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author:("began, Simone")
1.  In Vivo Noninvasive Detection of Brown Adipose Tissue through Intermolecular Zero-Quantum MRI 
PLoS ONE  2013;8(9):e74206.
The recent discovery of active Brown Adipose Tissue (BAT) in adult humans has opened new avenues for obesity research and treatment, as reduced BAT activity seem to be implicated in human energy imbalance, diabetes, and hypertension. However, clinical applications are currently limited by the lack of non-invasive tools for measuring mass and function of this tissue in humans. Here we present a new magnetic resonance imaging method based on the normally invisible intermolecular multiple-quantum coherence 1H MR signal. This method, which doesn’t require special hardware modifications, can be used to overcome partial volume effect, the major limitation of MR-based approaches that are currently being investigated for the detection of BAT in humans. With this method we can exploit the characteristic cellular structure of BAT to selectively image it, even when (as in humans) it is intimately mixed with other tissues. We demonstrate and validate this method in mice using PET scans and histology. We compare this methodology with conventional 1H MR fat fraction methods. Finally, we investigate its feasibility for the detection of BAT in humans.
PMCID: PMC3769256  PMID: 24040203
2.  SP-A Preserves Airway Homeostasis During Mycoplasma pneumoniae Infection in Mice 
The lung is constantly challenged during normal breathing by a myriad of environmental irritants and infectious insults. Pulmonary host defense mechanisms maintain homeostasis between inhibition/clearance of pathogens and regulation of inflammatory responses that could injure the airway epithelium. One component of this defense mechanism, surfactant protein-A (SP-A), exerts multifunctional roles in mediating host responses to inflammatory and infectious agents. SP-A has a bacteriostatic effect on Mycoplasma pneumoniae (Mp), which occurs by binding surface disaturated phosphatidylglycerols. SP-A can also bind the Mp membrane protein, MPN372. In this study we investigated the role of SP-A during acute phase pulmonary infection with Mp using mice deficient in SP-A. Biologic responses, inflammation and cellular infiltration, were much greater in Mp infected SP-A−/− mice than wild type mice. Likewise, physiologic responses (airway hyperresponsiveness and lung compliance) to Mp infection were more severely affected in SP-A−/− mice. Both Mp-induced biologic and physiologic changes were attenuated by pharmacologic inhibition of TNF-α. Our findings demonstrate that SP-A is vital to preserving lung homeostasis and host defense to this clinically relevant strain of Mp by curtailing inflammatory cell recruitment and limiting an overzealous TNF-α response.
PMCID: PMC3656438  PMID: 19494306
lung; inflammation; bacterial
3.  Optical clearing of archive-compatible paraffin embedded tissue for multiphoton microscopy: erratum 
Biomedical Optics Express  2013;4(2):219.
We correct an error in the methods reported in our previous paper [Biomed. Opt. Express 3, 2752 (2012)]; the liquid used for the final step of specimen preparation was 100% mineral oil, not a mixture of oil and glycerol.
PMCID: PMC3567708
(170.6900) Three-dimensional microscopy; (170.6930) Tissue; (110.0113) Imaging through turbid media
4.  Optical clearing of archive-compatible paraffin embedded tissue for multiphoton microscopy 
Biomedical Optics Express  2012;3(11):2752-2760.
Standard histopathology techniques (including paraffin embedding) are incompatible with thick tissue multiphoton imaging, and standard clearing techniques on those specimens destroy some molecular information. We demonstrate multiphoton imaging in specimens prepared according to standard histopathology techniques. This permits unlabeled 3-dimensional histology on archival tissue banks, which is of great value in evaluating prognostic indicators.
PMCID: PMC3493235  PMID: 23162713
(170.6900) Three-dimensional microscopy; (170.6930) Tissue; (110.0113) Imaging through turbid media
5.  The Role of Hyaluronan and Hyaluronan Binding Proteins in Human Asthma 
The characteristics of human asthma are chronic inflammation and airway remodeling. Hyaluronan (HA), a major extracellular matrix component, accumulates during inflammatory lung diseases including asthma. Hyaluronan fragments stimulate macrophages to produce inflammatory cytokines. We hypothesized that HA and its receptors would play a role in human asthma.
To investigate the role of HA and HA binding proteins in human asthma.
Twenty-one subjects with asthma and 25 normal control subjects underwent bronchoscopy with endobronchial biopsy and bronchoalveolar lavage (BAL). Fibroblasts were cultured, HA and HA synthase expression was determined at baseline and after exposure to several mediators relevant to asthma pathobiology. The expression of HA binding proteins, CD44, TLR2 and TLR4 on BAL macrophages was determined by flow cytometry. IL-8 production by macrophages in response to HA fragment stimulation was compared.
Airway fibroblasts from asthma patients produced significantly increased concentrations of lower molecular weight HA compared to those of normal fibroblasts. Hyaluronan synthase 2 mRNA was markedly increased in asthmatic fibroblasts. Asthmatic macrophages showed a decrease in cell surface CD44 expression and an increase in TLR2 and TLR4 expression. Macrophages from asthmatic subjects showed an increase in responsiveness to low molecular weight HA stimulation, as demonstrated by increased IL-8 production.
HA homeostasis is deranged in asthma with increased production by fibroblasts and decreased CD44 expression on alveolar macrophages. Upregulation of TLR2 and TLR4 on macrophages with increased sensitivity to HA fragments suggests a novel pro-inflammatory mechanism by which persistence of HA fragments could contribute to chronic inflammation and airway remodeling in asthma.
PMCID: PMC3149736  PMID: 21570715
Asthma; Hyaluronan; Cytokines; Fibroblasts; Macrophages
6.  Airway Fibroblasts in Asthma Manifest an Invasive Phenotype 
Rationale: Invasive cell phenotypes have been demonstrated in malignant transformation, but not in other diseases, such as asthma. Cellular invasiveness is thought to be mediated by transforming growth factor (TGF)-β1 