (1.2 × LCt50) is the minimum challenge recommended for evaluating therapeutic interventions. The predicted higher mortality in the phosgene-only controls will help demonstrate efficacy of candidate treatments and increase the probability that a change in survival rate is statistically significant.

  7. Ultrastructural features of the differentiating thyroid primordium in the sand lizard (Lacerta agilis L.) from the differentiation of the cellular cords to the formation of the follicular lumen.

    PubMed

    Rupik, Weronika; Kowalska, Magdalena; Swadźba, Elwira; Maślak, Robert

    2016-04-01

    The differentiation of the thyroid primordium of lacertilian species is poorly understood. The present study reports on the ultrastructural analysis of the developing thyroid primordium in the sand lizard (Lacerta agilis) during the early stages of differentiation. The early thyroid primordium of sand lizard embryos was composed of cellular cords that contained single cells with a giant lipid droplet, which were eliminated by specific autophagy (lipophagy). The follicular lumens at the periphery of the primordium differentiated even before the division of the cellular cords. When the single cells within the cords started to die through paraptosis, the adjacent cells started to polarise and junctional complexes began to form around them. After polarisation and clearing up after the formation of the lumens, the cellular cords divided into definitive follicles. The cellular cords in the central part of the primordium started to differentiate later than those at the periphery. The cellular cords divided into presumptive follicles first and only later differentiated into definitive follicles. During this process, a population of centrally located cells was removed through apoptosis to form the lumen. Although the follicular lumen in sand lizard embryos is differentiated by cavitation similar to that in the grass snake, there were very important differences during the early stages of the differentiation of the cellular cords and the formation of the thyroid follicles. Copyright © 2016 Elsevier GmbH. All rights reserved.

  8. Ventilation defects observed with hyperpolarized 3He magnetic resonance imaging in a mouse model of acute lung injury.

    PubMed

    Thomas, Abe C; Nouls, John C; Driehuys, Bastiaan; Voltz, James W; Fubara, Boma; Foley, Julie; Bradbury, J Alyce; Zeldin, Darryl C

    2011-05-01

    Regions of diminished ventilation are often evident during functional pulmonary imaging studies, including hyperpolarized gas magnetic resonance imaging (MRI), positron emission tomography, and computed tomography (CT). The objective of this study was to characterize the hypointense regions observed via (3)He MRI in a murine model of acute lung injury. LPS at doses ranging from 15-50 μg was intratracheally administered to C57BL/6 mice under anesthesia. Four hours after exposure to either LPS or saline vehicle, mice were imaged via hyperpolarized (3)He MRI. All images were evaluated to identify regions of hypointense signals. Lungs were then characterized by conventional histology, or used to obtain tissue samples from regions of normal and hypointense (3)He signals and analyzed for cytokine content. The characterization of (3)He MRI images identified three distinct types of hypointense patterns: persistent defects, atelectatic defects, and dorsal lucencies. Persistent defects were associated with the administration of LPS. The number of persistent defects depended on the dose of LPS, with a significant increase in mean number of defects in 30-50-μg LPS-dosed mice versus saline-treated control mice. Atelectatic defects predominated in LPS-dosed mice under conditions of low-volume ventilation, and could be reversed with deep inspiration. Dorsal lucencies were present in nearly all mice studied, regardless of the experimental conditions, including control animals that did not receive LPS. A comparison of (3)He MRI with histopathology did not identify tissue abnormalities in regions of low (3)He signal, with the exception of a single region of atelectasis in one mouse. Furthermore, no statistically significant differences were evident in concentrations of IL-1β, IL-6, macrophage inflammatory protein (MIP)-1α, MIP-2, chemokine (C-X-C motif) ligand 1 (KC), TNFα, and monocyte chemotactic protein (MCP)-1 between hypointense and normally ventilated lung regions in LPS

  9. Metabolite signatures in hydrophilic extracts of mouse lungs exposed to cigarette smoke revealed by 1H NMR metabolomics investigation

    SciTech Connect

    Hu, Jian Z.; Wang, Xuan; Feng, Ju; Webb-Robertson, Bobbie-Jo M.; Waters, Katrina M.; Tilton, Susan C.; Pounds, Joel G.; Corley, Richard A.; Liu, Maili; Hu, Mary Y.

    2015-05-12

    Herein, 1H-NMR metabolomics are carried out to evaluate the changes of metabolites in lungs of mice exposed to cigarette smoke. It is found that the concentrations of adenosine derivatives (i.e. ATP, ADP and AMP), inosine and uridine are significantly fluctuated in the lungs of mice exposed to cigarette smoke compared with those of controls regardless the mouse is obese or regular weight. The decreased ATP, ADP, AMP and elevated inosine predict that the deaminases in charge of adenosine derivatives to inosine derivatives conversion are altered in lungs of mice exposed to cigarette smoke. Transcriptional analysis reveals that the concentrations of adenosine monophosphate deaminase and adenosine deaminase are different in the lungs of mice exposed to cigarette smoke, confirming the prediction from metabolomics studies. We also found, for the first time, that the ratio of glycerophosphocholine (GPC) to phosphocholine (PC) is significantly increased in the lungs of obese mice compared with regular weight mice. The ratio of GPC/PC is further elevated in the lungs of obese group by cigarette smoke exposure. Since GPC/PC ratio is a known biomarker for cancer, these results may suggest that obese group is more susceptible to lung cancer when exposed to cigarette smoke.

  10. Studies using structural analogs and inbred strain differences to support a role for quinone methide metabolites of butylated hydroxytoluene (BHT) in mouse lung tumor promotion.

    PubMed

    Thompson, J A; Carlson, T J; Sun, Y; Dwyer-Nield, L D; Malkinson, A M

    2001-03-07

    Chronic treatment of BALB and GRS mice with BHT (2,6-di-tert-butyl-4-methylphenol) following a single urethane injection increases lung tumor multiplicity, but this does not occur in CXB4 mice. Previous data suggest that promotion requires the conversion of BHT to a tert-butyl-hydroxylated metabolite (BHTOH) in lung and the subsequent oxidation of this species to an electrophilic quinone methide. To obtain additional evidence for the importance of quinone methide formation, structural analogs that form less reactive quinone methides were tested and found to lack promoting activity in BHT-responsive mice. The possibility that promotion-unresponsive strains are unable to form BHTOH was tested by substituting this compound for BHT in the promotion protocol using CXB4 mice. No promotion occurred, and in-vitro work demonstrated that CXB4 mice are, in fact, capable of producing BHTOH and its quinone methide, albeit in smaller quantities. Incubations with BALB lung microsomes and radiolabeled substrates confirmed that more covalent binding to protein occurs with BHTOH than with BHT and, in addition, BHTOH quinone methide is considerably more toxic to mouse lung epithelial cells than BHT quinone methide. These data are consistent with the hypothesis that a two-step oxidation process, i.e. hydroxylation and quinone methide formation, is required for the promotion of mouse lung tumors by BHT.

  11. Gremlin-1 Overexpression in Mouse Lung Reduces Silica-Induced Lymphocyte Recruitment - A Link to Idiopathic Pulmonary Fibrosis through Negative Correlation with CXCL10 Chemokine.

    PubMed

    Koli, Katri; Sutinen, Eva; Rönty, Mikko; Rantakari, Pia; Fortino, Vittorio; Pulkkinen, Ville; Greco, Dario; Sipilä, Petra; Myllärniemi, Marjukka

    2016-01-01

    Idiopathic pulmonary fibrosis (IPF) is characterized by activation and injury of epithelial cells, the accumulation of connective tissue and changes in the inflammatory microenvironment. The bone morphogenetic protein (BMP) inhibitor protein gremlin-1 is associated with the progression of fibrosis both in human and mouse lung. We generated a transgenic mouse model expressing gremlin-1 in type II lung epithelial cells using the surfactant protein C (SPC) promoter and the Cre-LoxP system. Gremlin-1 protein expression was detected specifically in the lung after birth and did not result in any signs of respiratory insufficiency. Exposure to silicon dioxide resulted in reduced amounts of lymphocyte aggregates in transgenic lungs while no alteration in the fibrotic response was observed. Microarray gene expression profiling and analyses of bronchoalveolar lavage fluid cytokines indicated a reduced lymphocytic response and a downregulation of interferon-induced gene program. Consistent with reduced Th1 response, there was a downregulation of the mRNA and protein expression of the anti-fibrotic chemokine CXCL10, which has been linked to IPF. In human IPF patient samples we also established a strong negative correlation in the mRNA expression levels of gremlin-1 and CXCL10. Our results suggest that in addition to regulation of epithelial-mesenchymal crosstalk during tissue injury, gremlin-1 modulates inflammatory cell recruitment and anti-fibrotic chemokine production in the lung.

  12. Gremlin-1 Overexpression in Mouse Lung Reduces Silica-Induced Lymphocyte Recruitment – A Link to Idiopathic Pulmonary Fibrosis through Negative Correlation with CXCL10 Chemokine

    PubMed Central

    Koli, Katri; Sutinen, Eva; Rönty, Mikko; Rantakari, Pia; Fortino, Vittorio; Pulkkinen, Ville; Greco, Dario; Sipilä, Petra; Myllärniemi, Marjukka

    2016-01-01

    Idiopathic pulmonary fibrosis (IPF) is characterized by activation and injury of epithelial cells, the accumulation of connective tissue and changes in the inflammatory microenvironment. The bone morphogenetic protein (BMP) inhibitor protein gremlin-1 is associated with the progression of fibrosis both in human and mouse lung. We generated a transgenic mouse model expressing gremlin-1 in type II lung epithelial cells using the surfactant protein C (SPC) promoter and the Cre-LoxP system. Gremlin-1 protein expression was detected specifically in the lung after birth and did not result in any signs of respiratory insufficiency. Exposure to silicon dioxide resulted in reduced amounts of lymphocyte aggregates in transgenic lungs while no alteration in the fibrotic response was observed. Microarray gene expression profiling and analyses of bronchoalveolar lavage fluid cytokines indicated a reduced lymphocytic response and a downregulation of interferon-induced gene program. Consistent with reduced Th1 response, there was a downregulation of the mRNA and protein expression of the anti-fibrotic chemokine CXCL10, which has been linked to IPF. In human IPF patient samples we also established a strong negative correlation in the mRNA expression levels of gremlin-1 and CXCL10. Our results suggest that in addition to regulation of epithelial-mesenchymal crosstalk during tissue injury, gremlin-1 modulates inflammatory cell recruitment and anti-fibrotic chemokine production in the lung. PMID:27428020

  13. Characterization of the cell of origin and propagation potential of the fibroblast growth factor 9-induced mouse model of lung adenocarcinoma.

    PubMed

    Arai, Daisuke; Hegab, Ahmed E; Soejima, Kenzo; Kuroda, Aoi; Ishioka, Kota; Yasuda, Hiroyuki; Naoki, Katsuhiko; Kagawa, Shizuko; Hamamoto, Junko; Yin, Yongjun; Ornitz, David M; Betsuyaku, Tomoko

    2015-03-01

    Fibroblast growth factor 9 (FGF9) is essential for lung development and is highly expressed in a subset of human lung adenocarcinomas. We recently described a mouse model in which FGF9 expression in the lung epithelium caused proliferation of the airway epithelium at the terminal bronchioles and led to rapid development of adenocarcinoma. Here, we used this model to characterize the effects of prolonged FGF9 induction on the proximal and distal lung epithelia, and examined the propagation potential of FGF9-induced lung tumours. We showed that prolonged FGF9 over-expression in the lung resulted in the development of adenocarcinomas arising from both alveolar type II and airway secretory cells in the lung parenchyma and airways, respectively. We found that tumour cells harboured tumour-propagating cells that were able to form secondary tumours in recipient mice, regardless of FGF9 expression. However, the highest degree of tumour propagation was observed when unfractionated tumour cells were co-administered with autologous, tumour-associated mesenchymal cells. Although the initiation of lung adenocarcinomas was dependent on activation of the FGF9-FGF receptor 3 (FGFR3) signalling axis, maintenance and propagation of the tumour was independent of this signalling. Activation of an alternative FGF-FGFR axis and the interaction with tumour stromal cells is likely to be responsible for the development of this independence. This study demonstrates the complex role of FGF-FGFR signalling in the initiation, growth and propagation of lung cancer. Our findings suggest that analysing the expressions of FGF-FGFRs in human lung cancer will be a useful tool for guiding customized therapy.

  14. Characterization of azoxymethane-induced colon tumor metastasis to lung in a mouse model relevant to human sporadic colorectal cancer and evaluation of grape seed extract efficacy.

    PubMed

    Derry, Molly M; Raina, Komal; Agarwal, Rajesh; Agarwal, Chapla

    2014-08-01

    The second leading cause of cancer-related deaths (both genders combined) in the United States is colorectal cancer (CRC). This emphasizes the need to develop both effective therapies for CRC patients and pre-clinical models mimicking human disease that carry translational potential in drug-development. Notably, at present there are no in situ models of CRC metastasis to lung. In our azoxymethane-induced colon tumorigenesis study in A/J mice assessing grape seed extract (GSE) efficacy, during necropsy we also found multiple lung nodules suggestive of colon tumor metastasis to lung that were significantly inhibited in GSE fed group. Both histopathological and molecular studies were performed to characterize and establish the origin of these lesions in lung. Histologically these nodules were determined as adenocarcinoma of mucin origin. Molecular analyses by immunohistochemistry (IHC) and RT-PCR revealed strong protein and transcript levels of colon specific markers CDX2 and CK20 in these lung nodules compared to uninvolved control lung tissue. Vis-à-vis, these nodules also showed minimally expressed lung specific biomarkers, specifically surfactant D and TTF-1, in IHC analysis. Additionally, 0.25% GSE supplementation in diet (w/w) decreased the incidence of these lung nodules by 53% and their total number by 66%. Together, the characterization of this unique in situ mouse model of CRC metastasis to lung provides translational opportunities in developing effective therapies to clinically manage and treat CRC at the advanced stage. Moreover, GSE efficacy in inhibiting CRC metastasis to lung in this model further supports its translational potential in controlling CRC growth, progression and metastasis in patients.

  15. The effect of synthetic surfactant Exosurf on gene transfer in mouse lung in vivo.

    PubMed

    Raczka, E; Kukowska-Latallo, J F; Rymaszewski, M; Chen, C; Baker, J R

    1998-10-01

    Gene transfer in the lung holds promise for the treatment of diseases such as pulmonary fibrosis, cystic fibrosis and asthma. Pulmonary surfactant has been reported to enhance expression from endobronchial, adenovirus-mediated gene transfer in experimental animals. This study examines the effect of exogenous synthetic surfactant (Exosurf) on gene expression from naked plasmid DNA administered endobronchially to adult mice. Transfection efficiency was evaluated by quantifying the expression of chloramphenicol acetyltransferase (CAT) and luciferase (Luc) genes in the lung. Endobronchial administration of either CAT or Luc expression plasmid DNA resulted in detectable concentrations of each reporter protein. CAT expression from plasmid DNA was monitored after endobronchial administration with the maximal expression observed at 3-5 days after administration and decreasing for 5 days thereafter. When DNA was delivered in a 50% suspension of Exosurf, the expression of either CAT or Luc was significantly reduced by 89.6 +/- 1.4% and 82.7 +/- 10.5%, respectively. The decrease in Luc expression was closely correlated (r = 0.99, P < 0.001) to log concentration of surfactant in the plasmid buffer solution (IC50 = 8.6%). CAT expression was not altered when surfactant was administered either 2 h before or after plasmid DNA instillation. Examination of the components of Exosurf revealed that two compounds, DPPC and tyloxapol, showed inhibitory effects on CAT expression. However, the inhibition caused by Exosurf appeared greater than that of either component. Our results suggest that the lung surfactant is a barrier to transfection of the endobronchial airway and may be partly responsible for the low expression of exogenous DNA in vivo in the bronchial tree.

  16. Matrix Metalloproteinase 1 promotes tumor formation and lung metastasis in an intratibial injection osteosarcoma mouse model.

    PubMed

    Husmann, Knut; Arlt, Matthias J E; Muff, Roman; Langsam, Bettina; Bertz, Josefine; Born, Walter; Fuchs, Bruno

    2013-02-01

    Proteolytic degradation of the extracellular matrix (ECM) is an important process during tumor invasion. Matrix Metalloproteinase 1 (MMP-1) is one of the proteases that degrade collagen type I, a major component of bone ECM. In the present study, the biological relevance of MMP-1 in osteosarcoma (OS) tumor growth and metastasis was investigated in vitro and in vivo. Human OS cells in primary culture expressed MMP-1 encoding mRNA at considerably higher levels than normal human bone cells. In addition, MMP-1 mRNA and protein expression in the highly metastatic human osteosarcoma 143-B cell line was remarkably higher than in the non-metastatic parental HOS cell line. Stable shRNA-mediated downregulation of MMP-1 in 143-B cells impaired adhesion to collagen I and anchorage-independent growth, reflected by a reduced ability to grow in soft agar. Upon intratibial injection into SCID mice, 143-B cells with shRNA-downregulated MMP-1 expression formed smaller primary tumors and significantly lower numbers of lung micro- and macrometastases than control cells. Conversely, HOS cells stably overexpressing MMP-1 showed an enhanced adhesion capability to collagen I and accelerated anchorage-independent growth compared to empty vector-transduced control cells. Furthermore, and most importantly, individual MMP-1 overexpression in HOS cells enabled the formation of osteolytic primary tumors and lung metastasis while the HOS control cells did not develop any tumors or metastases after intratibial injection. The findings of the present study reveal an important role of MMP-1 in OS primary tumor and metastasis formation to the lung, the major organ of OS metastasis. Copyright © 2012 Elsevier B.V. All rights reserved.

  17. FIB-SEM imaging of carbon nanotubes in mouse lung tissue.

    PubMed

    Købler, Carsten; Saber, Anne Thoustrup; Jacobsen, Nicklas Raun; Wallin, Håkan; Vogel, Ulla; Qvortrup, Klaus; Mølhave, Kristian

    2014-06-01

    Ultrastructural characterisation is important for understanding carbon nanotube (CNT) toxicity and how the CNTs interact with cells and tissues. The standard method for this involves using transmission electron microscopy (TEM). However, in particular, the sample preparation, using a microtome to cut thin sample sections for TEM, can be challenging for investigation of regions with agglomerations of large and stiff CNTs because the CNTs cut with difficulty. As a consequence, the sectioning diamond knife may be damaged and the uncut CNTs are left protruding from the embedded block surface excluding them from TEM analysis. To provide an alternative to ultramicrotomy and subsequent TEM imaging, we studied focused ion beam scanning electron microscopy (FIB-SEM) of CNTs in the lungs of mice, and we evaluated the applicability of the method compared to TEM. FIB-SEM can provide serial section volume imaging not easily obtained with TEM, but it is time-consuming to locate CNTs in the tissue. We demonstrate that protruding CNTs after ultramicrotomy can be used to locate the region of interest, and we present FIB-SEM images of CNTs in lung tissue. FIB-SEM imaging was applied to lung tissue from mice which had been intratracheally instilled with two different multiwalled CNTs; one being short and thin, and the other longer and thicker. FIB-SEM was found to be most suitable for detection of the large CNTs (Ø ca. 70 nm), and to be well suited for studying CNT agglomerates in biological samples which is challenging using standard TEM techniques.

  18. Genetic Phagocyte NADPH Oxidase Deficiency Enhances Nonviable Candida albicans-Induced Inflammation in Mouse Lungs.

    PubMed

    Endo, Daiki; Fujimoto, Kenta; Hirose, Rika; Yamanaka, Hiroko; Homme, Mizuki; Ishibashi, Ken-Ichi; Miura, Noriko; Ohno, Naohito; Aratani, Yasuaki

    2017-02-01

    Patients with chronic granulomatous disease (CGD) have mutated phagocyte NADPH oxidase, resulting in reduced production of reactive oxygen species (ROS). While the mechanism underlying hyperinfection in CGD is well understood, the basis for inflammatory disorders that arise in the absence of evident infection has not been fully explained. This study aimed to evaluate the effect of phagocyte NADPH oxidase deficiency on lung inflammation induced by nonviable Candida albicans (nCA). Mice deficient in this enzyme (CGD mice) showed more severe neutrophilic pneumonia than nCA-treated wild-type mice, which exhibited significantly higher lung concentrations of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and keratinocyte-derived chemokine (KC). Neutralization of these proinflammatory mediators significantly reduced neutrophil infiltration. In vitro, production of IL-1β and TNF-α from neutrophils and that of KC from macrophages was enhanced in nCA-stimulated neutrophils from CGD mice. Expression of IL-1β mRNA was higher in the stimulated CGD neutrophils than in the stimulated wild-type cells, concomitant with upregulation of nuclear factor (NF)-κB and its upstream regulator extracellular-signal regulated kinase (ERK) 1/2. Pretreatment with an NADPH oxidase inhibitor significantly enhanced IL-1β production in the wild-type neutrophils stimulated with nCA. These results suggest that lack of ROS production because of NADPH oxidase deficiency results in the production of higher levels of proinflammatory mediators from neutrophils and macrophages, which may at least partly contribute to the exacerbation of nCA-induced lung inflammation in CGD mice.

  19. Vascular and epithelial damage in the lung of the mouse after X rays or neutrons

    SciTech Connect

    Law, M.P.; Ahier, R.G.

    1989-01-01

    The response of the lung was studied in CFLP mice after exposure of the whole thorax to X rays (250 kVp) or cyclotron neutrons (16 MeV deuterons on Be, mean energy 7.5 MeV). To measure blood volume and leakage of plasma proteins, 51Cr-labeled red blood cells and 125I-albumin were injected intravenously and 24 h later lungs were lavaged via the trachea. Radioactivities in lung tissue and lavage fluid were determined to estimate the accumulation of albumin in the interstitial and alveolar spaces indicating damage to blood vessels and alveolar epithelium respectively. Function of type II pneumonocytes was assessed by the amounts of surfactant (assayed as lipid phosphorous) released into the lavage fluid. During the first 6 weeks, lavage protein and surfactant were increased, the neutron relative biological effectiveness (RBE) being unity. During pneumonitis at 12-24 weeks, surfactant levels were normal, blood volume was decreased, and both interstitial and alveolar albumin were increased. Albumin levels then decreased. At late times after exposure (42-64 weeks) alveolar albumin returned to normal but interstitial albumin was still slightly elevated. Values of RBE for changes in blood volume and interstitial and alveolar albumin at 15 weeks and for changes in blood volume and interstitial albumin at 46 weeks were 1.4, comparable with that for animal survival at 180 days. The results indicate that surfactant production is not critical for animal survival. They suggest that changes in blood vessels and alveolar epithelium occur during acute pneumonitis; epithelial repair follows but some vascular damage may persist. The time course of the changes in albumin levels did not correlate with increases in collagen biosynthesis which have been observed as early as 1 month after exposure and persist for up to 1 year.

  20. Alternatively activated RAW264.7 macrophages enhance tumor lymphangiogenesis in mouse lung adenocarcinoma.

    PubMed

    Zhang, Bicheng; Wang, Jun; Gao, Juan; Guo, Yan; Chen, Xi; Wang, Baocheng; Gao, Jianfei; Rao, Zhiguo; Chen, Zhengtang

    2009-05-01

    Tumor-associated macrophages (TAMs) have been implicated in promoting tumor progression and invasion. The onset and maintenance of tumor angiogenesis and lymphangiogenesis also seem to be partly driven by a group of polarized alternatively activated macrophages (aaMphi) in lung adenocarcinoma. Here, the aaMphi and classically activated macrophages (caMphi) were obtained using RAW264.7 cells via IL-4 and IFN-gamma + LPS treatment, respectively. Co-inoculation of aaMphi with Lewis lung carcinoma (LLC) cells promoted tumor growth, increased lymph node metastasis, and reduced the survival in C57BL/6 mice bearing LLC. Furthermore, the effects of the activated macrophages on the lymphangiogenesis-related properties of lymphatic endothelial cells (LECs) were investigated in vitro. When LECs were cultured in macrophages conditioned medium or in a co-culture system of macrophages and LECs, aaMphi significantly promoted proliferation, migration, and tube-like formation of LECs. We identified high VEGF-C expression in aaMphi and low expression in caMphi as well as unactivated macrophages by ELISA and Western blotting. In LECs, co-culture with aaMphi resulted in a significant increase of mRNA levels of specific lymphatic marker VEGF receptor-3 and the homeobox gene Prox-1, as well as lymphangiogenic factor VEGF-C rather than VEGF-D by quantitative RT-PCR. Furthermore, enhanced LECs migration and capillary formation by co-culture with aaMphi were significantly inhibited by rVEGF receptor-3/Fc chimera. In conclusion, these data show that aaMphi play a critical role in tumor-induced lymphangiogenesis through up-regulating VEGF-C and increasing lymphangiogenesis-related behavior of LECs, which may contribute to lymphatic invasion in lung adenocarcinoma.

  1. Detection of Phenolic Metabolites of Styrene in Mouse Liver and Lung Microsomal IncubationsS⃞

    PubMed Central

    Shen, Shuijie; Zhang, Fan; Gao, Lingbo; Zeng, Su

    2010-01-01

    Metabolic activation is considered to be a critical step for styrene-induced pulmonary toxicity. Styrene-7,8-oxide is a primary oxidative metabolite generated by vinyl epoxidation of styrene. In addition, urinary 4-vinylphenol (4-VP), a phenolic metabolite formed by aromatic hydroxylation, has been detected in workers and experimental animals after exposure to styrene. In the present study, new oxidative metabolites of styrene, including 2-vinylphenol (2-VP), 3-vinylphenol (3-VP), vinyl-1,4-hydroquinone, and 2-hydroxystyrene glycol were detected in mouse liver microsomal incubations. The production rates of 2-VP, 3-VP, 4-VP, and styrene glycol were 0.0527 ± 0.0045, 0.0019 ± 0.0006, 0.0053 ± 0.0002, and 4.42 ± 0.33 nmol/(min · mg protein) in mouse liver microsomes, respectively. Both disulfiram (100 μM) and 5-phenyl-1-pentyne (5 μM) significantly inhibited the formation of the VPs and styrene glycol. 2-VP, 3-VP, and 4-VP were metabolized in mouse liver microsomes at rates of 2.50 ± 0.30, 2.63 ± 0.13, and 3.45 ± 0.11 nmol/(min · mg protein), respectively. The three VPs were further metabolized to vinylcatechols and/or vinyl-1,4-hydroquinone and the corresponding glycols. Pulmonary toxicity of 2-VP, 3-VP, and 4-VP was evaluated in CD-1 mice, and 4-VP was found to be more toxic than 2-VP and 3-VP. PMID:20724499

  2. Effects of smoke inhalation on surfactant phospholipids and phospholipase A2 activity in the mouse lung.

    PubMed Central

    Oulton, M.; Moores, H. K.; Scott, J. E.; Janigan, D. T.; Hajela, R.

    1991-01-01

    The effects of smoke inhalation on the pulmonary surfactant system were examined in mice exposed for 30 minutes to smoke generated from the burning of polyurethane foam. At 8 or 12 hours after exposure, surfactants were isolated separately from lung lavage (extracellular surfactant) and residual lung tissue (intracellular surfactant) for phospholipid analysis. Calcium-dependent phospholipase A2 (PLA2) was measured on a microsomal fraction prepared from the tissue homogenate. Smoke inhalation produced a twofold increase in extracellular surfactant total phospholipid. While there was no change in the total phospholipid or phosphatidylcholine (PC) content of the intracellular surfactant, smoke inhalation significantly decreased the disaturated species of PC (DSPC). The specific activity of PLA2 was reduced by more than 50% in both groups of exposed mice. Smoke inhalation appears to result in selective depletion of the DSPC of intracellular surfactant and PLA2 involved in its synthesis. This depletion may be compensated for by increased secretion or slower breakdown of the material present in the extracellular compartment. Images Figure 1 PMID:1987765

  3. Dexmedetomidine attenuates acute lung injury induced by lipopolysaccharide in mouse through inhibition of MAPK pathway.

    PubMed

    Xu, Yingzhen; Zhang, Ruyi; Li, Chunli; Yin, Xue; Lv, Changjun; Wang, Yaoqi; Zhao, Wenxiang; Zhang, Xiuli

    2015-10-01

    Dexmedetomidine (Dex) is widely used for sedation in intensive care units and can be used as an adjunct to anesthetics. Previous studies have demonstrated that Dex has anti-inflammatory property. In this study, we aim to explore the potential therapeutic effects and mechanisms of Dex on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. To induce ALI, mice were intraperitoneally injected with LPS, while Dex was treated 1 h before LPS injection. The inflammation of lung tissues was evaluated by HE stain, and bronchoalveolar lavage fluid (BALF) was obtained after 6 h to measure protein concentrations. We also used an enzyme-linked immunosorbent assay to detect the secretion levels of proinflammatory cytokines in the serum. Western blotting method was adopted to observe changes in mitogen-activated protein kinases and downstream nuclear transcription factors. The results showed that pretreatment with Dex considerably reduced neutrophil infiltration and pulmonary edema, and significantly reduced protein concentrations in the BALF, as well as suppressed LPS-induced elevation of proinflammatory cytokines (TNF-α and IL-1β) in the serum. In addition, we observed that the molecular mechanism of Dex-mediated anti-inflammation is associated with decreasing phosphorylation of MKK4, MMK3/6, ERK1/2, p38MAPK, and JNK, and diminishing activation of Elk-1, c-Jun, and ATF-2. Dex could attenuate ALI induced by LPS in mice, and this effect may be mediated through the inhibition of MAPK pathway.

  4. Protective effect of Jolkinolide B on LPS-induced mouse acute lung injury.

    PubMed

    Yang, Hailing; Li, Yan; Huo, Pengfei; Li, Xiao-Ou; Kong, Daliang; Mu, Wei; Fang, Wei; Li, Lingxia; Liu, Ning; Fang, Ling; Li, Hongjun; He, Chengyan

    2015-05-01

    Jolkinolide B (JB), an ent-abietane diterpenoid, isolated from the dried root of Euphorbia fischeriana, has been reported to have potent anti-tumor and anti-inflammatory activities. However, the effects of JB on acute lung injury (ALI) and underlying molecular mechanisms have not been investigated. The present study aimed to investigate the effect of JB on lipopolysaccharide (LPS)-induced ALI. Male C57BL/6 mice were pretreated with dexamethasone or JB 1h before intranasal instillation of LPS. The results showed that JB markedly attenuated LPS-induced histological alterations, lung edema, inflammatory cell infiltration, myeloperoxidase (MPO) activity as well as the production of TNF-α, IL-6 and IL-1β. Furthermore, JB also significantly inhibited LPS-induced the degradation of IκBα and phosphorylation of NF-κB p65 and MAPK. Therefore, our study provides the first line of evidence that pretreatment of JB has a protective effect on LPS-induced ALI in mice. The anti-inflammatory mechanism of JB may be attributed to its suppression of NF-κB and MAPK activation. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Detection of Sendai virus receptor, the ganglioside GDla, in target tissue (mouse lung)

    SciTech Connect

    Markwell, M.A.K.; Sato, E.

    1986-05-01

    Previously the authors had shown that the gangliosides GDla, GTlb, and GQlb derived from brain function as receptors for the paramyxovirus Sendai virus by their ability to induce infection when incubated with receptor-deficient cells. Analyses of MDBK, HeLa, and MDCK cells in culture demonstrated that these putative receptors were present in host cells in the quantities required for infection. The primary site of infection for Sendai virus in the whole animal is the respiratory tract, culminating in the lung. Therefore, the ganglioside content of this target organ was analyzed to determine the endogenous receptor population available to Sendai virus. The total ganglioside fraction of lung was resolved into individual species by HPTLC. Gangliosides of the gangliotetraose series were identified by the specific binding of /sup 125/I-labeled tetanus and cholera toxins before and after exposure with sialidase. In this manner one of the major resorcinol-positive bands was identified as GDla. Evidence of the more complex ganglioside receptors for Sendai virus was also seen.

  6. Early alterations in extracellular matrix and transforming growth factor [beta] gene expression in mouse lung indicative of late radiation fibrosis

    SciTech Connect

    Finkelstein, J.N.; Johnston, C.J.; Baggs, R.; Rubin, P. )

    1994-02-01

    Fibrosis, characterized by the accumulation of collagen, is a late result of thoracic irradiation. The expression of late radiation injury can be found immediately after irradiation by measuring messenger RNA (mRNA) abundance. To determine if extracellular matrix mRNA and transforming growth factor beta abundance was affected acutely after irradiation, the authors measured mRNA levels of collagen I (CI), collagen III (CIII), collagen IV (CIV), fibronectin (FN), and transforming growth factor [beta] (TGF[beta][sub 1,2 3]) in mouse lungs on day 1 and day 14 after graded doses of radiation. C57BL/6 female mice were irradiated with a single dose to the thorax of 5 or 12.5 Gy. Total lung RNA was prepared and immobilized by Northern and slot blotting and hybridized with radiolabelled cDNA probes for CI, CIII, CIV, FN, TGF[beta][sub 1,2 3] and a control probe encoding for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Autoradiographic data were quantified by video densitometry and results normalized to GAPDH. Changes in the expression of CI, CIII, CIV, FN and TGF[beta][sub 1,2 3] were observed as early as 1 day after exposure. Through 14 days, changes in mRNA up to 5-fold were seen for any one dose. Dose related changes as high as 10-fold were also evident. The CI:CIII ratio increased gradually for the 5 Gy dose at 14 days postirradiation while the CI:CII ratio for the 12.5 Gy dose decreased by approximately 4-fold as compared to the control. These studies suggest that alterations in expression of extracellular matrix and TGF[beta] mRNA occur very early after radiation injury even at low doses and may play a role in the development of chronic fibrosis. 37 refs., 6 figs.

  7. Spores of Aspergillus versicolor isolated from indoor air of a moisture-damaged building provoke acute inflammation in mouse lungs.

    PubMed

    Jussila, Juha; Komulainen, Hannu; Kosma, Veli-Matti; Nevalainen, Aino; Pelkonen, Jukka; Hirvonen, Maija-Riitta

    2002-12-01

    Microbial growth in moisture-damaged buildings has been associated with respiratory health effects, and the spores of the mycotoxin producing fungus Aspergillus versicolor are frequently present in the indoor air. To characterize the potential of these spores to cause harmful respiratory effects, mice were exposed via intratracheal instillation to a single dose of the spores of A. versicolor (1 x 10(5), 1 x 10(6), 5 x 10(6), 1 x 10(7), or 1 x 10(8) spores), isolated from the indoor air of a moisture-damaged building. Inflammation and toxicity in lungs were evaluated 24 h later by assessment of biochemical markers and histopathology. The time course of the effects was investigated with the dose of 5 x 10(6) spores for up to 28 days. The exposure to the spores increased transiently proinflammatory cytokine levels (tumor necrosis factor [TNF] alpha and interleukin [IL]-6) in bronchoalveolar lavage fluid (BALF). The cytokine responses were dose and time dependent. The highest cytokine concentrations were measured at 6 h after the dose, and they returned to the control level by 3 days. Moreover, the spores of A. versicolor recruited inflammatory cells into airways: Neutrophils peaked transiently at 24 h, macrophages at 3 days, and lymphocytes at 7 days after the dosing. The inflammatory cell response did not completely disappear during the subsequent 28 days, though no histopathological changes were seen at that time point. The spores did not induce expression of inducible nitric oxide synthase in lavaged cells. Only the highest spore dose (1 x 10(8)) markedly increased serum IL-6, increased vascular leakage, and caused cytotoxicity (i.e., increased levels of albumin, total protein, lactate dehydrogenase [LDH], and hemoglobin in BALF) in the airways. In summary, the spores of A. versicolor caused acute inflammation in mouse lungs. This indicates that they have potential to provoke adverse health effects in the occupants of moisture-damaged buildings.

  8. Antenatal maternal low protein diet: ACE-2 in the mouse lung and sexually dimorphic programming of hypertension.

    PubMed

    Goyal, Ravi; Van-Wickle, Jonathan; Goyal, Dipali; Longo, Lawrence D

    2015-05-14

    Elevated blood pressure is an important global health problem, and in-utero under-nutrition may be an important factor in the pathogenesis of hypertension. In the present study, we tested the hypothesis that antenatal maternal low protein diet (MLPD) leads to sexually dimorphic developmental programming of the components of the pulmonary renin-angiotensin system. This may be important in the antenatal MLPD-associated development of hypertension. In pregnant mice, we administered normal (control) and isocaloric 50% protein restricted diet, commencing one week before mating and continuing until delivery of the pups. From the 18th to 24th week postnatal, we measured blood pressure in the offspring by use of a non-invasive tail-cuff method. In the same mice, we examined the mRNA and protein expression of the key components of the pulmonary renin-angiotensin system. Also, we examined microRNA complementary to angiotensin converting enzymes (ACE) 2 in the offspring lungs. Our results demonstrate that as a consequence of antenatal MLPD: 1) pup birthweight was significantly reduced in both sexes. 2) female offspring developed hypertension, but males did not. 3) In female offspring, ACE-2 protein expression was significantly reduced without any change in the mRNA levels. 4) miRNA 429, which has a binding site on ACE-2 - 3' UTR was significantly upregulated in the female antenatal MLPD offspring. 5) In males, ACE-2 mRNA and protein expression were unaltered. We conclude that in the mouse, antenatal MLPD-induced reduction of ACE-2 in the female offspring lung may be an important mechanisms in sexually dimorphic programming of hypertension.

  9. Stimulation of immature lung macrophages with intranasal interferon gamma in a novel neonatal mouse model of respiratory syncytial virus infection.

    PubMed

    Empey, Kerry M; Orend, Jacob G; Peebles, R Stokes; Egaña, Loreto; Norris, Karen A; Oury, Tim D; Kolls, Jay K

    2012-01-01

    Respiratory syncytial virus (RSV) is the leading cause of bronchiolitis and viral death in infants. Reduced CD8 T-cells and negligible interferon gamma (IFNγ) in the airway are associated with severe infant RSV disease, yet there is an abundance of alveolar macrophages (AM) and neutrophils. However, it is unclear, based on our current understanding of macrophage functional heterogeneity, if immature AM improve viral clearance or contribute to inflammation and airway obstruction in the IFNγ-deficient neonatal lung environment. The aim of the current study was to define the age-dependent AM phenotype during neonatal RSV infection and investigate their differentiation to classically activated macrophages (CAM) using i.n. IFNγ in the context of improving viral clearance. Neonatal and adult BALB/cJ mice were infected with 1×10(6) plaque forming units (PFU)/gram (g) RSV line 19 and their AM responses compared. Adult mice showed a rapid and robust CAM response, indicated by increases in major histocompatibility complex class II (MHC II), CD86, CCR7, and a reduction in mannose receptor (MR). Neonatal mice showed a delayed and reduced CAM response, likely due to undetectable IFNγ production. Intranasal (i.n.) treatment with recombinant mouse IFNγ (rIFNγ) increased the expression of CAM markers on neonatal AM, reduced viral lung titers, and improved weight gain compared to untreated controls with no detectable increase in CD4 or CD8 T-cell infiltration. In vitro infection of J774A.1 macrophages with RSV induced an alternatively activated macrophage (AAM) phenotype however, when macrophages were first primed with IFNγ, a CAM phenotype was induced and RSV spread to adjacent Hep-2 cells was reduced. These studies demonstrate that the neonatal AM response to RSV infection is abundant and immature, but can be exogenously stimulated to express the antimicrobial phenotype, CAM, with i.n. rIFNγ.

  10. Effects of nickel-oxide nanoparticle pre-exposure dispersion status on bioactivity in the mouse lung.

    PubMed

    Sager, Tina; Wolfarth, Michael; Keane, Michael; Porter, Dale; Castranova, Vincent; Holian, Andrij

    2016-01-01

    Nanotechnology is emerging as one of the world's most promising new technologies. From a toxicology perspective, nanoparticles possess two features that promote their bioactivity. The first involves physical-chemical characteristics of the nanoparticle, which include the surface area of the nanoparticle. The second feature is the ability of the nanoparticle to traverse cell membranes. These two important nanoparticle characteristics are greatly influenced by placing nanoparticles in liquid medium prior to animal exposure. Nanoparticles tend to agglomerate and clump in suspension, making it difficult to reproducibly deliver them for in vivo or in vitro experiments, possibly affecting experimental variability. Thus, we hypothesize that nanoparticle dispersion status will correlate with the in vivo bioactivity/toxicity of the particle. To test our hypothesis, nano-sized nickel oxide was suspended in four different dispersion media (phosphate-buffered saline (PBS), dispersion medium (DM), a combination of dipalmitoyl-phosphatidyl choline (DPPC) and albumin in concentrations that mimic diluted alveolar lining fluid), Survanta®, or pluronic (Pluronic F-68). Well-dispersed and poorly dispersed suspensions were generated in each media by varying sonication time on ice utilizing a Branson Sonifer 450 (25W continuous output, 20 min or 5 min, respectively). Mice (male, C57BL/6J, 7-weeks-old) were given 0-80 µg/mouse of nano-sized nickel oxide in the different states of dispersion via pharyngeal aspiration. At 1 and 7 d post-exposure, mice underwent whole lung lavage to assess pulmonary inflammation and injury as a function of dispersion status, dose and time. The results show that pre-exposure dispersion status correlates with pulmonary inflammation and injury. These results indicate that a greater degree of pre-exposure dispersion increases pulmonary inflammation and cytotoxicity, as well as decreases in the integrity of the blood-gas barrier in the lung.

  11. Rosiglitazone is a superior bronchodilator compared to chloroquine and β-adrenoceptor agonists in mouse lung slices

    PubMed Central

    2014-01-01

    Background Current therapy for relieving bronchoconstriction may be ineffective in severe asthma, particularly in the small airways. The aim of this study was to further characterise responses to the recently identified novel bronchodilators rosiglitazone (RGZ) and chloroquine (CQ) under conditions where β-adrenoceptor agonist efficacy was limited or impaired in mouse small airways within lung slices. Methods Relaxation to RGZ and CQ was assessed following submaximal methacholine (MCh) pre-contraction, in slices treated overnight with either RGZ, CQ or albuterol (ALB) (to induce β-adrenoceptor desensitization), and in slices treated with caffeine/ryanodine in which contraction is associated with increases in Ca2+ sensitivity in the absence of contractile agonist-induced Ca2+ oscillations. Furthermore, the effects of RGZ, CQ, ALB and isoproterenol (ISO) on the initiation and development of methacholine-induced contraction were also compared. Results RGZ and CQ, but not ALB or ISO, elicited complete relaxation with increasing MCh pre-contraction and maintained their potency and efficacy following β-adrenoceptor desensitization. RGZ, CQ and ALB maintained efficacy following overnight incubation with RGZ or CQ. Relaxation responses to all dilators were generally maintained but delayed after caffeine/ryanodine. Pre-treatment with RGZ, but not CQ, ALB or ISO, reduced MCh potency. Conclusions This study demonstrates the superior effectiveness of RGZ in comparison to CQ and β-adrenoceptor agonists as a dilator of mouse small airways. Further investigation of the mechanisms underlying the relatively greater efficacy of RGZ under these conditions are warranted and should be extended to include studies in human asthmatic airways. PMID:24621080

  12. Comparative transcriptomics of Pleurotus eryngii reveals blue-light regulation of carbohydrate-active enzymes (CAZymes) expression at primordium differentiated into fruiting body stage.

    PubMed

    Xie, Chunliang; Gong, Wenbing; Zhu, Zuohua; Yan, Li; Hu, Zhenxiu; Peng, Yuande

    2017-09-29

    Blue light is an important environmental factor which could induce mushroom primordium differentiation and fruiting body development. However, the mechanisms of Pleurotus eryngii primordium differentiation and development induced by blue light are still unclear. The CAZymes (carbohydrate-active enzymes) play important roles in degradation of renewable lignocelluloses to provide carbohydrates for fungal growth, development and reproduction. In the present research, the expression profiles of genes were measured by comparison between the Pleurotus eryngii at primordium differentiated into fruiting body stage after blue light stimulation and dark using high-throughput sequencing approach. After assembly and compared to the Pleurotus eryngii reference genome, 11,343 unigenes were identified. 539 differentially expressed genes including white collar 2 type of transcription factor gene, A mating type protein gene, MAP kinase gene, oxidative phosphorylation associated genes, CAZymes genes and other metabolism related genes were identified during primordium differentiated into fruiting body stage after blue light stimulation. KEGG results showed that carbon metabolism, glycolysis/gluconeogenesis and biosynthesis of amino acids pathways were affected during blue light inducing primordia formation. Most importantly, 319 differentially expressed CAZymes participated in carbon metabolism were identified. The expression patterns of six representative CAZymes and laccase genes were further confirmed by qRT-PCR. Enzyme activity results indicated that the activities of CAZymes and laccase were affected in primordium differentiated into fruiting body under blue light stimulation. In conclusion, the comprehensive transcriptome and CAZymes of Pleurotus eryngii at primordium differentiated into fruiting body stage after blue light stimulation were obtained. The biological insights gained from this integrative system represent a valuable resource for future genomic studies on this

  13. Mouse lung inflammation after instillation of particulate matter collected from a working dairy barn

    SciTech Connect

    Wegesser, Teresa C.; Last, Jerold A.

    2009-05-01

    Coarse and fine particulate matter (PM{sub 2.5-10} and PM{sub 2.5}, respectively) are regulated ambient air pollutants thought to have major adverse health effects in exposed humans. The role of endotoxin and other bioaerosol components in the toxicity of PM from ambient air is controversial. This study evaluated the inflammatory lung response in mice instilled intratracheally with PM{sub 2.5-10} and PM{sub 2.5} emitted from a working dairy barn, a source presumed to have elevated concentrations of endotoxin. PM{sub 2.5-10} was more pro-inflammatory on an equal weight basis than was PM{sub 2.5}; both fractions elicited a predominantly neutrophilic response. The inflammatory response was reversible, with a peak response to PM{sub 2.5-10} observed at 24 h after instillation, and a return to control values by 72 h after instillation. The major active pro-inflammatory component in whole PM{sub 2.5-10}, but not in whole PM{sub 2.5}, is heat-labile, consistent with it being endotoxin. A heat treatment protocol for the gradual inactivation of biological materials in the PM fractions over a measurable time course was developed and optimized in this study using pure lipopolysaccharide (LPS) as a model system. The time course of heat inactivation of pure LPS and of endotoxin activity in PM{sub 2.5-10} as measured by Limulus bioassay is identical. The active material in both PM{sub 2.5-10} and PM{sub 2.5} remained in the insoluble fraction when the whole PM samples were extracted with physiological saline solution. Histological analysis of lung sections from mice instilled with PM{sub 2.5-10} or PM{sub 2.5} showed evidence of inflammation consistent with the cellular responses observed in lung lavage fluid. The major pro-inflammatory components present in endotoxin-rich PM were found in the insoluble fraction of PM{sub 2.5-10}; however, in contrast with PM{sub 2.5-10} isolated from ambient air in the Central Valley of California, the active components in the insoluble

  14. IDENTIFICATION OF STEREOCHEMICAL CONFIGURATIONS OF CYCLOPENTA[CD]PYRENE-DNA ADDUCTS IN STRAIN A/J MOUSE LUNG AND C3H10T1/2CL8 CELLS

    EPA Science Inventory

    Identification of Sterochemical Configurations of Cyclopent A[cd]Pyrene DNA Adducts in Strain A/J Mouse Lung and C3H10T1/2CL8 Cells.

    Four major and several minor DNA adducts were resolved by 32P-postlabeling analysis of DNA from strain A/J mouse lung and C3H10T1/2CL8 (C3H...

  15. IDENTIFICATION OF STEREOCHEMICAL CONFIGURATIONS OF CYCLOPENTA[CD]PYRENE-DNA ADDUCTS IN STRAIN A/J MOUSE LUNG AND C3H10T1/2CL8 CELLS

    EPA Science Inventory

    Identification of Sterochemical Configurations of Cyclopent A[cd]Pyrene DNA Adducts in Strain A/J Mouse Lung and C3H10T1/2CL8 Cells.

    Four major and several minor DNA adducts were resolved by 32P-postlabeling analysis of DNA from strain A/J mouse lung and C3H10T1/2CL8 (C3H...

  16. Fibroblast radiosensitivity in vitro and lung fibrosis in vivo: Comparison between a fibrosis-prone and fibrosis-resistant mouse strain

    SciTech Connect

    Dileto, C.L.; Travis, E.L.

    1996-07-01

    Radiation-induced pneumonitis and fibrosis in the lung after treatment to the thoracic cavity for malignant disease currently limit the maximum tolerated dose to that region. It has been suggested that heterogeneity in susceptibility to radiation-induced fibrosis exists in the population, implying that the lung tolerance dose is defined by a sensitive subset of the patient population. Studies of radiotherapy patients have indicated that the survival at 2 Gy (SF2) of cultured skin fibroblasts correlates with the incidence and severity of postirradiation damage in a number of tissues, suggesting that this assay may be a useful predictor of late tissue effects. The goal of the studies presented here was to determine if the radiosensitivity of fibroblasts in vitro isolated form mouse lungs was correlated with the severity of radiation induced fibrosis in the lungs of two in red strains of mice previously shown to differ markedly in their susceptibility to radiation-induced lung fibrosis: the C3Hf/Kam strain, classified as fibrosis-resistant, and the C57BL/6J strain, classified as fibrosisprone. Quantitative measurements of lung fibrosis after irradiation were compared to SF 2 values for fibroblasts of skin and lung cultured form each strain. Lung fibrosis was quantified, using computerized image analysis, as the percentage of fibrosis on Masson`s Trichrome-stained lung sections from both strains after single doses of radiation to the thorax. For the measurements of SF2, fibroblasts plated at the second passage and grown to confluence were given single doses of radiation ranging from 0 to 6 Gy. 30 refs., 4 figs., 1 tab.

  17. PR-Set7 is degraded in a conditional Cul4A transgenic mouse model of lung cancer

    SciTech Connect

    Wang, Yang; Xu, Zhidong; Mao, Jian -Hua; Hsieh, David; Au, Alfred; Jablons, David M.; Li, Hui; You, Lian

    2015-06-01

    Background and objective. Maintenance of genomic integrity is essential to ensure normal organismal development and to prevent diseases such as cancer. PR-Set7 (also known as Set8) is a cell cycle regulated enzyme that catalyses monomethylation of histone 4 at Lys20 (H4K20me1) to promote chromosome condensation and prevent DNA damage. Recent studies show that CRL4CDT2-mediated ubiquitylation of PR-Set7 leads to its degradation during S phase and after DNA damage. This might occur to ensure appropriate changes in chromosome structure during the cell cycle or to preserve genome integrity after DNA damage. Methods. We developed a new model of lung tumor development in mice harboring a conditionally expressed allele of Cul4A. We have therefore used a mouse model to demonstrate for the first time that Cul4A is oncogenic in vivo. With this model, staining of PR-Set7 in the preneoplastic and tumor lesions in AdenoCre-induced mouse lungs was performed. Meanwhile we identified higher protein level changes of γ-tubulin and pericentrin by IHC. Results. The level of PR-Set7 down-regulated in the preneoplastic and adenocarcinomous lesions following over-expression of Cul4A. We also identified higher levels of the proteins pericentrin and γ-tubulin in Cul4A mouse lungs induced by AdenoCre. Conclusion. PR-Set7 is a direct target of Cul4A for degradation and involved in the formation of lung tumors in the conditional Cul4A transgenic mouse model.

  18. Histochemical analyses and quantum dot imaging of microvascular blood flow with pulmonary edema in living mouse lungs by "in vivo cryotechnique".

    PubMed

    Saitoh, Yurika; Terada, Nobuo; Saitoh, Sei; Ohno, Nobuhiko; Jin, Takashi; Ohno, Shinichi

    2012-02-01

    Light microscopic imaging of blood vessels and distribution of serum proteins is essential to analyze hemodynamics in living animal lungs under normal respiration or respiratory diseases. In this study, to demonstrate dynamically changing morphology and immunohistochemical images of their living states, "in vivo cryotechnique" (IVCT) combined with freeze-substitution fixation was applied to anesthetized mouse lungs. By hematoxylin-eosin staining, morphological features, such as shapes of alveolar septum and sizes of alveolar lumen, reflected their respiratory conditions in vivo, and alveolar capillaries were filled with variously shaped erythrocytes. Albumin was usually immunolocalized in the capillaries, which was confirmed by double-immunostaining for aquaporin-1 of endothelium. To capture accurate time-courses of blood flow in peripheral pulmonary alveoli, glutathione-coated quantum dots (QDs) were injected into right ventricles, and then IVCT was performed at different time-points after the QD injection. QDs were localized in most arterioles and some alveolar capillaries at 1 s, and later in venules at 2 s, reflecting a typical blood flow direction in vivo. Three-dimensional QD images of microvascular networks were reconstructed by confocal laser scanning microscopy. It was also applied to lungs of acute pulmonary hypertension mouse model. Erythrocytes were crammed in blood vessels, and some serum components leaked into alveolar lumens, as confirmed by mouse albumin immunostaining. Some separated collagen fibers and connecting elastic fibers were still detected in edematous tunica adventitia near terminal bronchioles. Thus, IVCT combined with histochemical approaches enabled us to capture native images of dynamically changing structures and microvascular hemodynamics of living mouse lungs.

  19. Multiple cells-of-origin of mutant K-Ras–induced mouse lung adenocarcinoma

    PubMed Central

    Sutherland, Kate D.; Song, Ji-Ying; Kwon, Min Chul; Proost, Natalie; Zevenhoven, John; Berns, Anton

    2014-01-01

    Much controversy surrounds the cell-of-origin of mutant K-Ras (K-RasG12D)–induced lung adenocarcinoma. To shed light on this issue, we have used technology that enables us to conditionally target K-RasG12D expression in Surfactant Protein C (SPC)+ alveolar type 2 cells and in Clara cell antigen 10 (CC10)+ Clara cells by use of cell-type–restricted recombinant Adeno-Cre viruses. Experiments were performed both in the presence and absence of the tumor suppressor gene p53, enabling us to assess what effect the cell-of-origin and the introduced genetic lesions have on the phenotypic characteristics of the resulting adenocarcinomas. We conclude that both SPC-expressing alveolar type 2 cells and CC10-expressing Clara cells have the ability to initiate malignant transformation following the introduction of these genetic alterations. The lungs of K-Raslox–Stop–lox–G12D/+ and K-Raslox–Stop–lox–G12D/+;tumor suppressor gene Trp53F/F mice infected with Adeno5–SPC–Cre and Adeno5–CC10–Cre viruses displayed differences in their tumor spectrum, indicating distinct cellular routes of tumor initiation. Moreover, using a multicolor Cre reporter line, we demonstrate that the resulting tumors arise from a clonal expansion of switched cells. Taken together, these results indicate that there are multiple cellular paths to K-RasG12D–induced adenocarcinoma and that the initiating cell influences the histopathological phenotype of the tumors that arise. PMID:24586047

  20. Correlation between macrolide lung pharmacokinetics and therapeutic efficacy in a mouse model of pneumococcal pneumonia.

    PubMed

    Veber, B; Vallée, E; Desmonts, J M; Pocidalo, J J; Azoulay-Dupuis, E

    1993-09-01

    The correlation between the pharmacokinetics of erythromycin, roxithromycin, clarithromycin, spiramycin and azithromycin and their efficacy was investigated in two pneumococcal pneumonia models. Female Swiss and C57B1/6 mice were infected with Streptococcus pneumoniae strain P4241 by the intratracheal per oral route. This virulent strain produces acute pneumonia with death within 3-4 days (Swiss mice), or subacute pneumonia with death within 10 days (C57B1/6 mice) in untreated mice and the outcome of the disease is closely related to progressive weight loss. Swiss mice received three doses of each macrolide 50 mg/kg bd beginning 18 h post-infection. C57B1/6 mice received three doses of each macrolide 25 mg/kg, bd (except azithromycin was 12.5 mg/kg bd) beginning 48 h post-infection. Cure rates were evaluated on the basis of body weight variations recorded daily after the end of treatment. Pharmacokinetic parameters were determined in infected and non-infected mice after a single dose of each macrolide 50 mg/kg sc. The pharmacokinetics of azithromycin was also determined in leucopenic Swiss mice. We observed a hierarchy of in-vivo efficacy as follows: azithromycin > spiramycin = clarithromycin > roxithromycin = erythromycin which did not correlate with in-vitro MIC or MBC. The same hierarchy was found in terms of the lung T1/2. Lung T1/2s of macrolides could thus be predictive of their efficacy in respiratory tract infections. A reduced tissue AUC of azithromycin was seen in leucopenic mice suggesting leucocytes may help transport macrolides to sites of infection.

  1. In vivo treatment with aflatoxin B1 increases DNA oxidation, base excision repair activity and 8-oxoguanine DNA glycosylase 1 levels in mouse lung.

    PubMed

    Guindon-Kezis, Katherine A; Mulder, Jeanne E; Massey, Thomas E

    2014-07-03

    Carcinogenicity of the mycotoxin aflatoxin B1 (AFB1), which is produced by Aspergillus fungi, is associated with bioactivation of AFB1 to AFB1-8,9-exo-epoxide and formation of DNA adducts. However, AFB1 also causes 8-hydroxy-2'-deoxyguanosine (8-OHdG) formation in mouse lung DNA, suggesting that oxidative DNA damage may also contribute to AFB1 carcinogenicity. The oxidative DNA damage 5-hydroxy-2'-deoxycytidine (5-OHdC) may also contribute to AFB1 carcinogenicity. The objective of the present study was to determine the effect of treatment of mice with AFB1 on pulmonary and hepatic: 8-OHdG and 5-OHdC levels; base excision repair (BER, which repairs oxidative DNA damage) activities; and on levels of 8-oxoguanine DNA glycosylase (OGG1, the rate-limiting enzyme in the BER of 8-OHdG). Female A/J mice were treated with vehicle (dimethyl sulfoxide) or 50 mg/kg AFB1 ip. Oxidative DNA damage was measured using HPLC with electrochemical detection, BER activity was assessed using an in vitro assay that employs a substrate plasmid DNA with 8-OHdG lesions, and OGG1 protein levels were determined by immunoblotting. Two hours post treatment, AFB1 increased 8-OHdG levels in mouse lung DNA by approximately 69% relative to control (p<0.05), but did not alter 8-OHdG levels in liver or 5-OHdC levels in lung or liver (p>0.05). AFB1 treatment also increased BER activity in mouse lung by approximately 87% (p<0.05) but did not affect hepatic BER activity (p>0.05). Levels of OGG1 immunoreactive protein were increased in both lung (20%) and liver (60%) (p<0.05). These results are consistent with oxidative DNA damage contributing to the carcinogenicity of AFB1 in this model.

  2. Anti-metastatic effect of the TM4SF5-specific peptide vaccine and humanized monoclonal antibody on colon cancer in a mouse lung metastasis model

    PubMed Central

    Park, Byoung Kwon; Park, Sangkyu; Ha, Ji-Hee; Kim, Te Ha; Gautam, Avishekh; Kim, Jung Nam; Lee, Su In; Park, Han-Bum; Kim, Yong-Sung; Kwon, Hyung-Joo; Lee, Younghee

    2016-01-01

    Transmembrane 4 superfamily member 5 protein (TM4SF5) is a potential therapeutic target for hepatocellular carcinoma (HCC) and colon cancer. In a previous study, we demonstrated the prophylactic and therapeutic effects of a TM4SF5-specific peptide vaccine and monoclonal antibody in HCC and colon cancer in a mouse model. Here, we designed a cyclic peptide targeting TM4SF5. Cyclic peptide-specific antibodies were produced in mice after immunization with a complex of the peptide, CpG-DNA, and liposomes. Intravenous injection of the CT-26 mouse colon cancer cell line into mice induced tumors in the lung. Immunization with the peptide vaccine improved the survival rate and reduced the growth of lung tumors. We established a monoclonal antibody specific to the cyclic TM4SF5-based peptide and humanized the antibody sequence by complementarity determining region-grafting. The humanized antibody was reactive to the cyclic peptide and TM4SF5 protein. Treatment of CT-26 cells with the humanized antibody reduced cell motility in vitro. Furthermore, direct injection of the humanized anti-TM4SF5 antibody in vivo reduced growth of lung tumors in mouse metastasis model. Therefore, we conclude that the immunization with the cyclic peptide vaccine and injection of the TM4SF5-specifc humanized antibody have an anti-metastatic effect against colon cancer in mice. Importantly, the humanized antibody may serve as a starting platform for further development and application in clinical settings. PMID:27816969

  3. Granzyme A Is Expressed in Mouse Lungs during Mycobacterium tuberculosis Infection but Does Not Contribute to Protection In Vivo.

    PubMed

    Uranga, Santiago; Marinova, Dessislava; Martin, Carlos; Pardo, Julián; Aguilo, Nacho

    2016-01-01

    Granzyme A, a serine protease expressed in the granules of cytotoxic T and Natural Killer cells, is involved in the generation of pro-inflammatory cytokines by macrophages. Granzyme A has been described to induce in macrophages in vitro the activation of pro-inflammatory pathways that impair intracellular mycobacterial replication. In the present study, we explored the physiological relevance of Granzyme A in the control of pulmonary Mycobacterium tuberculosis infection in vivo. Our results show that, even though Granzyme A is expressed by cytotoxic cells from mouse lungs during pulmonary infection, its deficiency in knockout mice does not have an effect in the control of M. tuberculosis infection. In addition our findings indicate that absence of Granzyme A does not affect the protection conferred by the live-attenuated M. tuberculosis vaccine MTBVAC. Altogether, our findings are in apparent contradiction with previously published in vitro results and suggest that Granzyme A does not have a crucial role in vivo in the protective response to tuberculosis.

  4. Mathematical modeling of tumor cell proliferation kinetics and label retention in a mouse model of lung cancer.

    PubMed

    Zheng, Yanyan; Moore, Helen; Piryatinska, Alexandra; Solis, Trinidad; Sweet-Cordero, E Alejandro

    2013-06-15

    Slowly cycling tumor cells that may be present in human tumors may evade cytotoxic therapies, which tend to be more efficient at destroying cells with faster growth rates. However, the proportion and growth rate of slowly cycling tumor cells is often unknown in preclinical model systems used for drug discovery. Here, we report a quantitative approach to quantitate slowly cycling malignant cells in solid tumors, using a well-established mouse model of Kras-induced lung cancer (Kras(G12D/+)). 5-Bromo-2-deoxyuridine (BrdUrd) was administered to tumor-bearing mice, and samples were collected at defined times during pulse and chase phases. Mathematical and statistical modeling of the label-retention data during the chase phase supported the existence of a slowly cycling label-retaining population in this tumor model and permitted the estimation of its proportion and proliferation rate within a tumor. The doubling time of the slowly cycling population was estimated at approximately 5.7 weeks, and this population represented approximately 31% of the total tumor cells in this model system. The mathematical modeling techniques implemented here may be useful in other tumor models where direct observation of cell-cycle kinetics is difficult and may help evaluate tumor cell subpopulations with distinct cell-cycling rates.

  5. The role of Intelectin-2 in resistance to Ascaris suum lung larval burdens in susceptible and resistant mouse strains.

    PubMed

    Dold, Christina; Pemberton, Alan D; Stafford, Peter; Holland, Celia V; Behnke, Jerzy M

    2011-04-01

    The underlying mechanism of predisposition to Ascaris infection is not yet understood but host genetics are thought to play a fundamental role. We investigated the association between the Intelectin-2 gene and resistance in F2 mice derived from mouse strains known to be susceptible and resistant to infection. Ascaris larvae were isolated from murine lungs and the number of copies of the Intelectin-2 gene was determined in F2 mice. Intelectin-2 gene copy number was not significantly linked to larval burden. In a pilot experiment, the response to infection in parental mice of both sexes was observed in order to address the suitability of female F2 mice. No overall significant sex effect was detected. However, a divergence in resistance/susceptibility status was observed between male and, female hybrid offspring. The responsiveness to Ascaris in mice is likely to be controlled by multiple genes and, despite a unique absence from the susceptible C57BL/6j strain, the Intelectin-2 gene does not play a significant role in resistance. The observed intra-strain variation in larval burden requires further investigation but we hypothesize that it stems from social/dominance hierarchies created by the presence of female mice and possibly subsequent hormonal perturbations that modify the intensity of the immune response.

  6. Granzyme A Is Expressed in Mouse Lungs during Mycobacterium tuberculosis Infection but Does Not Contribute to Protection In Vivo

    PubMed Central

    Uranga, Santiago; Marinova, Dessislava; Martin, Carlos; Pardo, Julián; Aguilo, Nacho

    2016-01-01

    Granzyme A, a serine protease expressed in the granules of cytotoxic T and Natural Killer cells, is involved in the generation of pro-inflammatory cytokines by macrophages. Granzyme A has been described to induce in macrophages in vitro the activation of pro-inflammatory pathways that impair intracellular mycobacterial replication. In the present study, we explored the physiological relevance of Granzyme A in the control of pulmonary Mycobacterium tuberculosis infection in vivo. Our results show that, even though Granzyme A is expressed by cytotoxic cells from mouse lungs during pulmonary infection, its deficiency in knockout mice does not have an effect in the control of M. tuberculosis infection. In addition our findings indicate that absence of Granzyme A does not affect the protection conferred by the live-attenuated M. tuberculosis vaccine MTBVAC. Altogether, our findings are in apparent contradiction with previously published in vitro results and suggest that Granzyme A does not have a crucial role in vivo in the protective response to tuberculosis. PMID:27055232

  7. Nanotitanium dioxide toxicity in mouse lung is reduced in sanding dust from paint.

    PubMed

    Saber, Anne Thoustrup; Jacobsen, Nicklas Raun; Mortensen, Alicia; Szarek, Józef; Jackson, Petra; Madsen, Anne Mette; Jensen, Keld Alstrup; Koponen, Ismo K; Brunborg, Gunnar; Gützkow, Kristine Bjerve; Vogel, Ulla; Wallin, Håkan

    2012-02-02

    Little is known of how the toxicity of nanoparticles is affected by the incorporation in complex matrices. We compared the toxic effects of the titanium dioxide nanoparticle UV-Titan L181 (NanoTiO2), pure or embedded in a paint matrix. We also compared the effects of the same paint with and without NanoTiO2. Mice received a single intratracheal instillation of 18, 54 and 162 μg of NanoTiO2 or 54, 162 and 486 μg of the sanding dust from paint with and without NanoTiO2. DNA damage in broncheoalveolar lavage cells and liver, lung inflammation and liver histology were evaluated 1, 3 and 28 days after intratracheal instillation. Printex 90 was included as positive control. There was no additive effect of adding NanoTiO2 to paints: Therefore the toxicity of NanoTiO2 was reduced by inclusion into a paint matrix. NanoTiO2 induced inflammation in mice with severity similar to Printex 90. The inflammatory response of NanoTiO2 and Printex 90 correlated with the instilled surface area. None of the materials, except of Printex 90, induced DNA damage in lung lining fluid cells. The highest dose of NanoTiO2 caused DNA damage in hepatic tissue 1 day after intratracheal instillation. Exposure of mice to the dust from paints with and without TiO2 was not associated with hepatic histopathological changes. Exposure to NanoTiO2 or to Printex 90 caused slight histopathological changes in the liver in some of the mice at different time points. Pulmonary inflammation and DNA damage and hepatic histopathology were not changed in mice instilled with sanding dust from NanoTiO2 paint compared to paint without NanoTiO2. However, pure NanoTiO2 caused greater inflammation than NanoTiO2 embedded in the paint matrix.

  8. Nanotitanium dioxide toxicity in mouse lung is reduced in sanding dust from paint

    PubMed Central

    2012-01-01

    Background Little is known of how the toxicity of nanoparticles is affected by the incorporation in complex matrices. We compared the toxic effects of the titanium dioxide nanoparticle UV-Titan L181 (NanoTiO2), pure or embedded in a paint matrix. We also compared the effects of the same paint with and without NanoTiO2. Methods Mice received a single intratracheal instillation of 18, 54 and 162 μg of NanoTiO2 or 54, 162 and 486 μg of the sanding dust from paint with and without NanoTiO2. DNA damage in broncheoalveolar lavage cells and liver, lung inflammation and liver histology were evaluated 1, 3 and 28 days after intratracheal instillation. Printex 90 was included as positive control. Results There was no additive effect of adding NanoTiO2 to paints: Therefore the toxicity of NanoTiO2 was reduced by inclusion into a paint matrix. NanoTiO2 induced inflammation in mice with severity similar to Printex 90. The inflammatory response of NanoTiO2 and Printex 90 correlated with the instilled surface area. None of the materials, except of Printex 90, induced DNA damage in lung lining fluid cells. The highest dose of NanoTiO2 caused DNA damage in hepatic tissue 1 day after intratracheal instillation. Exposure of mice to the dust from paints with and without TiO2 was not associated with hepatic histopathological changes. Exposure to NanoTiO2 or to Printex 90 caused slight histopathological changes in the liver in some of the mice at different time points. Conclusions Pulmonary inflammation and DNA damage and hepatic histopathology were not changed in mice instilled with sanding dust from NanoTiO2 paint compared to paint without NanoTiO2. However, pure NanoTiO2 caused greater inflammation than NanoTiO2 embedded in the paint matrix. PMID:22300483

  9. Role of Aquaporin-4 in Airspace-to-Capillary Water Permeability in Intact Mouse Lung Measured by a Novel Gravimetric Method

    PubMed Central

    Song, Yuanlin; Ma, Tonghui; Matthay, Michael A.; Verkman, A.S.

    2000-01-01

    The mammalian peripheral lung contains at least three aquaporin (AQP) water channels: AQP1 in microvascular endothelia, AQP4 in airway epithelia, and AQP5 in alveolar epithelia. In this study, we determined the role of AQP4 in airspace-to-capillary water transport by comparing water permeability in wild-type mice and transgenic null mice lacking AQP1, AQP4, or AQP1/AQP4 together. An apparatus was constructed to measure lung weight continuously during pulmonary artery perfusion of isolated mouse lungs. Osmotically induced water flux (Jv) between the airspace and capillary compartments was measured from the kinetics of lung weight change in saline-filled lungs in response to changes in perfusate osmolality. Jv in wild-type mice varied linearly with osmotic gradient size (4.4 × 10−5 cm3 s−1 mOsm−1) and was symmetric, independent of perfusate osmolyte size, weakly temperature dependent, and decreased 11-fold by AQP1 deletion. Transcapillary osmotic water permeability was greatly reduced by AQP1 deletion, as measured by the same method except that the airspace saline was replaced by an inert perfluorocarbon. Hydrostatically induced lung edema was characterized by lung weight changes in response to changes in pulmonary arterial inflow or pulmonary venous outflow pressure. At 5 cm H2O outflow pressure, the filtration coefficient was 4.7 cm3 s−1 mOsm−1 and reduced 1.4-fold by AQP1 deletion. To study the role of AQP4 in lung water transport, AQP1/AQP4 double knockout mice were generated by crossbreeding of AQP1 and AQP4 null mice. Jv were (cm3 s−1 mOsm−1 × 10−5, SEM, n = 7–12 mice): 3.8 ± 0.4 (wild type), 0.35 ± 0.02 (AQP1 null), 3.7 ± 0.4 (AQP4 null), and 0.25 ± 0.01 (AQP1/AQP4 null). The significant reduction in Pf in AQP1 vs. AQP1/AQP4 null mice was confirmed by an independent pleural surface fluorescence method showing a 1.6 ± 0.2-fold (SEM, five mice) reduced Pf in the AQP1/AQP4 double knockout mice vs. AQP1 null mice. These results establish a

  10. Mesothelial cell proliferation after instillation of long or short asbestos fibers into mouse lung.

    PubMed Central

    Adamson, I. Y.; Bakowska, J.; Bowden, D. H.

    1993-01-01

    The relationship of asbestos deposition in the lung to subsequent cell proliferation at the pleural surface is not clear. The present study examines DNA synthesis by various pulmonary cells, particularly those at the pleura after intratracheal injection of 0.1 mg crocidolite to mice using: 1) long fibers (> 20 mu), which are deposited in bronchiolar regions and induce fibrosis; 2) short fibers (< 1 mu), which reach alveoli but do not induce fibrosis. Mice also received 2 microCi/g tritiated thymidine 1 hour before death at intervals to 16 weeks. Short fibers induced only a small increase in labeling of bronchiolar epithelial and interstitial cells, which subsided by 5 days, when a small increase in labeled mesothelial and subpleural cells was seen. In contrast, long fibers damaged the bronchiolar epithelium and became incorporated into connective tissue. During regeneration, 12% of cells were labeled at 3 days and labeling was greater than controls to 4 weeks. Increased peribronchiolar labeling of fibroblasts and interstitial macrophages was seen around long fibers, and increased DNA synthesis by mesothelial and subpleural cells was found. Up to 2% of mesothelial cells were labeled 1 week after long fibers compared to near zero in controls. No long fibers were found at the pleura. Activation of interstitial macrophages in response to long crocidolite fibers is associated with fibroblast proliferation. It is now suggested that mesothelial cells may also be stimulated by cytokines from activated interstitial macrophages that diffuse across the interstitium, without requiring actual fiber translocation to the pleura. Images Figure 1 Figure 2 Figure 3 Figure 6 Figure 9 Figure 11 PMID:8475994

  11. Beta-adrenergic regulation of secretion in Clara cell adenomas of the mouse lung

    SciTech Connect

    Palmer, K.C.; Grammas, P.

    1986-03-05

    Certain chemically-induced pulmonary adenomas of the mouse have been characterized as being Clara cell in origin. In examining these Clara cell tumors as a model of normal Clara cell biology, they studied the response of tumor cells in vivo to the secretory agonist, isoproterenol (10 mg/kg) and the antagonist propranolol (2.0 mg/kg) 1 hr. following intraperitoneal injection of 120 day old tumor-bearing mice. Ultrastructural morphometry was used to quantitate the secretory response of tumor cells by measuring the volume density of the secretory granules. In the intact animal, isoproterenol stimulated secretion in the Clara cell adenomas (40% decrease in vol. dens. with no change in surface to volume ratio of granules) while propranolol prevented this effect. Beta-adrenergic receptors on isolated tumor Clara cells were demonstrated by radioligand binding assay using /sup 125/I-iodocyanopindolol (ICYP). Scatchard analysis of data derived from whole cells indicates a maximum receptor binding capacity of 27 fmol/mg protein and a K/sub D/ of 0.029 nM. Isoproterenol displacement of ICYP binding yields an IC/sub 50/ of 8 x 10/sup -7/M and a calculated K/sub D/ of 3.36 x 10/sup -7/M. The beta/sub 2/ identity of these receptors was determined utilizing the relatively specific beta/sub 1/ and beta/sub 2/ antagonists practolol and ICI-118,551, respectively. Practolol failed to displace more than 30% of ICYP binding even at 100 ..mu..M, while ICI-118,551 displacement of ICYP yielded a linear (r = 0.93) Hofstee plot and a K/sub D/ of 5.04 x 10/sup -9/M. These findings suggest that the secretory activity of adenoma-derived Clara cells is under beta-adrenergic control similar to that of normal bronchiolar Clara cells.

  12. Lack of contribution of covalent benzo[a]pyrene-7,8-quinone-DNA adducts in benzo[a]pyrene-induced mouse lung tumorigenesis.

    PubMed

    Nesnow, Stephen; Nelson, Garret; Padgett, William T; George, Michael H; Moore, Tanya; King, Leon C; Adams, Linda D; Ross, Jeffrey A

    2010-07-30

    Benzo[a]pyrene (B[a]P) is a potent human and rodent lung carcinogen. This activity has been ascribed in part to the formation of anti-trans-7,8-dihydroxy-7,8-dihydroB[a]P-9,10-epoxide (BPDE)-DNA adducts. Other carcinogenic mechanisms have been proposed: (1) the induction of apurinic sites from radical cation processes, and (2) the metabolic formation of B[a]P-7,8-quinone (BPQ) that can form covalent DNA adducts or reactive oxygen species which can damage DNA. The studies presented here sought to examine the role of stable BPQ-DNA adducts in B[a]P-induced mouse lung tumorigenesis. Male strain A/J mice were injected intraperitoneally once with BPQ or trans-7,8-dihydroxy-7,8-dihydroB[a]P (BP-7,8-diol) at 30, 10, 3, or 0mg/kg. Lungs and livers were harvested after 24h, the DNA extracted and subjected to (32)P-postlabeling analysis. Additional groups of mice were dosed once with BPQ or BP-7,8-diol each at 30 mg/kg and tissues harvested 48 and 72 h later, or with B[a]P (50mg/kg, a tumorigenic dose) and tissues harvested 72 h later. No BPQ or any other DNA adducts were observed in lung or liver tissues 24, 48, or 72 h after the treatment with 30 mg/kg BPQ. BP-7,8-diol gave BPDE-DNA adducts at all time points in both tissues and B[a]P treatment gave BPDE-DNA adducts in the lung. In each case, no BPQ-DNA adducts were detected. Mouse body weights significantly decreased over time after BPQ or BP-7,8-diol treatments suggesting that systemic toxicity was induced by both agents. Model studies with BPQ and N-acetylcysteine suggested that BPQ is rapidly inactivated by sulfhydryl-containing compounds and not available for DNA adduction. We conclude that under these treatment conditions BPQ does not form stable covalent DNA adducts in the lungs or livers of strain A/J mice, suggesting that stable BPQ-covalent adducts are not a part of the complex of mechanisms involved in B[a]P-induced mouse lung tumorigenesis.

  13. Myoblast cytonemes mediate Wg signaling from the wing imaginal disc and Delta-Notch signaling to the air sac primordium

    PubMed Central

    Huang, Hai; Kornberg, Thomas B

    2015-01-01

    The flight muscles, dorsal air sacs, wing blades, and thoracic cuticle of the Drosophila adult function in concert, and their progenitor cells develop together in the wing imaginal disc. The wing disc orchestrates dorsal air sac development by producing decapentaplegic and fibroblast growth factor that travel via specific cytonemes in order to signal to the air sac primordium (ASP). Here, we report that cytonemes also link flight muscle progenitors (myoblasts) to disc cells and to the ASP, enabling myoblasts to relay signaling between the disc and the ASP. Frizzled (Fz)-containing myoblast cytonemes take up Wingless (Wg) from the disc, and Delta (Dl)-containing myoblast cytonemes contribute to Notch activation in the ASP. Wg signaling negatively regulates Dl expression in the myoblasts. These results reveal an essential role for cytonemes in Wg and Notch signaling and for a signal relay system in the myoblasts. DOI: http://dx.doi.org/10.7554/eLife.06114.001 PMID:25951303

  14. 5D imaging via light sheet microscopy reveals cell dynamics during the eye-antenna disc primordium formation in Drosophila

    NASA Astrophysics Data System (ADS)

    Huang, Yu Shan; Ku, Hui Yu; Tsai, Yun Chi; Chang, Chin Hao; Pao, Sih Hua; Sun, Y. Henry; Chiou, Arthur

    2017-03-01

    5D images of engrailed (en) and eye gone (eyg) gene expressions during the course of the eye-antenna disc primordium (EADP) formation of Drosophila embryos from embryonic stages 13 through 16 were recorded via light sheet microscopy and analyzed to reveal the cell dynamics involved in the development of the EADP. Detailed analysis of the time-lapsed images revealed the process of EADP formation and its invagination trajectory, which involved an inversion of the EADP anterior-posterior axis relative to the body. Furthermore, analysis of the en-expression pattern in the EADP provided strong evidence that the EADP is derived from one of the en-expressing head segments.

  15. 5D imaging via light sheet microscopy reveals cell dynamics during the eye-antenna disc primordium formation in Drosophila

    PubMed Central

    Huang, Yu Shan; Ku, Hui Yu; Tsai, Yun Chi; Chang, Chin Hao; Pao, Sih Hua; Sun, Y. Henry; Chiou, Arthur

    2017-01-01

    5D images of engrailed (en) and eye gone (eyg) gene expressions during the course of the eye-antenna disc primordium (EADP) formation of Drosophila embryos from embryonic stages 13 through 16 were recorded via light sheet microscopy and analyzed to reveal the cell dynamics involved in the development of the EADP. Detailed analysis of the time-lapsed images revealed the process of EADP formation and its invagination trajectory, which involved an inversion of the EADP anterior-posterior axis relative to the body. Furthermore, analysis of the en-expression pattern in the EADP provided strong evidence that the EADP is derived from one of the en-expressing head segments. PMID:28322328

  16. Phosphate starvation of maize inhibits lateral root formation and alters gene expression in the lateral root primordium zone

    PubMed Central

    2012-01-01

    Background Phosphorus (P) is an essential macronutrient for all living organisms. Maize (Zea mays) is an important human food, animal feed and energy crop throughout the world, and enormous quantities of phosphate fertilizer are required for maize cultivation. Thus, it is important to improve the efficiency of the use of phosphate fertilizer for maize. Results In this study, we analyzed the maize root response to phosphate starvation and performed a transcriptomic analysis of the 1.0-1.5 cm lateral root primordium zone. In the growth of plants, the root-to-shoot ratio (R/L) was reduced in both low-phosphate (LP) and sufficient-phosphate (SP) solutions, but the ratio (R/L) exhibited by the plants in the LP solution was higher than that of the SP plants. The growth of primary roots was slightly promoted after 6 days of phosphate starvation, whereas the numbers of lateral roots and lateral root primordia were significantly reduced, and these differences were increased when associated with the stress caused by phosphate starvation. Among the results of a transcriptomic analysis of the maize lateral root primordium zone, there were two highlights: 1) auxin signaling participated in the response and the modification of root morphology under low-phosphate conditions, which may occur via local concentration changes due to the biosynthesis and transport of auxin, and LOB domain proteins may be an intermediary between auxin signaling and root morphology; and 2) the observed retardation of lateral root development was the result of co-regulation of DNA replication, transcription, protein synthesis and degradation and cell growth. Conclusions These results indicated that maize roots show a different growth pattern than Arabidopsis under low-phosphate conditions, as the latter species has been observed to halt primary root growth when the root tip comes into contact with low-phosphate media. Moreover, our findings enrich our understanding of plant responses to phosphate

  17. Shape-based tracking allows functional discrimination of two immune cell subsets expressing the same fluorescent tag in mouse lung explant.

    PubMed

    Fiole, Daniel; Touvrey, Cédric; Quesnel-Hellmann, Anne; Douady, Julien; Tournier, Jean-Nicolas

    2012-01-01

    Dendritic Cells (DC) represent a key lung immune cell population, which play a critical role in the antigen presenting process and initiation of the adaptive immune response. The study of DCs has largely benefited from the joint development of fluorescence microscopy and knock-in technology, leading to several mouse strains with constitutively labeled DC subsets. However, in the lung most transgenic mice do express fluorescent protein not only in DCs, but also in closely related cell lineages such as monocytes and macrophages. As an example, in the lungs of CX(3)CR1(+/gfp) mice the green fluorescent protein is expressed mostly by both CD11b conventional DCs and resident monocytes. Despite this non-specific staining, we show that a shape criterion can discriminate these two particular subsets. Implemented in a cell tracking code, this quantified criterion allows us to analyze the specific behavior of DCs under inflammatory conditions mediated by lipopolysaccharide on lung explants. Compared to monocytes, we show that DCs move slower and are more confined, while both populations do not have any chemotactism-associated movement. We could generalize from these results that DCs can be automatically discriminated from other round-shaped cells expressing the same fluorescent protein in various lung inflammation models.

  18. Persistence of lung CD8 T cell oligoclonal expansions upon smoking cessation in a mouse model of cigarette smoke-induced emphysema.

    PubMed

    Motz, Gregory T; Eppert, Bryan L; Sun, Guangyun; Wesselkamper, Scott C; Linke, Michael J; Deka, Ranjan; Borchers, Michael T

    2008-12-01

    The role of adaptive immunity in the development or progression of chronic obstructive pulmonary disease (COPD) remains undefined. Recently, the presence of autoantibodies and autoreactive T cells has been demonstrated in COPD patients. In addition, oligoclonal expansions of lung T cells have been observed in COPD patients, but the overlapping incidence of infections, tumors, and cigarette smoke exposure obscures the antigenic stimulus. We analyzed the TCR Vbeta repertoire of CD4 and CD8 T cells purified from the lungs and spleens of mice chronically exposed to cigarette smoke. In a mouse model of COPD, we demonstrate that chronic cigarette smoke exposure causes oligoclonal expansions of T cells isolated from the lungs, but not spleens. TCR Vbeta repertoire analyses revealed oligoclonal expansions predominantly occurred in lung CD8 T cells, with preferential usage of Vbeta7, Vbeta9, Vbeta13, and Vbeta14. Using nucleotide sequence analysis based on Jbeta analyses, we demonstrate selection of CDR3 amino acid motifs, which strongly suggests Ag-driven oligoclonal T cell expansion. Analysis of the lung TCR Vbeta repertoire of mice with cigarette smoke-induced emphysema, which had undergone smoking cessation for 6 mo, revealed that oligoclonal expansions persisted. This study formally demonstrates that chronic cigarette smoke exposure, alone, causes a persistent adaptive T cell immune response. These findings have important implications for therapeutic approaches in the treatment of COPD, and provide insight into potential mechanisms involved in disease pathogenesis.

  19. Shape-Based Tracking Allows Functional Discrimination of Two Immune Cell Subsets Expressing the Same Fluorescent Tag in Mouse Lung Explant

    PubMed Central

    Fiole, Daniel; Touvrey, Cédric; Quesnel-Hellmann, Anne

    2012-01-01

    Dendritic Cells (DC) represent a key lung immune cell population, which play a critical role in the antigen presenting process and initiation of the adaptive immune response. The study of DCs has largely benefited from the joint development of fluorescence microscopy and knock-in technology, leading to several mouse strains with constitutively labeled DC subsets. However, in the lung most transgenic mice do express fluorescent protein not only in DCs, but also in closely related cell lineages such as monocytes and macrophages. As an example, in the lungs of CX3CR1+/gfp mice the green fluorescent protein is expressed mostly by both CD11b conventional DCs and resident monocytes. Despite this non-specific staining, we show that a shape criterion can discriminate these two particular subsets. Implemented in a cell tracking code, this quantified criterion allows us to analyze the specific behavior of DCs under inflammatory conditions mediated by lipopolysaccharide on lung explants. Compared to monocytes, we show that DCs move slower and are more confined, while both populations do not have any chemotactism-associated movement. We could generalize from these results that DCs can be automatically discriminated from other round-shaped cells expressing the same fluorescent protein in various lung inflammation models. PMID:22745831

  20. Response of the mouse lung transcriptome to welding fume: effects of stainless and mild steel fumes on lung gene expression in A/J and C57BL/6J mice

    PubMed Central

    2010-01-01

    Background Debate exists as to whether welding fume is carcinogenic, but epidemiological evidence suggests that welders are an at risk population for the development of lung cancer. Recently, we found that exposure to welding fume caused an acutely greater and prolonged lung inflammatory response in lung tumor susceptible A/J versus resistant C57BL/6J (B6) mice and a trend for increased tumor incidence after stainless steel (SS) fume exposure. Here, our objective was to examine potential strain-dependent differences in the regulation and resolution of the lung inflammatory response induced by carcinogenic (Cr and Ni abundant) or non-carcinogenic (iron abundant) metal-containing welding fumes at the transcriptome level. Methods Mice were exposed four times by pharyngeal aspiration to 5 mg/kg iron abundant gas metal arc-mild steel (GMA-MS), Cr and Ni abundant GMA-SS fume or vehicle and were euthanized 4 and 16 weeks after the last exposure. Whole lung microarray using Illumina Mouse Ref-8 expression beadchips was done. Results Overall, we found that tumor susceptibility was associated with a more marked transcriptional response to both GMA-MS and -SS welding fumes. Also, Ingenuity Pathway Analysis revealed that gene regulation and expression in the top molecular networks differed between the strains at both time points post-exposure. Interestingly, a common finding between the strains was that GMA-MS fume exposure altered behavioral gene networks. In contrast, GMA-SS fume exposure chronically upregulated chemotactic and immunomodulatory genes such as CCL3, CCL4, CXCL2, and MMP12 in the A/J strain. In the GMA-SS-exposed B6 mouse, genes that initially downregulated cellular movement, hematological system development/function and immune response were involved at both time points post-exposure. However, at 16 weeks, a transcriptional switch to an upregulation for neutrophil chemotactic genes was found and included genes such as S100A8, S100A9 and MMP9. Conclusions

  1. A3 and P2Y2 receptors control the recruitment of neutrophils to the lungs in a mouse model of sepsis.

    PubMed

    Inoue, Yoshiaki; Chen, Yu; Hirsh, Mark I; Yip, Linda; Junger, Wolfgang G

    2008-08-01

    We have recently shown that A3 adenosine receptors and P2Y2 purinergic receptors play an important role in neutrophil chemotaxis. Chemotaxis of neutrophils to sites of infections is critical for immune defense. However, excessive accumulation of neutrophils in the lungs can cause acute lung tissue damage. Here we assessed the role of A3 and P2Y2 receptors in neutrophil sequestration to the lungs in a mouse model of sepsis. Sepsis was induced by cecal ligation and puncture (CLP) using adult male C57BL/6J mice (wild type [WT]), homozygous A3 receptor knockout (A3KO) mice, and P2Y2 receptor knockout (P2Y2KO) mice. Animals were killed 2, 4, 6, or 8 h after CLP, and peritoneal lavage fluid and blood were collected. Lungs were removed, and neutrophil infiltration was evaluated using elastase as a marker. Leukocyte and bacterial counts in peritoneal lavage fluid and blood samples were determined. Survival after sepsis was determined in a separate group. Leukocyte counts in the peritoneum were lower in A3KO and P2Y2KO mice than in WT mice. Conversely, initial leukocyte counts in the peripheral blood were higher in KO mice than in WT mice. Neutrophil sequestration to the lungs reached a maximum 2 h after CLP and remained significantly higher in WT mice compared with A3KO and P2Y2KO mice (P < 0.001). Survival after 24 h was significantly lower in WT mice (37.5%) than in A3KO or P2Y2KO mice (82.5%; P < 0.05). These data suggest that A3 and P2Y2 receptors are involved in the influx of neutrophils into the lungs after sepsis. Thus, pharmaceutical approaches that target these receptors might be useful to control acute lung tissue injury in sepsis.

  2. siRNA-Mediated RNA Interference in Precision-Cut Tissue Slices Prepared from Mouse Lung and Kidney.

    PubMed

    Ruigrok, Mitchel J R; Maggan, Nalinie; Willaert, Delphine; Frijlink, Henderik W; Melgert, Barbro N; Olinga, Peter; Hinrichs, Wouter L J

    2017-09-11

    Small interfering RNA (siRNA)-mediated RNAi interference (RNAi) is a powerful post-transcriptional gene silencing mechanism which can be used to study the function of genes in vitro (cell cultures) and in vivo (animal models). However, there is a translational gap between these models. Hence, there is a need for novel experimental models that combine the advantages of in vitro and in vivo models (e.g., simplicity, flexibility, throughput, and representability) to study the effects of siRNA. This need may be addressed by precision-cut tissue slices (PCTS), which represent an ex vivo model that mimics the structural and functional characteristics of a whole organ. The goal of this study was to investigate whether self-deliverable siRNA (Accell siRNA) can be used in precision-cut lung slices (PCLuS) and precision-cut kidney slices (PCKS) to achieve RNAi ex vivo. PCLuS and PCKS were prepared from mouse tissue, and they were subsequently incubated up to 48 h with no siRNA (untransfected), non-targeting Accell siRNA, or Gapdh-targeting Accell siRNA. Significant Gapdh mRNA silencing was achieved (PCLuS ~ 55%; PCKS ~ 40%) without compromising the viability and morphology of slices. Fluorescence microscopy confirmed that Accell siRNA diffused into PCLuS and PCKS. Spontaneous inflammation upon incubation was observed in PCLuS and PCKS as shown by a higher mRNA expression of pro-inflammatory cytokines Il1b, Il6, and Tnfa, although Accell siRNA appeared to diminish this response in PCLuS after 24 h. In conclusion, this ex vivo transfection model can be used to evaluate the effects of siRNA in relevant biological environments.

  3. The complement C3a receptor is critical in defense against Chlamydia psittaci in mouse lung infection and required for antibody and optimal T cell response.

    PubMed

    Dutow, Pavel; Fehlhaber, Beate; Bode, Jenny; Laudeley, Robert; Rheinheimer, Claudia; Glage, Silke; Wetsel, Rick A; Pabst, Oliver; Klos, Andreas

    2014-04-15

    The complement system protects against extracellular pathogens and links innate and adaptive immunity. In this study, we investigated the anaphylatoxin C3a receptor (C3aR) in Chlamydia psittaci lung infection and elucidated C3a-dependent adaptive immune mechanisms. Survival, body weight, and clinical score were monitored in primary mouse infection and after serum transfer. Bacterial load, histology, cellular distribution, cytokines, antibodies, and lymphocytes were analyzed. C3aR(-/-) mice showed prolonged pneumonia with decreased survival, lower weight, and higher clinical score. Compared to wild-type mice bacterial clearance was impaired, and inflammatory parameters were increased. In lung-draining lymph nodes of C3aR(-/-) mice the total number of B cells, CD4(+) T cells, and Chlamydia-specific IFN-γ(+) (CD4(+) or CD8(+)) cells was reduced upon infection, and the mice were incapable of Chlamydia-specific immunoglobulin M or immunoglobulin G production. Performed before infection, transfer of hyperimmune serum prolonged survival of C3aR(-/-) mice. C3a and its receptor are critical for defense against C. psittaci in mouse lung infection. In this model, C3a acts via its receptor as immune modulator. Enhancement of specific B and T cell responses upon infection with an intracellular bacterium were identified as hitherto unknown features of C3a/C3aR. These new functions might be of general immunological importance.

  4. Optical coherence tomography-based contact indentation for diaphragm mechanics in a mouse model of transforming growth factor alpha induced lung disease.

    PubMed

    Wang, Kimberley C W; Astell, Chrissie J; Wijesinghe, Philip; Larcombe, Alexander N; Pinniger, Gavin J; Zosky, Graeme R; Kennedy, Brendan F; Berry, Luke J; Sampson, David D; James, Alan L; Le Cras, Timothy D; Noble, Peter B

    2017-05-04

    This study tested the utility of optical coherence tomography (OCT)-based indentation to assess mechanical properties of respiratory tissues in disease. Using OCT-based indentation, the elastic modulus of mouse diaphragm was measured from changes in diaphragm thickness in response to an applied force provided by an indenter. We used a transgenic mouse model of chronic lung disease induced by the overexpression of transforming growth factor-alpha (TGF-α), established by the presence of pleural and peribronchial fibrosis and impaired lung mechanics determined by the forced oscillation technique and plethysmography. Diaphragm elastic modulus assessed by OCT-based indentation was reduced by TGF-α at both left and right lateral locations (p < 0.05). Diaphragm elastic modulus at left and right lateral locations were correlated within mice (r = 0.67, p < 0.01) suggesting that measurements were representative of tissue beyond the indenter field. Co-localised images of diaphragm after TGF-α overexpression revealed a layered fibrotic appearance. Maximum diaphragm force in conventional organ bath studies was also reduced by TGF-α overexpression (p < 0.01). Results show that OCT-based indentation provided clear delineation of diseased diaphragm, and together with organ bath assessment, provides new evidence suggesting that TGF-α overexpression produces impairment in diaphragm function and, therefore, an increase in the work of breathing in chronic lung disease.

  5. The role of hypoxia and neurogenic genes (Mash-1 and Prox-1) in the developmental programming and maturation of pulmonary neuroendocrine cells in fetal mouse lung.

    PubMed

    McGovern, Suzanne; Pan, Jie; Oliver, Guillermo; Cutz, Ernest; Yeger, Herman

    2010-02-01

    Pulmonary neuroendocrine cells (PNECs) are the first cell type to differentiate within the primitive airway epithelium, suggesting a possible role in lung development. The differentiation of PNECs in fetal lung is governed by proneural genes such as the mammalian homolog of the achaete-scute complex (Mash-1) and a related transcription factor, hairy and enhancer of split1 (Hes-1). We examined the expression of Mash-1 and a downstream transcription factor Prox-1 in the developing mouse lung of wild-type and respective knockout mouse models. During early stages (embryonic day 12, E12) of development, only some PNECs expressed Mash-1 and Prox-1, but by E15, all PNECs coexpressed both transcription factors. PNECs failed to develop in Mash-1 but not in Prox-1-null mice, indicating that Mash-1 is essential for the initiation of the PNEC phenotype, whereas Prox-1 is associated with the development of this phenotype. As lung develops within a low O(2) environment (fetal euoxia, pO(2) approximately 20 to 30 mm Hg), we examined the effects of hypoxia on PNEC differentiation. Organ cultures of fetal mouse lungs at E12 and E16 were maintained under either 20% O(2) (normoxia, Nox) or 5% O(2) (hypoxia, Hox) and were examined every 24 h for up to 6 days in culture. In E12 explants, Hox enhanced branching morphogenesis and increased cell proliferation, but PNEC numbers and Mash-1 expression were significantly reduced. This effect could be reversed by switching the explants back to Nox. In contrast, Hox had no apparent effect on Hes-1 expression. Similarly, Hox had no effect on airway branching, PNEC numbers, or Mash-1 expression in E16 explants, indicating locked-in developmental programming. We suggest that during early stages of lung development, pO(2) concentration in concert with neurogenic gene expression modulates PNEC phenotype. Thus, disturbances in intrauterine pO(2) homeostasis could alter the functional maturation of the PNEC system and hence be involved in the

  6. Up-regulation of nucleotide excision repair in mouse lung and liver following chronic exposure to aflatoxin B{sub 1} and its dependence on p53 genotype

    SciTech Connect

    Mulder, Jeanne E.; Bondy, Genevieve S.; Mehta, Rekha; Massey, Thomas E.

    2014-03-01

    Aflatoxin B{sub 1} (AFB{sub 1}) is biotransformed in vivo into an epoxide metabolite that forms DNA adducts that may induce cancer if not repaired. p53 is a tumor suppressor gene implicated in the regulation of global nucleotide excision repair (NER). Male heterozygous p53 knockout (B6.129-Trp53{sup tm1Brd}N5, Taconic) and wild-type mice were exposed to 0, 0.2 or 1.0 ppm AFB{sub 1} for 26 weeks. NER activity was assessed with an in vitro assay, using AFB{sub 1}-epoxide adducted plasmid DNA as a substrate. For wild-type mice, repair of AFB{sub 1}–N7-Gua adducts was 124% and 96% greater in lung extracts from mice exposed to 0.2 ppm and 1.0 ppm AFB{sub 1} respectively, and 224% greater in liver extracts from mice exposed to 0.2 ppm AFB{sub 1} (p < 0.05). In heterozygous p53 knockout mice, repair of AFB{sub 1}–N7-Gua was only 45% greater in lung extracts from mice exposed to 0.2 ppm AFB{sub 1} (p < 0.05), and no effect was observed in lung extracts from mice treated with 1.0 ppm AFB{sub 1} or in liver extracts from mice treated with either AFB{sub 1} concentration. p53 genotype did not affect basal levels of repair. AFB{sub 1} exposure did not alter repair of AFB{sub 1}-derived formamidopyrimidine adducts in lung or liver extracts of either mouse genotype nor did it affect XPA or XPB protein levels. In summary, chronic exposure to AFB{sub 1} increased NER activity in wild-type mice, and this response was diminished in heterozygous p53 knockout mice, indicating that loss of one allele of p53 limits the ability of NER to be up-regulated in response to DNA damage. - Highlights: • Mice are chronically exposed to low doses of the mycotoxin aflatoxin B{sub 1} (AFB{sub 1}). • The effects of AFB{sub 1} and p53 status on nucleotide excision repair are investigated. • AFB{sub 1} increases nucleotide excision repair in wild type mouse lung and liver. • This increase is attenuated in p53 heterozygous mouse lung and liver. • Results portray the role of p53 in

  7. Impact and mechanism of non-steroidal anti-inflammatory drugs combined with chemotherapeutic drugs on human lung cancer-nude mouse transplanted tumors

    PubMed Central

    SUN, WEIYI; CHEN, GANG

    2016-01-01

    The present study aimed to investigate the impact of indomethacin treatment combined with oxaliplatin treatment on the expression of cluster of differentiation 44 variant 6 (CD44v6), matrix metalloproteinase-2 (MMP-2) and survivin in human lung cancer-nude mouse transplanted tumors. The human lung adenocarcinoma (A549)-nude mouse transplanted tumor model was established, and the mice were divided into a control group, an indomethacin treatment group, an oxaliplatin treatment group and an indomethacin-oxaliplatin combination treatment group. The tumor inhibition rate was calculated following sacrificing of the mice. Immunohistochemical staining and fluorescence reverse transcription-quantitative polymerase chain reaction were utilized to detect the protein and messenger (m)RNA expression of CD44v6, MMP-2 and survivin. The tumor inhibition rates of the indomethacin group, the oxaliplatin group and the combination group were 26.67, 47.70 and 68.88%, respectively. The protein and mRNA expression levels of CD44v6, MMP-2 and survivin in the transplanted tumors of each treatment group were reduced compared with the control group (P<0.05), and those of the combination group were lower compared with the single-drug treatment groups (P<0.05). Survivin and MMP-2, MMP-2 and CD44v6, and MMP-2 and CD44v6 all exhibited linear positive correlation. The present study provides evidence that the administration of indomethacin alone, or in combination with oxaliplatin, may significantly inhibit the growth of lung cancer-nude mouse transplanted tumors and the expression of CD44v6, MMP-2 and survivin inside the tumor. The combination of non-steroidal anti-inflammatory drugs with chemotherapeutic drugs may improve the antitumor effects. PMID:27313765

  8. Tumor growth affects the metabonomic phenotypes of multiple mouse non-involved organs in an A549 lung cancer xenograft model

    PubMed Central

    Xu, Shan; Tian, Yuan; Hu, Yili; Zhang, Nijia; Hu, Sheng; Song, Dandan; Wu, Zhengshun; Wang, Yulan; Cui, Yanfang; Tang, Huiru

    2016-01-01

    The effects of tumorigenesis and tumor growth on the non-involved organs remain poorly understood although many research efforts have already been made for understanding the metabolic phenotypes of various tumors. To better the situation, we systematically analyzed the metabolic phenotypes of multiple non-involved mouse organ tissues (heart, liver, spleen, lung and kidney) in an A549 lung cancer xenograft model at two different tumor-growth stages using the NMR-based metabonomics approaches. We found that tumor growth caused significant metabonomic changes in multiple non-involved organ tissues involving numerous metabolic pathways, including glycolysis, TCA cycle and metabolisms of amino acids, fatty acids, choline and nucleic acids. Amongst these, the common effects are enhanced glycolysis and nucleoside/nucleotide metabolisms. These findings provided essential biochemistry information about the effects of tumor growth on the non-involved organs. PMID:27329570

  9. The roles of diol epoxide and o-quinone pathways in mouse lung tumorigenesis induced by benzo(a)pyrene: relevance to human lung carcinogenesis

    EPA Science Inventory

    There is sufficient epidemiological evidence supported by experimental data that some PAH-containing complex environmental mixtures pose risks to human health by increasing lung cancer incidence. The International Agency for Research on Cancer has determined that human respirator...

  10. The roles of diol epoxide and o-quinone pathways in mouse lung tumorigenesis induced by benzo(a)pyrene: relevance to human lung carcinogenesis

    EPA Science Inventory

    There is sufficient epidemiological evidence supported by experimental data that some PAH-containing complex environmental mixtures pose risks to human health by increasing lung cancer incidence. The International Agency for Research on Cancer has determined that human respirator...

  11. Growth and Metastases of Human Lung Cancer Are Inhibited in Mouse Xenografts by a Transition State Analogue of 5′-Methylthioadenosine Phosphorylase*

    PubMed Central

    Basu, Indranil; Locker, Joseph; Cassera, Maria B.; Belbin, Thomas J.; Merino, Emilio F.; Dong, Xinyuan; Hemeon, Ivan; Evans, Gary B.; Guha, Chandan; Schramm, Vern L.

    2011-01-01

    The S-adenosylmethionine (AdoMet) salvage enzyme 5′-methylthioadenosine phosphorylase (MTAP) has been implicated as both a cancer target and a tumor suppressor. We tested these hypotheses in mouse xenografts of human lung cancers. AdoMet recycling from 5′-methylthioadenosine (MTA) was blocked by inhibition of MTAP with methylthio-DADMe-Immucillin-A (MTDIA), an orally available, nontoxic, picomolar transition state analogue. Blood, urine, and tumor levels of MTA increased in response to MTDIA treatment. MTDIA treatment inhibited A549 (human non-small cell lung carcinoma) and H358 (human bronchioloalveolar non-small cell lung carcinoma cells) xenograft tumor growth in immunodeficient Rag2−/−γC−/− and NCr-nu mice. Systemic MTA accumulation is implicated as the tumor-suppressive metabolite because MTDIA is effective for in vivo treatment of A549 MTAP−/− and H358 MTAP+/+ tumors. Tumors from treated mice showed increased MTA and decreased polyamines but little alteration in AdoMet, methionine, or adenine levels. Gene expression profiles of A549 tumors from treated and untreated mice revealed only modest alterations with 62 up-regulated and 63 down-regulated mRNAs (≥3-fold). MTDIA antitumor activity in xenografts supports MTAP as a target for lung cancer therapy. PMID:21135097

  12. Sucrose stimulates branching morphogenesis of embryonic mouse lung in vitro: a problem of osmotic balance between lumen fluid and culture medium.

    PubMed

    Nogawa, Hiroyuki; Hasegawa, Yoko

    2002-10-01

    In organ cultures of lung rudiments from 11-day mouse embryos, it was found that addition of sucrose to the culture medium stimulated branching morphogenesis and reduced lumen distension. Two possible roles of sucrose were postulated: one as a nutrient and another as a generator of osmotic pressure inducing osmosis of water from the lumen fluid to the culture medium across a simple columnar epithelial cell layer. To assess which was the case, branching morphogenesis was investigated in lung rudiments cultured in medium in which osmotic pressure was increased by the addition of lactose or NaCl rather than sucrose: similar acceleration of branching was observed in both. In another experiment, lumen fluid of cultured lung rudiments was mechanically drained each day, and significantly stimulated branching morphogenesis was observed even when sucrose was not added to the culture medium. Heparin is known to induce abnormal lumen distension and inhibits branching morphogenesis. Heparin-induced abnormal morphogenesis was prevented either by the addition of sucrose to the culture medium or by the mechanical drainage of lumen fluid. These results suggest that lumen distension caused by the accumulation of lumen fluid disrupts lung branching morphogenesis in vitro, even when the mechanism of branching morphogenesis is intact.

  13. Benzo[b]fluoranthene: tumorigenicity in strain A/J mouse lungs, DNA adducts and mutations in the Ki-ras oncogene.

    PubMed

    Mass, M J; Abu-Shakra, A; Roop, B C; Nelson, G; Galati, A J; Stoner, G D; Nesnow, S; Ross, J A

    1996-08-01

    The polycyclic aromatic hydrocarbon benzo[b]fluoranthene (B[b]F) is a pervasive constituent of environmental combustion products. We sought to examine the lung tumorigenic activity of B[b]F in strain A/J mice, to study the relationship between formation and decay of B[b]F-DNA adducts and to examine mutations in the Ki-ras proto-oncogene in DNA from B[b]F-induced tumors. Mice were given i.p. injections of 0, 10, 50, 100 or 200 mg/kg body wt and lung adenomas were scored after 8 months. B[b]F induced significant numbers of mouse lung adenomas in a dose-related fashion, with the highest dose (200 mg/kg) yielding 6.95 adenomas/ mouse, with 100% of the mice exhibiting an adenoma. In mice given tricaprylin, the vehicle control, there were 0.60 adenomas/mouse, with 55% of the mice exhibiting an adenoma. Based on dose, B[b]F was less active than benzo[a]pyrene. DNA adducts were analyzed qualitatively and quantitatively by 32P-post-labeling in lungs of strain A/J mice 1, 3, 5, 7, 14 and 21 days after i.p. injection. Maximal levels of adduction occurred 5 days after treatment with the 200 mg/kg dose group, producing 1230 amol B[b]F-DNA adducts/microgram DNA. The major B[b]F-DNA adduct was identified by co-chromatography as trans-9, 10-dihydroxy-anti-11, 12-epoxy-5-hydroxy-9, 10, 11, 12-tetra-hydro-B[b]F-deoxyguanosine. Approximately 86% of the tumors had a mutation in codon 12 of the Ki-ras oncogene, as determined by direct DNA sequencing of PCR-amplified exon 1 and single-stranded conformation polymorphism analysis. Analysis of the Ki-ras mutation spectrum in 25 of 29 B[b]F-induced tumors revealed the predominant mutation to be a G-->T transversion in the first or second base of codon 12, congruous with the DNA adduct data. Our data are consistent with previous reports in mouse skin implicating a phenolic diol epoxide as the proximate carcinogenic form of B[b]F that binds to guanine.

  14. Decoding c-Myc networks of cell cycle and apoptosis regulated genes in a transgenic mouse model of papillary lung adenocarcinomas

    PubMed Central

    Ciribilli, Yari; Singh, Prashant; Spanel, Reinhard; Inga, Alberto; Borlak, Jürgen

    2015-01-01

    The c-Myc gene codes for a basic-helix-loop-helix-leucine zipper transcription factor protein and is reported to be frequently over-expressed in human cancers. Given that c-Myc plays an essential role in neoplastic transformation we wished to define its activity in lung cancer and therefore studied its targeted expression to respiratory epithelium in a transgenic mouse disease model. Using histological well-defined tumors, transcriptome analysis identified novel c-Myc responsive cell cycle and apoptosis genes that were validated as direct c-Myc targets using EMSA, Western blotting, gene reporter and ChIP assays. Through computational analyses c-Myc cooperating transcription factors emerged for repressed and up-regulated genes in cancer samples, namely Klf7, Gata3, Sox18, p53 and Elf5 and Cebpα, respectively. Conversely, at promoters of genes regulated in transgenic but non-carcinomatous lung tissue enriched binding sites for c-Myc, Hbp1, Hif1 were observed. Bioinformatic analysis of tumor transcriptomic data revealed regulatory gene networks and highlighted mortalin and moesin as master regulators while gene reporter and ChIP assays in the H1299 lung cancer cell line as well as cross-examination of published ChIP-sequence data of 7 human and 2 mouse cell lines provided strong evidence for the identified genes to be c-Myc targets. The clinical significance of findings was established by evaluating expression of orthologous proteins in human lung cancer. Taken collectively, a molecular circuit for c-Myc-dependent cellular transformation was identified and the network analysis broadened the perspective for molecularly targeted therapies. PMID:26427040

  15. Decoding c-Myc networks of cell cycle and apoptosis regulated genes in a transgenic mouse model of papillary lung adenocarcinomas.

    PubMed

    Ciribilli, Yari; Singh, Prashant; Spanel, Reinhard; Inga, Alberto; Borlak, Jürgen

    2015-10-13

    The c-Myc gene codes for a basic-helix-loop-helix-leucine zipper transcription factor protein and is reported to be frequently over-expressed in human cancers. Given that c-Myc plays an essential role in neoplastic transformation we wished to define its activity in lung cancer and therefore studied its targeted expression to respiratory epithelium in a transgenic mouse disease model. Using histological well-defined tumors, transcriptome analysis identified novel c-Myc responsive cell cycle and apoptosis genes that were validated as direct c-Myc targets using EMSA, Western blotting, gene reporter and ChIP assays.Through computational analyses c-Myc cooperating transcription factors emerged for repressed and up-regulated genes in cancer samples, namely Klf7, Gata3, Sox18, p53 and Elf5 and Cebpα, respectively. Conversely, at promoters of genes regulated in transgenic but non-carcinomatous lung tissue enriched binding sites for c-Myc, Hbp1, Hif1 were observed. Bioinformatic analysis of tumor transcriptomic data revealed regulatory gene networks and highlighted mortalin and moesin as master regulators while gene reporter and ChIP assays in the H1299 lung cancer cell line as well as cross-examination of published ChIP-sequence data of 7 human and 2 mouse cell lines provided strong evidence for the identified genes to be c-Myc targets. The clinical significance of findings was established by evaluating expression of orthologous proteins in human lung cancer. Taken collectively, a molecular circuit for c-Myc-dependent cellular transformation was identified and the network analysis broadened the perspective for molecularly targeted therapies.

  16. Do variations in mast cell hyperplasia account for differences in radiation-induced lung injury among different mouse strains, rats and nonhuman primates?

    PubMed

    Down, Julian D; Medhora, Meetha; Jackson, Isabel L; Cline, J Mark; Vujaskovic, Zeljko

    2013-08-01

    The role of mast cell infiltrates in the pathology of radiation damage to the lung has been a subject of continuing investigation over the past four decades. This has been accompanied by a number of proposals as to how mast cells and the secretory products thereof participate in the generation of acute inflammation (pneumonitis) and the chronic process of collagen deposition (fibrosis). An additional pathophysiology examines the possible connection between mast cell hyperplasia and pulmonary hypertension through the release of vasoactive mediators. The timing and magnitude of pneumonitis and fibrosis are known to vary tremendously among different genetic mouse strains and animal species. Therefore, we have systematically compared mast cell numbers in lung sections from nine mouse strains, two rat strains and nonhuman primates (NHP) after whole thorax irradiation (WTI) at doses ranging from 10-15 Gy and at the time of entering respiratory distress. Mice of the BALB/c strain had a dramatic increase in interstitial mast cell numbers, similar to WAG/Rij and August rats, while relatively low levels of mast cell infiltrate were observed in other mouse strains (CBA, C3H, B6, C57L, WHT and TO mice). Enumeration of mast cell number in five NHPs (rhesus macaque), exhibiting severe pneumonitis at 17 weeks after 10 Gy WTI, also indicated a low response shared by the majority of mouse strains. There appeared to be no relationship between the mast cell response and the strain-dependent susceptibility towards pneumonitis or fibrosis. Further investigations are required to explore the possible participation of mast cells in mediating specific vascular responses and whether a genetically diverse mast cell response occurs in humans.

  17. Do Variations in Mast Cell Hyperplasia Account for Differences in Radiation-Induced Lung Injury among Different Mouse Strains, Rats and Nonhuman Primates?

    PubMed Central

    Down, Julian D.; Medhora, Meetha; Jackson, Isabel L.; Cline, J. Mark; Vujaskovic, Zeljko

    2013-01-01

    The role of mast cell infiltrates in the pathology of radiation damage to the lung has been a subject of continuing investigation over the past four decades. This has been accompanied by a number of proposals as to how mast cells and the secretory products thereof participate in the generation of acute inflammation (pneumonitis) and the chronic process of collagen deposition (fibrosis). An additional pathophysiology examines the possible connection between mast cell hyperplasia and pulmonary hypertension through the release of vasoactive mediators. The timing and magnitude of pneumonitis and fibrosis are known to vary tremendously among different genetic mouse strains and animal species. Therefore, we have systematically compared mast cell numbers in lung sections from nine mouse strains, two rat strains and nonhuman primates (NHP) after whole thorax irradiation (WTI) at doses ranging from 10–15 Gy and at the time of entering respiratory distress. Mice of the BALB/c strain had a dramatic increase in interstitial mast cell numbers, similar to WAG/Rij and August rats, while relatively low levels of mast cell infiltrate were observed in other mouse strains (CBA, C3H, B6, C57L, WHT and TO mice). Enumeration of mast cell number in five NHPs (rhesus macaque), exhibiting severe pneumonitis at 17 weeks after 10 Gy WTI, also indicated a low response shared by the majority of mouse strains. There appeared to be no relationship between the mast cell response and the strain-dependent susceptibility towards pneumonitis or fibrosis. Further investigations are required to explore the possible participation of mast cells in mediating specific vascular responses and whether a genetically diverse mast cell response occurs in humans. PMID:23819595

  18. DIETARY FLAXSEED PREVENTS RADIATION-INDUCED OXIDATIVE LUNG DAMAGE, INFLAMMATION AND FIBROSIS IN A MOUSE MODEL OF THORACIC RADIATION INJURY

    PubMed Central

    Lee, James C.; Krochak, Ryan; Blouin, Aaron; Kanterakis, Stathis; Chatterjee, Shampa; Arguiri, Evguenia; Vachani, Anil; Solomides, Charalambos C.; Cengel, Keith A.; Christofidou-Solomidou, Melpo

    2009-01-01

    Flaxseed (FS) has high contents of omega-3 fatty acids and lignans with antioxidant properties. Its use in preventing thoracic X-ray radiation therapy (XRT)-induced pneumonopathy has never been evaluated. We evaluated FS supplementation given to mice given before and post-XRT. FS-derived lignans, known for their direct antioxidant properties, were evaluated in abrogating ROS generation in cultured endothelial cells following gamma radiation exposure. Mice were fed 10% FS or isocaloric control diet for three weeks and given 13.5 Gy thoracic XRT. Lungs were evaluated at 24 hours for markers of radiation-induced injury, three weeks for acute lung damage (lipid peroxidation, lung edema and inflammation), and at four months for late lung damage (inflammation and fibrosis). FS-Lignans blunted ROS generation in vitro, resulting from radiation in a dose-dependent manner. FS-fed mice had reduced expression of lung injury biomarkers (Bax, p21, and TGF-beta1) at 24 hours following XRT and reduced oxidative lung damage as measured by malondialdehyde (MDA) levels at 3 weeks following XRT. In addition, FS-fed mice had decreased lung fibrosis as determined by hydroxyproline content and decreased inflammatory cell influx into lungs at 4 months post XRT. Importantly, when Lewis Lung carcinoma cells were injected systemically in mice, FS dietary supplementation did not appear to protect lung tumors from responding to thoracic XRT. Dietary FS is protective against pulmonary fibrosis, inflammation and oxidative lung damage in a murine model. Moreover, in this model, tumor radioprotection was not observed. FS lignans exhibited potent radiation-induced ROS scavenging action. Taken together, these data suggest that dietary flaxseed may be clinically useful as an agent to increase the therapeutic index of thoracic XRT by increasing the radiation tolerance of lung tissues. PMID:18981722

  19. Acrylamide-induced carcinogenicity in mouse lung involves mutagenicity: cII gene mutations in the lung of big blue mice exposed to acrylamide and glycidamide for up to 4 weeks.

    PubMed

    Manjanatha, Mugimane G; Guo, Li-Wu; Shelton, Sharon D; Doerge, Daniel R

    2015-06-01

    Potential health risks for humans from exposure to acrylamide (AA) and its epoxide metabolite glycidamide (GA) have garnered much attention lately because substantial amounts of AA are present in a variety of fried and baked starchy foods. AA is tumorigenic in rodents, and a large number of in vitro and in vivo studies indicate that AA is genotoxic. A recent cancer bioassay on AA demonstrated that the lung was one of the target organs for tumor induction in mice; however, the mutagenicity of AA in this tissue is unclear. Therefore, to investigate whether or not gene mutation is involved in the etiology of AA- or GA-induced mouse lung carcinogenicity, we screened for cII mutant frequency (MF) in lungs from male and female Big Blue (BB) mice administered 0, 1.4, and 7.0 mM AA or GA in drinking water for up to 4 weeks (19-111 mg/kg bw/days). Both doses of AA and GA produced significant increases in cII MFs, with the high doses producing responses 2.7-5.6-fold higher than the corresponding controls (P ≤ 0.05; control MFs = 17.2 ± 2.2 and 15.8 ± 3.5 × 10(-6) in males and females, respectively). Molecular analysis of the mutants from high doses indicated that AA and GA produced similar mutation spectra and that these spectra were significantly different from the spectra in control mice (P ≤ 0.01). The predominant types of mutations in the lung cII gene from AA- and GA-treated mice were A:T → T:A, and G:C → C:G transversions, and -1/+1 frameshifts at a homopolymeric run of Gs. The MFs and types of mutations induced by AA and GA in the lung are consistent with AA exerting its genotoxicity via metabolism to GA. These results suggest that AA is a mutagenic carcinogen in mouse lungs and therefore further studies on its potential health risk to humans are warranted. Environ. Mol. Mutagen. 56:446-456, 2015. © 2015 Wiley Periodicals, Inc.

  20. Comparison of ribavirin and oseltamivir in reducing mortality and lung injury in mice infected with mouse adapted A/California/04/2009 (H1N1)

    PubMed Central

    Zarogiannis, Sotirios G.; Noah, James W.; Jurkuvenaite, Asta; Steele, Chad; Matalon, Sadis; Noah, Diana L.

    2016-01-01

    Aim To compare the efficacy of ribavirin and oseltamivir in reducing mortality, lung injury and cytokine response profile in pandemic influenza H1N1 (2009) infection. Main Methods We assessed survival, weight loss, lung viral load (by RT-PCR), lung injury (by protein content in bronchoalveolar lavage), and inflammation (cell counts, differentials and cytokines in the bronchoalveolar lavage) in BALB/c mice after infection with mouse-adapted pandemic influenza strain A/California/04/2009. Key Findings Our results indicate that ribavirin (80 mg kg−1) and oseltamivir (50 mg kg−1) are equally effective in improving survival (100% vs. 0% in water treated controls), while ribavirin proved to be more effective in significantly preventing weight loss. Both drugs diminished the injury of the alveolar-capillary barrier by decreasing the protein detected in the BAL to baseline levels, and they were also equally effective in reduction lung viral loads by 100-fold. Administration of either drug did not decrease the amount of inflammatory infiltrate in the lung, but ribavirin significantly reduced the percentage comprised of lymphocytes. This study shows that these antivirals differentially regulate inflammatory cytokines and chemokines with ribavirin significantly reducing most of the cytokines/chemokines measured. Ribavirin treatment leads to a Th1 cytokine response while oseltamivir leads to a Th2 cytokine response with significant increase in the levels of the anti-inflammatory cytokine IL-10. Significance This study reveals new mechanistic insights in the way that ribavirin and oseltamivir exert their antiviral activity and supports the theory that ribavirin could potentially serve as an efficacious therapeutic alternative for oseltamivir resistant pandemic H1N1 strains. PMID:22269828

  1. Comparison of ribavirin and oseltamivir in reducing mortality and lung injury in mice infected with mouse adapted A/California/04/2009 (H1N1).

    PubMed

    Zarogiannis, Sotirios G; Noah, James W; Jurkuvenaite, Asta; Steele, Chad; Matalon, Sadis; Noah, Diana L

    2012-03-10

    To compare the efficacy of ribavirin and oseltamivir in reducing mortality, lung injury and cytokine response profile in pandemic influenza H1N1 (2009) infection. We assessed survival, weight loss, lung viral load (by RT-PCR), lung injury (by protein content in bronchoalveolar lavage), and inflammation (cell counts, differentials and cytokines in the bronchoalveolar lavage) in BALB/c mice after infection with mouse-adapted pandemic influenza strain A/California/04/2009. Our results indicate that ribavirin (80 mg kg(-1)) and oseltamivir (50 mg kg(-1)) are equally effective in improving survival (100% vs. 0% in water treated controls), while ribavirin proved to be more effective in significantly preventing weight loss. Both drugs diminished the injury of the alveolar-capillary barrier by decreasing the protein detected in the BAL to baseline levels, and they were also equally effective in reduction lung viral loads by 100-fold. Administration of either drug did not decrease the amount of inflammatory infiltrate in the lung, but ribavirin significantly reduced the percentage comprised of lymphocytes. This study shows that these antivirals differentially regulate inflammatory cytokines and chemokines with ribavirin significantly reducing most of the cytokines/chemokines measured. Ribavirin treatment leads to a Th1 cytokine response while oseltamivir leads to a Th2 cytokine response with significant increase in the levels of the anti-inflammatory cytokine IL-10. This study reveals new mechanistic insights in the way that ribavirin and oseltamivir exert their antiviral activity and supports the theory that ribavirin could potentially serve as an efficacious therapeutic alternative for oseltamivir resistant pandemic H1N1 strains. Copyright © 2012 Elsevier Inc. All rights reserved.

  2. Nuclear erythroid 2 p45-related factor-2 Nrf2 ameliorates cigarette smoking-induced mucus overproduction in airway epithelium and mouse lungs.

    PubMed

    Ying, Ying-Hua; Lin, Xiao-Ping; Zhou, Hong-bin; Wu, Yin-fang; Yan, Fu-gui; Hua, Wen; Xia, Li-Xia; Qiu, Zhang-wei; Chen, Zhi-Hua; Li, Wen; Shen, Hua-Hao

    2014-10-01

    Nuclear erythroid 2 p45-related factor-2 (Nrf2) is known to play important roles in airway disorders, whereas little has been investigated about its direct role in airway mucus hypersecretion. The aim of this study is to determine whether this factor could protect pulmonary epithelium and mouse airway from cigarette-induced mucus overproduction. Using genetic approaches, the role of Nrf2 on cigarette smoking extracts (CSE) induced MUC5AC expression was investigated in lung A549 cells. Nrf2 deficiency mice were smoked for various periods, and the airway inflammation and mucus production was characterized. Acute smoking exposure induced expression of MUC5AC and Nrf2 in both A549 cells and mouse lungs. Genetic ablation of Nrf2 augmented, whereas overexpression of this molecule ameliorated CSE-induced expression of MUC5AC. Nrf2 knockout mice, after exposure to cigarette smoking, displayed enhanced airway inflammation and mucus production. Nrf2 negatively regulated smoking-induced mucus production in vitro and in vivo, suggesting therapeutic potentials of this factor in airway diseases with hypersecreted mucus. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  3. Involvement of EZH2, SUV39H1, G9a and associated molecules in pathogenesis of urethane induced mouse lung tumors: Potential targets for cancer control

    SciTech Connect

    Pandey, Manuraj; Sahay, Satya; Tiwari, Prakash; Upadhyay, Daya S.; Sultana, Sarwat; Gupta, Krishna P.

    2014-10-15

    In the present study, we showed the correlation of EZH2, SUV39H1 or G9a expression and histone modifications with the urethane induced mouse lung tumorigenesis in the presence or absence of antitumor agent, inositol hexaphosphate (IP6). Tumorigenesis and the molecular events involved therein were studied at 1, 4, 12 or 36 weeks after the exposure. There were no tumors at 1 or 4 weeks but tumors started appearing at 12 weeks and grew further till 36 weeks after urethane exposure. Among the molecular events, upregulation of EZH2 and SUV39H1 expressions appeared to be time dependent, but G9a expression was altered significantly only at later stages of 12 or 36 weeks. Alteration in miR-138 expression supports the upregulation of its target, EZH2. H3K9me2, H3K27me3 or H4K20me3 was found to be altered at 12 or 36 weeks. However, ChIP analysis of p16 and MLH1 promoters showed their binding with H3K9me2 and H3K27me3 which was maximum at 36 weeks. Thus, histone modification and their interactions with gene promoter resulted in the reduced expression of p16 and MLH1. IP6 prevented the incidence and the size of urethane induced lung tumors. IP6 also prevented the urethane induced alterations in EZH2, SUV39H1, G9a expressions and histone modifications. Our results suggest that the alterations in the histone modification pathways involving EZH2 and SUV39H1 expressions are among the early events in urethane induced mouse lung tumorigenesis and could be exploited for cancer control. - Highlights: • Urethane induces mouse lung tumor in a time dependent manner. • EZH2, SUV39H1, G9a induced by urethane and progress with time • Downregulation of miRNA-138 supports the EZH2 upregulation. • Methylation of histones showed a consequence of upregulated EZH2, SUV39H1 and G9a. • IP6 inhibits urethane induced changes and prevents tumor development.

  4. Multicellular genesis of leaf primordium was demonstrated via chimaeric transgenic plant of maize (Zea mays L.) regenerated from Type II calli.

    PubMed

    Xu, Zi-Qin; Huang, Xuan; Feng, Chao; Tian, Na; Xu, Dan; Feng, Shu-Zhen

    2010-10-01

    Type-II embryonic calli were induced from immature embryos of maize (Zea mays L.) genotype YD and bombarded with beta-glucuronidase gene. Bombarded calli were proliferated on normal N6 medium for 2 weeks at 26°C in the dark and selected on N6 medium containing 1 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 5 mg/l phosphinothricin (PPT) but without casamino acids and proline under the same conditions for 14 days. Regeneration was carried out on hormone-free MS medium containing 5 mg/l phosphinothricin at 26°C under 3000 lux illumination. Plants over 8 cm were transplanted into soil and sprayed with 250 mg/l phosphinothricin when two new leaves appeared. Except normal transgenic plants, chimaeric transgenics also were regenerated in the present work. The expression pattern of beta-glucuronidase gene in leaves of chimaeric transgenic plant revealed that more than one cell formed leaf primordium at the initial stage, and filial cells stemed from each cell in leaf primordium arranged in a row longitudinally from leaf base to leaf apex. There was a clear boundary as a straight line between the area formed by transformed cells and the area formed by normal cells. A hypothesis was put forward that the primitive cells in leaf primordium divided in a longitudinal style, resulted in leaf elongation, then the filial cells divided transversally and synchronously toward the outside to broaden the leaf.

  5. A change in the number of CCSP(pos)/SPC(pos) cells in mouse lung during development, growth, and repair.

    PubMed

    Sun, Ruowen; Zhou, Qiliang; Ye, Xulu; Takahata, Takenori; Ishiguro, Atsushi; Kijima, Hiroshi; Nukiwa, Toshihiro; Saijo, Yasuo

    2013-12-01

    Putative resident stem/progenitor cells have been identified in the bronchoalveolar duct junction (BADJ) of the murine lung. However, the contribution of stem cells expressing both Clara cell secretory protein (CCSP) and pro-surfactant protein C (SP-C) to the repair and maintenance of normal homeostasis is still unclear. In this study, we identified and then quantified CD45(neg)/CCSP(pos)/SP-C(pos) cell numbers in normal and lung-injured mice. Normal lung tissues of fetal, newborn, and adult mice were used to evaluate lung progenitor cells during development and growth. Mice treated with naphthalene were used for the bronchiolar epithelium injury model, and mice treated with bleomycin were used for the alveolar epithelium injury model. These lung tissues were stained with CD45, CCSP, and SP-C antibodies by immunofluorescence. The number of lung progenitor cells was counted as CD45(neg)/CCSP(pos)/SP-C(pos) cells by flow cytometry. CCSP(pos)/SP-C(pos) epithelial cells in the BADJ were identified from E18 to 7 months after birth. The percentage of CD45(neg)/CCSP(pos)/SP-C(pos) cells was relatively stable to 7 months (between 0.3±0.04% and 1.28±0.11%). When lungs were treated with naphthalene, the proliferation of CCSP(pos)/SP-C(pos) cells was observed as patches of double-positive cells and preceded the recovery of bronchioles. In contrast, when lungs were treated with bleomycin, the proliferation of CCSP(pos)/SP-C(pos) cells was observed, but the type II alveolar epithelial cells never recovered to baseline. CCSP(pos)/SP-C(pos) lung cells were stable until 7 months after birth. These cells in the BADJ primarily regenerate bronchiolar epithelial cells and not alveolar epithelial cells. Copyright © 2013 The Japanese Respiratory Society. Published by Elsevier B.V. All rights reserved.

  6. The Complement C3a Receptor Is Critical in Defense against Chlamydia psittaci in Mouse Lung Infection and Required for Antibody and Optimal T Cell Response

    PubMed Central

    Dutow, Pavel; Fehlhaber, Beate; Bode, Jenny; Laudeley, Robert; Rheinheimer, Claudia; Glage, Silke; Wetsel, Rick A.; Pabst, Oliver; Klos, Andreas

    2014-01-01

    Background. The complement system protects against extracellular pathogens and links innate and adaptive immunity. In this study, we investigated the anaphylatoxin C3a receptor (C3aR) in Chlamydia psittaci lung infection and elucidated C3a-dependent adaptive immune mechanisms. Methods. Survival, body weight, and clinical score were monitored in primary mouse infection and after serum transfer. Bacterial load, histology, cellular distribution, cytokines, antibodies, and lymphocytes were analyzed. Results. C3aR−/− mice showed prolonged pneumonia with decreased survival, lower weight, and higher clinical score. Compared to wild-type mice bacterial clearance was impaired, and inflammatory parameters were increased. In lung-draining lymph nodes of C3aR−/− mice the total number of B cells, CD4+ T cells, and Chlamydia-specific IFN-γ+ (CD4+ or CD8+) cells was reduced upon infection, and the mice were incapable of Chlamydia-specific immunoglobulin M or immunoglobulin G production. Performed before infection, transfer of hyperimmune serum prolonged survival of C3aR−/− mice. Conclusions. C3a and its receptor are critical for defense against C. psittaci in mouse lung infection. In this model, C3a acts via its receptor as immune modulator. Enhancement of specific B and T cell responses upon infection with an intracellular bacterium were identified as hitherto unknown features of C3a/C3aR. These new functions might be of general immunological importance. PMID:24273177

  7. Silica Triggers Inflammation and Ectopic Lymphoid Neogenesis in the Lungs in Parallel with Accelerated Onset of Systemic Autoimmunity and Glomerulonephritis in the Lupus-Prone NZBWF1 Mouse

    PubMed Central

    Bates, Melissa A.; Brandenberger, Christina; Langohr, Ingeborg; Kumagai, Kazuyoshi; Harkema, Jack R.; Holian, Andrij; Pestka, James J.

    2015-01-01

    Genetic predisposition and environmental factors influence the development of human autoimmune disease. Occupational exposure to crystalline silica (cSiO2) has been etiologically linked to increased incidence of autoimmunity, including systemic lupus erythematosus (SLE), but the underlying mechanisms are poorly understood. The purpose of this study was to test the hypothesis that early repeated short-term cSiO2 exposure will modulate both latency and severity of autoimmunity in the lupus-prone female NZBWF1 mouse. Weekly intranasal exposure to cSiO2 (0.25 and 1.0 mg) for 4 wk beginning at 9 wk of age both reduced latency and increased intensity of glomerulonephritis. cSiO2 elicited robust inflammatory responses in the lungs as evidenced by extensive perivascular and peribronchial lymphoplasmacytic infiltration consisting of IgG-producing plasma cells, and CD45R+ and CD3+ lymphocytes that were highly suggestive of ectopic lymphoid tissue (ELT). In addition, there were elevated concentrations of immunoglobulins and the cytokines MCP-1, TNF-α and IL-6 in bronchoalveolar lavage fluid. cSiO2-associated kidney and lung effects paralleled dose-dependent elevations of autoantibodies and proinflammatory cytokines in plasma. Taken together, cSiO2-induced pulmonary inflammation and ectopic lymphoid neogenesis in the NZBWF1 mouse corresponded closely to systemic inflammatory and autoimmune responses as well as the early initiation of pathological outcomes in the kidney. These findings suggest that following airway exposure to crystalline silica, in mice genetically prone to SLE, the lung serves as a platform for triggering systemic autoimmunity and glomerulonephritis. PMID:25978333

  8. Validation of syngeneic mouse models of melanoma and non-small cell lung cancer for investigating the anticancer effects of the soy-derived peptide Lunasin

    PubMed Central

    Devapatla, Bharat; Shidal, Chris; Yaddanapudi, Kavitha; Davis, Keith R.

    2017-01-01

    Background: Lunasin is a naturally occurring peptide present in soybean that has both chemopreventive and therapeutic activities that can prevent cellular transformation and inhibit the growth of several human cancer types. Recent studies indicate that Lunasin has several distinct potential modes of action including suppressing integrin signaling and epigenetic effects driven by modulation of histone acetylation. In addition to direct effects on cancer cells, Lunasin also has effects on innate immunity that may contribute to its ability to inhibit tumor growth in vivo. Methods : Standard assays for cell proliferation and colony formation were used to assess Lunasin’s in vitro activity against murine Lewis lung carcinoma (LLC) and B16-F0 melanoma cells.  Lunasin’s in vivo activity was assessed by comparing the growth of tumors initiated by subcutaneous implantation of LLC or B16-F0 cells in Lunasin-treated and untreated C57BL/6 mice. Results : Lunasin was found to inhibit growth of murine LLC cells and murine B16-F0 melanoma cells in vitro and in wild-type C57BL/6 mice.  The effects of Lunasin in these two mouse models were very similar to those previously observed in studies of human non-small cell lung cancer and melanoma cell lines. Conclusions : We have now validated two established syngeneic mouse models as being responsive to Lunasin treatment.  The validation of these two in vivo syngeneic models will allow detailed studies on the combined therapeutic and immune effects of Lunasin in a fully immunocompetent mouse model. PMID:28299174

  9. Electroporation-mediated in vivo gene delivery of the Na+/K+-ATPase pump reduced lung injury in a mouse model of lung contusion.

    PubMed

    Machado-Aranda, David A; Suresh, M V; Yu, Bi; Raghavendran, Krishnan

    2012-01-01

    Lung contusion (LC) is an independent risk factor for acute respiratory distress syndrome. The final common pathway in ARDS involves accumulation of fluid in the alveoli. In this study, we demonstrate the application of a potential gene therapy approach by delivering the Na+/K+-ATPase pump subunits in a murine model of LC. We hypothesized that restoring the activity of the pump will result in removal of excess alveolar fluid and additionally reduce inflammation. Under anesthesia, C57/BL6 mice were struck along the right posterior axillary line 1 cm above the costal margin with a cortical contusion impactor. Immediately afterward, 100 μg of plasmid DNA coding for the α,β of the Na+/K+-ATPase pump were instilled into the lungs (LC-electroporation-pump group). Contusion only (LC-only) and a sham saline instillation group after contusion were used as controls (LC-electroporation-sham). By using a BTX 830 electroporator, eight electrical pulses of 200 V/cm field strength were applied transthoracically. Mice were killed at 24 hours, 48 hours, and 72 hours after delivery. Bronchial alveolar lavage was recollected to measure albumin and cytokines by enzyme-linked immunosorbent assay. Pulmonary compliance was measured, and lungs were subject to histopathologic analysis. After the electroporation and delivery of genes coding for the α,β subunits of the Na+/K+-ATPase pump, there was a significant mitigation of acute lung injury as evidenced by reduction in bronchial alveolar lavage levels of albumin, improved pressure volume curves, and reduced inflammation seen on histology. Electroporation-mediated gene transfer of the subunits of the Na+/K+-ATPase pump enhanced recovery from acute inflammatory lung injury after LC.

  10. Electroporation mediated in-vivo Gene Delivery of the Na+/K+-ATPase pump Reduced Lung Injury in a mouse model of Lung Contusion

    PubMed Central

    Machado-Aranda, David A; Madathilparambil, Suresh; Yu, Bi; Raghavendran, Krishnan

    2011-01-01

    Objective Lung contusion (LC) is an independent risk factor for Adult Respiratory Distress Syndrome (ARDS). The final common pathway in ARDS involves accumulation of fluid in the alveoli. In this study we demonstrate the application of a potential gene therapy approach by delivering the Na+/K+-ATPase pump sub-units in a murine model of LC. We hypothesized that restoring the activity of the pump will result in removal of excess alveolar fluid and additionally reduce inflammation. Methods Under anesthesia, C57/BL6 mice were struck along the right posterior axillary line 1 cm above the costal margin with a cortical contusion impactor. Immediately afterwards; 100 μg of plasmid DNA coding for the α,β of the Na+/K+-ATPase pump were instilled into the lungs (LC EP-pump group). Contusion only (LC only) and a sham saline instillation group after contusion were used as controls (LC EP-sham). Using a BTX 830 Electroporator, 8 electrical pulses of 200-V/cm field strength were applied transthoracically. Mice were sacrificed at 24hr, 48hr and 72hr post-delivery. Bronchial alveolar lavage (BAL) was recollected to measure albumin and cytokines by ELISA. Pulmonary compliance was measured and lungs were subject to histopathologic analysis. Results Following the electroporation and delivery of genes coding for the α,β subunits of the Na+/K+-ATPase pump, there was a significant mitigation of acute lung injury as evidenced by reduction in BAL levels of albumin, improved P-V curves and reduced inflammation seen on histology. Conclusion Electroporation mediated gene transfer of the subunits of the Na+/K+-ATPase pump enhanced recovery from acute inflammatory lung injury following LC. PMID:22310113

  11. Genome-wide analysis of the mouse lung transcriptome reveals novel molecular gene interaction networks and cell-specific expression signatures.

    PubMed

    Alberts, Rudi; Lu, Lu; Williams, Robert W; Schughart, Klaus

    2011-05-02

    The lung is critical in surveillance and initial defense against pathogens. In humans, as in mice, individual genetic differences strongly modulate pulmonary responses to infectious agents, severity of lung disease, and potential allergic reactions. In a first step towards understanding genetic predisposition and pulmonary molecular networks that underlie individual differences in disease vulnerability, we performed a global analysis of normative lung gene expression levels in inbred mouse strains and a large family of BXD strains that are widely used for systems genetics. Our goal is to provide a key community resource on the genetics of the normative lung transcriptome that can serve as a foundation for experimental analysis and allow predicting genetic predisposition and response to pathogens, allergens, and xenobiotics. Steady-state polyA+ mRNA levels were assayed across a diverse and fully genotyped panel of 57 isogenic strains using the Affymetrix M430 2.0 array. Correlations of expression levels between genes were determined. Global expression QTL (eQTL) analysis and network covariance analysis was performed using tools and resources in GeneNetwork http://www.genenetwork.org. Expression values were highly variable across strains and in many cases exhibited a high heritability factor. Several genes which showed a restricted expression to lung tissue were identified. Using correlations between gene expression values across all strains, we defined and extended memberships of several important molecular networks in the lung. Furthermore, we were able to extract signatures of immune cell subpopulations and characterize co-variation and shared genetic modulation. Known QTL regions for respiratory infection susceptibility were investigated and several cis-eQTL genes were identified. Numerous cis- and trans-regulated transcripts and chromosomal intervals with strong regulatory activity were mapped. The Cyp1a1 P450 transcript had a strong trans-acting eQTL (LOD 11

  12. FORMATION OF NON-INFECTIOUS INFLUENZA VIRUS IN MOUSE LUNGS: ITS DEPENDENCE UPON EXTENSIVE PULMONARY CONSOLIDATION INITIATED BY THE VIRAL INOCULUM

    PubMed Central

    Ginsberg, Harold S.

    1954-01-01

    Formation of non-infectious virus—particles which hemagglutinate red blood cells and react with antibody to fix complement but do not infect the chick embryo or mouse—occurred when large quantities of certain strains of influenza viruses were inoculated intranasally into mice. Dependent upon the agent employed, 106.5 to 108.5 E.I.D. was essential to elicit this phenomenon. To accomplish this unusual multiplication it was essential to use a strain of virus which effected extensive pulmonary consolidation; strains of virus which did not produce marked lung lesions, even when as much as 108.5 E.I.D. was inoculated, did not form non-infectious virus. The development of this viral form was directly dependent upon the extent of cell damage obtained: consolidation of more than 50 per cent of the lung volume was required. The majority of non-infectious particles developed during the initial cycle of viral multiplication, and concurrently with the formation of non-infectious virus there was a corresponding decrease in the number of infectious viral particles. Non-infectious virus could not be propagated on serial passage in mouse lungs: on second lung passage only fully infectious virus was detectable. The formation of the non-infectious viral form was not the result of interference with synthesis of infectious virus by inactivated virus in the inoculum; for inoculation of heated infected allantoic fluid which contained more than 99 per cent of non-infectious virus did not result in the development of new non-infectious virus. Although inoculation of a large quantity of virus resulted in infection which yielded a relatively low titer of infectious and high titer of non-infectious virus, inoculation of a small quantity of the agent resulted in a high yield of infectious virus and no non-infectious that was detectable. In both instances the total quantity of antigenic viral material synthesized in the mouse lungs was the same. These data do not support the hypothesis that

  13. Short-term cigarette smoke exposure induces reversible changes in energy metabolism and cellular redox status independent of inflammatory responses in mouse lungs.

    PubMed

    Agarwal, Amit R; Zhao, Liqin; Sancheti, Harsh; Sundar, Isaac K; Rahman, Irfan; Cadenas, Enrique

    2012-11-15

    Cigarette smoking leads to alteration in cellular redox status, a hallmark in the pathogenesis of chronic obstructive pulmonary disease. This study examines the role of cigarette smoke (CS) exposure in the impairment of energy metabolism and, consequently, mitochondrial dysfunction. Male A/J mice were exposed to CS generated by a smoking machine for 4 or 8 wk. A recovery group was exposed to CS for 8 wk and allowed to recover for 2 wk. Acute CS exposure altered lung glucose metabolism, entailing a decrease in the rate of glycolysis and an increase in the pentose phosphate pathway, as evidenced by altered expression and activity of GAPDH and glucose-6-phosphate dehydrogenase, respectively. Impairment of GAPDH was found to be due to glutathionylation of its catalytic site cysteines. Metabolic changes were associated with changes in cellular and mitochondrial redox status, assessed in terms of pyridine nucleotides and glutathione. CS exposure elicited an upregulation of the expression of complexes II, III, IV, and V and of the activity of complexes II, IV, and V. Microarray analysis of gene expression in mouse lungs after exposure to CS for 8 wk revealed upregulation of a group of genes involved in metabolism, electron transfer chain, oxidative phosphorylation, mitochondrial transport and dynamics, and redox regulation. These changes occurred independently of inflammatory responses. These findings have implications for the early onset of alterations in energy and redox metabolism upon acute lung exposure to CS.

  14. A quantitative histological study of strain-dependent differences in the effects of irradiation on mouse lung during the early phase

    SciTech Connect

    Sharplin, J.; Franko, A.J. )

    1989-07-01

    Strain differences in the radiation response of mouse lung during the early phase (before 28 weeks postirradiation) were investigated histologically. The nine strains tested were divided into three groups on the basis of the nature of the edema present, the occurrence of hyaline membranes, and the presence of fibrosis. Group 1 mice, three C57 strains, developed hyaline membranes, focal fibrosis, and a protein-rich edema containing fibrin. Group 3, CBA and two C3H strains, had only a protein-poor edema with little fibrin and developed no visible fibrosis. Group 2 mice had both types of edema and small quantities of focal fibrosis. The degree of lung impairment in mice dying of respiratory insufficiency was assessed by scoring lung acini as nonfunctional or open and presumably functional. Over 70% of acini were nonfunctional as a result of airflow obstruction. This was considered sufficient to account for death. Carbon perfusion immediately before sacrifice indicated that all types of lesions were at least partially perfused with blood. Pleural effusions were found in some individuals of two strains. The proportion of nonfunctional acini was similar in mice of the same strain with and without effusions, which would not be expected if the effusions contributed appreciably to respiratory distress in the early phase.

  15. Vapors Produced by Electronic Cigarettes and E-Juices with Flavorings Induce Toxicity, Oxidative Stress, and Inflammatory Response in Lung Epithelial Cells and in Mouse Lung

    PubMed Central

    Lerner, Chad A.; Sundar, Isaac K.; Yao, Hongwei; Gerloff, Janice; Ossip, Deborah J.; McIntosh, Scott; Robinson, Risa; Rahman, Irfan

    2015-01-01

    Oxidative stress and inflammatory response are the key events in the pathogenesis of chronic airway diseases. The consumption of electronic cigarettes (e-cigs) with a variety of e-liquids/e-juices is alarmingly increasing without the unrealized potential harmful health effects. We hypothesized that electronic nicotine delivery systems (ENDS)/e-cigs pose health concerns due to oxidative toxicity and inflammatory response in lung cells exposed to their aerosols. The aerosols produced by vaporizing ENDS e-liquids exhibit oxidant reactivity suggesting oxidants or reactive oxygen species (OX/ROS) may be inhaled directly into the lung during a “vaping” session. These OX/ROS are generated through activation of the heating element which is affected by heating element status (new versus used), and occurs during the process of e-liquid vaporization. Unvaporized e-liquids were oxidative in a manner dependent on flavor additives, while flavors containing sweet or fruit flavors were stronger oxidizers than tobacco flavors. In light of OX/ROS generated in ENDS e-liquids and aerosols, the effects of ENDS aerosols on tissues and cells of the lung were measured. Exposure of human airway epithelial cells (H292) in an air-liquid interface to ENDS aerosols from a popular device resulted in increased secretion of inflammatory cytokines, such as IL-6 and IL-8. Furthermore, human lung fibroblasts exhibited stress and morphological change in response to treatment with ENDS/e-liquids. These cells also secrete increased IL-8 in response to a cinnamon flavored e-liquid and are susceptible to loss of cell viability by ENDS e-liquids. Finally, exposure of wild type C57BL/6J mice to aerosols produced from a popular e-cig increase pro-inflammatory cytokines and diminished lung glutathione levels which are critical in maintaining cellular redox balance. Thus, exposure to e-cig aerosols/juices incurs measurable oxidative and inflammatory responses in lung cells and tissues that could lead to

  16. MiR-196a regulates heme oxygenase-1 by silencing Bach1 in the neonatal mouse lung.

    PubMed

    Go, Hayato; La, Ping; Namba, Fumihiko; Ito, Masato; Yang, Guang; Brydun, Andrey; Igarashi, Kazuhiko; Dennery, Phyllis A

    2016-08-01

    In the lung, heme oxygenase-1 (HO-1) is developmentally regulated, with its highest expression in the first days of life. In addition, neonatal mice have limited HO-1 induction in hyperoxia compared with adults. However, few reports have addressed the functional effect of microRNAs (miRNAs) in the regulation of HO-1 in vivo. The aims of the present study were to characterize changes in lung miRNA expression during postnatal development and in response to hyperoxic exposure, and to identify miRNAs that target lung HO-1 gene expression. Neonatal (<12 h old) and adult (2 mo old) mice were exposed to room air or hyperoxia (95% oxygen) for 72 h. TaqMan low-density array rodent miRNA assays were used to calculate miRNA expression changes between control and hyperoxia groups in neonatal and adult lungs. In neonates, we identified miR-196a, which binds to the 3'-untranslated region of the transcriptional repressor BTB and CNC homology 1 (Bach1) and regulates its expression, and subsequently leads to higher levels of lung HO-1 mRNA compared with levels in adults. Despite the increase at baseline, miR-196a was degraded in hyperoxia resulting in limited HO-1 induction in neonatal mice lungs. Furthermore, the developmental differences in lung HO-1 gene expression can be explained in part by the variation in miRNA-196a and its effect on Bach1. This report is the first to show developmental differences in lung miR-196a and its effect on Bach1 and HO-1 expression at baseline and in hyperoxia. Copyright © 2016 the American Physiological Society.

  17. Impedance in isolated mouse lungs for the determination of site of action of vasoactive agents and disease.

    PubMed

    Vanderpool, Rebecca R; Naeije, Robert; Chesler, Naomi C

    2010-05-01

    Hypoxic pulmonary hypertension is a disease of the lung vasculature that is usually quantified by pulmonary vascular resistance (PVR). However, a more complete description of lung vascular function and right ventricular afterload is provided by pulmonary vascular impedance (PVZ) from spectral analysis of pulsatile pressure-flow relationships. We studied pulsatile pressure-flow relationships in isolated, perfused lungs of mice in normoxia, after induction of hypoxic pulmonary hypertension by 10 days of hypoxic exposure, and after the administration of the vasoactive agents sodium nitroprusside and serotonin in order to gain insight into the effects of disease and vasoactive agents on afterload. Chronic hypoxia exposure increased 0 Hz impedance (Z(0)) from 2.0 +/- 0.2 to 3.3 +/- 0.2 mmHg min/mL but decreased characteristic impedance (Z(C)) from 0.21 +/- 0.02 to 0.18 +/- 0.01 mmHg min/mL (both p < 0.05). Sodium nitroprusside only slightly decreased Z(0) but increased Z(C) in normal lungs (p < 0.05) and did not affect Z(C) and decreased Z(0) in hypertensive lungs (p < 0.05). Serotonin increased Z(C) in normal and hypertensive lungs but decreased Z(0) in hypertensive lungs (p < 0.05). There was an inverse correlation between mean pulmonary artery pressure and Z(C) in all circumstances. These findings demonstrate that vasoactive interventions can have different sites of action (i.e., proximal vs. distal segments) in the normal and chronically hypoxic pulmonary vasculature, and the pressure-dependency of Z(C) and R(W). The measurement of PVZ in isolated lungs allows for an improved understanding of the modes of action of drugs and hypoxia on the pulmonary circulation.

  18. Suppression of basal and carbon nanotube-induced oxidative stress, inflammation and fibrosis in mouse lungs by Nrf2.

    PubMed

    Dong, Jie; Ma, Qiang

    2016-08-01

    The lungs are susceptible to oxidative damage by inhaled pathogenic agents, including multi-walled carbon nanotubes (MWCNT). The nuclear factor erythroid 2-related factor 2 (Nrf2) has been implicated in regulating the body's defense against oxidative stress. Here, we analyzed the function of Nrf2 in the lungs. Under a basal condition, Nrf2 knockout (KO) mice showed apparent pulmonary infiltration of granulocytes, macrophages and B and T lymphocytes, and elevated deposition of collagen fibers. Exposure to MWCNT (XNRI MWNT-7, Mitsui, Tokyo, Japan) by pharyngeal aspiration elicited rapid inflammatory and fibrotic responses in a dose (0, 5, 20 and 40 μg) and time (1, 3, 7 and 14 d)-dependent manner. The responses reached peak levels on day 7 post-exposure to 40 μg MWCNT, evidenced by massive inflammatory infiltration and formation of inflammatory and fibrotic foci, which were more evident in Nrf2 KO than wild-type (WT) lungs. At the molecular level, Nrf2 protein was detected at a low level under a basal condition, and was dramatically increased by MWCNT in WT, but not Nrf2 KO, lungs. Activation of Nrf2 was inversely correlated with induced expression of fibrosis marker genes and profibrotic cytokines. Furthermore, the levels of ROS and oxidative stress were remarkably higher in Nrf2 KO than WT lungs under a physiological condition, and were dramatically increased by MWCNT, with the increase significantly more striking in KO lungs. The findings reveal that Nrf2 plays an important role in suppressing the basal and MWCNT-induced oxidant production, inflammation and fibrosis in the lungs, thereby protecting against MWCNT lung toxicity.

  19. Respective Roles of CYP2A5 and CYP2F2 in the Bioactivation of 3-Methylindole in Mouse Olfactory Mucosa and Lung: Studies Using Cyp2a5-Null and Cyp2f2-Null Mouse Models

    PubMed Central

    Zhou, Xin; D'Agostino, Jaime; Li, Lei; Moore, Chad D.; Yost, Garold S.

    2012-01-01

    The aim of this study was to determine whether mouse CYP2A5 and CYP2F2 play critical roles in the bioactivation of 3-methylindole (3MI), a tissue-selective toxicant, in the target tissues, the nasal olfactory mucosa (OM) and lung. Five metabolites of 3MI were identified in NADPH- and GSH-fortified microsomal reactions, including 3-glutathionyl-S-methylindole (GS-A1), 3-methyl-2-glutathionyl-S-indole (GS-A2), 3-hydroxy-3-methyleneindolenine (HMI), indole-3-carbinol (I-3-C), and 3-methyloxindole (MOI). The metabolite profiles and enzyme kinetics of the reactions were compared between OM and lung, and among wild-type, Cyp2a5-null, and Cyp2f2-null mice. In lung reactions, GS-A1, GS-A2, and HMI were detected as major products, and I-3-C and MOI, as minor metabolites. In OM reactions, all five metabolites were detected in ample amounts. The loss of CYP2F2 affected formation of all 3MI metabolites in the lung and formation of HMI, GS-A1, and GS-A2 in the OM. In contrast, loss of CYP2A5 did not affect formation of 3MI metabolites in the lung but caused substantial decreases in I-3-C and MOI formation in the OM. Thus, whereas CYP2F2 plays a critical role in the 3MI metabolism in the lung, both CYP2A5 and CYP2F2 play important roles in 3MI metabolism in the OM. Furthermore, the fate of the reactive metabolites produced by the two enzymes through common dehydrogenation and epoxidation pathways seemed to differ with CYP2A5 supporting direct conversion to stable metabolites and CYP2F2 supporting further formation of reactive iminium ions. These results provide the basis for understanding the respective roles of CYP2A5 and CYP2F2 in 3MI's toxicity in the respiratory tract. PMID:22228748

  20. X-Ray based Lung Function measurement-a sensitive technique to quantify lung function in allergic airway inflammation mouse models

    NASA Astrophysics Data System (ADS)

    Dullin, C.; Markus, M. A.; Larsson, E.; Tromba, G.; Hülsmann, S.; Alves, F.

    2016-11-01

    In mice, along with the assessment of eosinophils, lung function measurements, most commonly carried out by plethysmography, are essential to monitor the course of allergic airway inflammation, to examine therapy efficacy and to correlate animal with patient data. To date, plethysmography techniques either use intubation and/or restraining of the mice and are thus invasive, or are limited in their sensitivity. We present a novel unrestrained lung function method based on low-dose planar cinematic x-ray imaging (X-Ray Lung Function, XLF) and demonstrate its performance in monitoring OVA induced experimental allergic airway inflammation in mice and an improved assessment of the efficacy of the common treatment dexamethasone. We further show that XLF is more sensitive than unrestrained whole body plethysmography (UWBP) and that conventional broncho-alveolar lavage and histology provide only limited information of the efficacy of a treatment when compared to XLF. Our results highlight the fact that a multi-parametric imaging approach as delivered by XLF is needed to address the combined cellular, anatomical and functional effects that occur during the course of asthma and in response to therapy.

  1. X-Ray based Lung Function measurement–a sensitive technique to quantify lung function in allergic airway inflammation mouse models

    PubMed Central

    Dullin, C.; Markus, M. A.; Larsson, E.; Tromba, G.; Hülsmann, S.; Alves, F.

    2016-01-01

    In mice, along with the assessment of eosinophils, lung function measurements, most commonly carried out by plethysmography, are essential to monitor the course of allergic airway inflammation, to examine therapy efficacy and to correlate animal with patient data. To date, plethysmography techniques either use intubation and/or restraining of the mice and are thus invasive, or are limited in their sensitivity. We present a novel unrestrained lung function method based on low-dose planar cinematic x-ray imaging (X-Ray Lung Function, XLF) and demonstrate its performance in monitoring OVA induced experimental allergic airway inflammation in mice and an improved assessment of the efficacy of the common treatment dexamethasone. We further show that XLF is more sensitive than unrestrained whole body plethysmography (UWBP) and that conventional broncho-alveolar lavage and histology provide only limited information of the efficacy of a treatment when compared to XLF. Our results highlight the fact that a multi-parametric imaging approach as delivered by XLF is needed to address the combined cellular, anatomical and functional effects that occur during the course of asthma and in response to therapy. PMID:27805632

  2. X-Ray based Lung Function measurement-a sensitive technique to quantify lung function in allergic airway inflammation mouse models.

    PubMed

    Dullin, C; Markus, M A; Larsson, E; Tromba, G; Hülsmann, S; Alves, F

    2016-11-02

    In mice, along with the assessment of eosinophils, lung function measurements, most commonly carried out by plethysmography, are essential to monitor the course of allergic airway inflammation, to examine therapy efficacy and to correlate animal with patient data. To date, plethysmography techniques either use intubation and/or restraining of the mice and are thus invasive, or are limited in their sensitivity. We present a novel unrestrained lung function method based on low-dose planar cinematic x-ray imaging (X-Ray Lung Function, XLF) and demonstrate its performance in monitoring OVA induced experimental allergic airway inflammation in mice and an improved assessment of the efficacy of the common treatment dexamethasone. We further show that XLF is more sensitive than unrestrained whole body plethysmography (UWBP) and that conventional broncho-alveolar lavage and histology provide only limited information of the efficacy of a treatment when compared to XLF. Our results highlight the fact that a multi-parametric imaging approach as delivered by XLF is needed to address the combined cellular, anatomical and functional effects that occur during the course of asthma and in response to therapy.

  3. Expression of genes involved in mouse lung cell differentiation/regulation after acute exposure to photons and protons with or without low-dose preirradiation.

    PubMed

    Tian, Jian; Zhao, WeiLing; Tian, Sisi; Slater, James M; Deng, Zhiyong; Gridley, Daila S

    2011-11-01

    The goal of this study was to compare the effects of acute 2 Gy irradiation with photons (0.8 Gy/min) or protons (0.9 Gy/min), both with and without pre-exposure to low-dose/low-dose-rate γ rays (0.01 Gy at 0.03 cGy/h), on 84 genes involved in stem cell differentiation or regulation in mouse lungs on days 21 and 56. Genes with a ≥1.5-fold difference in expression and P < 0.05 compared to 0 Gy controls are emphasized. Two proteins specific for lung stem cells/progenitors responsible for local tissue repair were also compared. Overall, striking differences were present between protons and photons in modulating the genes. More genes were affected by protons than by photons (22 compared to 2 and 6 compared to 2 on day 21 and day 56, respectively) compared to 0 Gy. Preirradiation with low-dose-rate γ rays enhanced the acute photon-induced gene modulation on day 21 (11 compared to 2), and all 11 genes were significantly downregulated on day 56. On day 21, seven genes (aldh2, bmp2, cdc2a, col1a1, dll1, foxa2 and notch1) were upregulated in response to most of the radiation regimens. Immunoreactivity of Clara cell secretory protein was enhanced by all radiation regimens. The number of alveolar type 2 cells positive for prosurfactant protein C in irradiated groups was higher on day 56 (12.4-14.6 cells/100) than on day 21 (8.5-11.2 cells/100) (P < 0.05). Taken together, these results showed that acute photons and protons induced different gene expression profiles in the lungs and that pre-exposure to low-dose-rate γ rays sometimes had modulatory effects. In addition, proteins associated with lung-specific stem cells/progenitors were highly sensitive to radiation.

  4. Radiation-induced lung metastasis development is MT1-MMP-dependent in a triple-negative breast cancer mouse model.

    PubMed

    Bouchard, Gina; Therriault, Hélène; Geha, Sameh; Bujold, Rachel; Saucier, Caroline; Paquette, Benoit

    2017-02-14

    The prognosis of triple-negative breast cancer (TNBC) is still difficult to establish. Some TNBC benefit from radiotherapy (RT) and are cured, while in other patients metastases appear during the first 3 years after treatment. In this study, an animal model of TNBC was used to determine whether the expression of the cell membrane protease MT1-MMP in cancer cells was associated with radiation-stimulated development of lung metastases. Using invasion chambers, irradiated fibroblasts were used as chemoattractants to assess the invasiveness of TNBC D2A1 cell lines showing downregulated expression of MT1-MMP, which were compared with D2A1-wt (wild-type) and D2A1 shMT1-mock (empty vector) cell lines. In a mouse model, a mammary gland was irradiated followed by the implantation of the downregulated MT1-MMP D2A1, D2A1-wt or D2A1 shMT1-mock cell lines. Migration of D2A1 cells in the mammary gland, number of circulating tumour cells and development of lung metastases were assessed. The reduction of MT1-MMP expression decreased the invasiveness of D2A1 cells and blocked the radiation enhancement of cancer cell invasion. In BALB/c mice, irradiation of the mammary gland has stimulated the invasion of cancer cells, which was associated with a higher number of circulating tumour cells and of lung metastases. These adverse effects of radiation were prevented by downregulating the MT1-MMP. This study shows that the MT1-MMP is necessary for the radiation enhancement of lung metastasis development, and that its expression level and/or localisation could be evaluated as a biomarker for predicting the early recurrence observed in some TNBC patients.

  5. Endostar enhances the antitumor effects of radiation by affecting energy metabolism and alleviating the tumor microenvironment in a Lewis lung carcinoma mouse model

    PubMed Central

    ZHENG, YONG-FA; GE, WEI; XU, HUI-LIN; CAO, DE-DONG; LIU, LIANG; MING, PING-PO; LI, CHANG-HU; XU, XI-MING; TAO, WEI-PING; TAO, ZE-ZHANG

    2015-01-01

    Lung cancer is a leading cause of morbidity and mortality. Previous studies have identified that an improvement in treatment efficacy was achieved using Endostar; however, the role of Endostar in lung cancer remains poorly understood. The present study investigated whether the enhanced antitumor effects of Endostar in combination with radiation involved changes in the metabolism and microenvironment in non-small cell lung cancer. A Lewis lung carcinoma mouse model was used, including the control, Endostar (ES), radiotherapy (RT) and Endostar plus radiotherapy (ES + RT) groups. The tumor inhibition rates and growth were described based on changes in tumor volume. In addition, ultraviolet enzymatic analysis was performed to determine the lactate level and reverse transcription-polymerase chain reaction was used to measure the mRNA expression of lactate dehydrogenase (LDH). A Meph-3 pH meter was used to detect the ranges of tumor interstitial tissue pH, and immunohistochemical analysis was adopted to examine hypoxia within the tumor microenvironment. The tumor inhibition rate of the ES + RT group was significantly higher compared with the other three groups (P<0.05). Following treatment, the lactate levels decreased in all three treatment groups compared with the control, particularly in the ES + RT group (P<0.05). Reduced LDH expression and hypoxic fraction in the tumor microenvironment were also observed in the ES + RT group (P<0.05). Furthermore, changes from acidic to alkaline pH in the tumor microenvironment were detected in the ES + RT group. The present study suggested that Endostar is involved in the regulation of metabolism and tumor microenvironment hypoxia, which may be responsible for the enhanced antitumor effect of Endostar in combination with radiotherapy. PMID:26722291

  6. Aspect Ratio Plays a Role in the Hazard Potential of CeO2 Nanoparticles in Mouse Lung and Zebrafish Gastrointestinal Tract

    PubMed Central

    Lin, Sijie; Wang, Xiang; Ji, Zhaoxia; Chang, Chong Hyun; Dong, Yuan; Meng, Huan; Liao, Yu-Pei; Wang, Meiying; Song, Tze-Bin; Kohan, Sirus; Xia, Tian; Zink, Jeffrey I.; Lin, Shuo; Nel, André E.

    2014-01-01

    We have previously demonstrated that there is a relationship between the aspect ratio (AR) of CeO2 nanoparticles and in vitro hazard potential. CeO2 nanorods with AR ≥ 22 induced lysosomal damage and progressive effects on IL-1β production and cytotoxicity in the human myeloid cell line, THP-1. In order to determine whether this toxicological paradigm for long aspect ratio (LAR) CeO2 is also relevant in vivo, we performed comparative studies in the mouse lung and gastrointestinal tract (GIT) of zebrafish larvae. Although oropharyngeal aspiration could induce acute lung inflammation for CeO2 nanospheres and nanorods, only the nanorods with the highest AR (C5) induced significant IL-1β and TGF-β1 production in the bronchoalveolar lavage fluid (BALF) at 21 days but not inducing pulmonary fibrosis. However, after a longer duration (44 days) exposure to 4 mg/kg of the C5 nanorods, more collagen production was seen with CeO2 nanorods vs. nanospheres after correcting for Ce lung burden. Using an oral-exposure model in zebrafish larvae, we demonstrated that C5 nanorods also induced significant growth inhibition, a decrease in body weight, and delayed vertebral calcification. In contrast, CeO2 nanospheres and shorter nanorods had no effect. Histological and transmission electron microscopy (TEM) analyses showed that the key injury mechanism of C5 was in the epithelial lining of the GIT, which demonstrated blunted microvilli and compromised digestive function. All considered, these data demonstrate that, similar to cellular studies, LAR CeO2 nanorods exhibit more toxicity in the lung and GIT, which could be relevant to inhalation and environmental hazard potential. PMID:24720650

  7. Aspect ratio plays a role in the hazard potential of CeO2 nanoparticles in mouse lung and zebrafish gastrointestinal tract.

    PubMed

    Lin, Sijie; Wang, Xiang; Ji, Zhaoxia; Chang, Chong Hyun; Dong, Yuan; Meng, Huan; Liao, Yu-Pei; Wang, Meiying; Song, Tze-Bin; Kohan, Sirus; Xia, Tian; Zink, Jeffrey I; Lin, Shuo; Nel, André E

    2014-05-27

    We have previously demonstrated that there is a relationship between the aspect ratio (AR) of CeO2 nanoparticles and in vitro hazard potential. CeO2 nanorods with AR ≥ 22 induced lysosomal damage and progressive effects on IL-1β production and cytotoxicity in the human myeloid cell line, THP-1. In order to determine whether this toxicological paradigm for long aspect ratio (LAR) CeO2 is also relevant in vivo, we performed comparative studies in the mouse lung and gastrointestinal tract (GIT) of zebrafish larvae. Although oropharyngeal aspiration could induce acute lung inflammation for CeO2 nanospheres and nanorods, only the nanorods with the highest AR (C5) induced significant IL-1β and TGF-β1 production in the bronchoalveolar lavage fluid at 21 days but did not induce pulmonary fibrosis. However, after a longer duration (44 days) exposure to 4 mg/kg of the C5 nanorods, more collagen production was seen with CeO2 nanorods vs nanospheres after correcting for Ce lung burden. Using an oral-exposure model in zebrafish larvae, we demonstrated that C5 nanorods also induced significant growth inhibition, a decrease in body weight, and delayed vertebral calcification. In contrast, CeO2 nanospheres and shorter nanorods had no effect. Histological and transmission electron microscopy analyses showed that the key injury mechanism of C5 was in the epithelial lining of the GIT, which demonstrated blunted microvilli and compromised digestive function. All considered, these data demonstrate that, similar to cellular studies, LAR CeO2 nanorods exhibit more toxicity in the lung and GIT, which could be relevant to inhalation and environmental hazard potential.

  8. Inactivation of CD11b in a mouse transgenic model protects against sepsis-induced lung PMN infiltration and vascular injury.

    PubMed

    Gao, Xiao-Pei; Liu, Qinghui; Broman, Michael; Predescu, Dan; Frey, Randall S; Malik, Asrar B

    2005-04-14

    To inactivate chronically the beta2-integrin CD11b (Mac-1), we made a transgenic model in mice in which we expressed the CD11b antagonist polypeptide neutrophil inhibitory factor (NIF). Using these mice, we determined the in vivo effects of CD11b inactivation on polymorphonuclear leukocyte (PMN) function and acute lung injury (ALI) induced by Escherichia coli septicemia. In wild-type PMNs, CD11b expression was induced within 1 h after E. coli challenge, whereas this response was significantly reduced in NIF(+/+) PMNs. Coimmunoprecipitation studies showed that NIF associated with CD11b in NIF(+/+) PMNs. To validate the effectiveness of CD11b blockade, we compared PMN function in NIF(+/+) and Mac-1-deficient (Mac-1(-/-)) mice. Adhesion of both Mac-1(-/-) and NIF(+/+) PMNs to endothelial cells in response to LPS was reduced in both types of PMNs and fully blocked only by the addition of anti-CD11a monoclonal antibody. This finding is indicative of intact CD11a function in the NIF(+/+) PMNs but the blockade of CD11b function. CD11b inactivation in NIF(+/+) mice interfered with lung PMN infiltration induced by E. coli and prevented the increase in lung microvessel permeability and edema formation, with most of the protection seen in the 1-h period after the E. coli. Thus our results demonstrate that CD11b plays a crucial role in mediating lung PMN sequestration and vascular injury in the early phase of gram-negative septicemia. The NIF(+/+) mouse model, in which CD11b is inactivated by binding to NIF, is a potentially useful model for in vivo assessment of the role of PMN CD11b in the mechanism of vascular inflammation.

  9. Comparison of cell cycle components, apoptosis and cytoskeleton-related molecules and therapeutic effects of flavopiridol and geldanamycin on the mouse fibroblast, lung cancer and embryonic stem cells.

    PubMed

    Aktug, Huseyin; Acikgoz, Eda; Uysal, Aysegul; Oltulu, Fatih; Oktem, Gulperi; Yigitturk, Gurkan; Demir, Kenan; Yavasoglu, Altug; Bozok Cetintas, Vildan

    2016-09-01

    Similarities and differences in the cell cycle components, apoptosis and cytoskeleton-related molecules among mouse skin fibroblast cells (MSFs), mouse squamous cell lung carcinomas (SqCLCs) and mouse embryonic stem cells (mESCs) are important determinants of the behaviour and differentiation capacity of these cells. To reveal apoptotic pathways and to examine the distribution and the role of cell cycle-cell skeleton comparatively would necessitate tumour biology and stem cell biology to be assessed together in terms of oncogenesis and embryogenesis. The primary objectives of this study are to investigate the effects of flavopiridol, a cell cycle inhibitor, and geldanamycin, a heat shock protein inhibitor on mouse somatic, tumour and embryonic stem cells, by specifically focusing on alterations in cytoskeletal proteins, cell polarity and motility as well as cell cycle regulators. To meet these objectives, expression of several genes, cell cycle analysis and immunofluorescence staining of intracellular cytoskeletal molecules were performed in untreated and flavopiridol- or geldanamycin-treated cell lines. Cytotoxicity assays showed that SqCLCs are more sensitive to flavopiridol than MSFs and mESCs. Keratin-9 and keratin-2 expressions increased dramatically whereas cell cycle regulatory genes decreased significantly in the flavopiridol-treated MSFs. Flavopiridol-treated SqCLCs displayed a slight increase in several cell cytoskeleton regulatory genes as well as cell cycle regulatory genes. However, gene expression profiles of mESCs were not affected after flavopiridol treatment except th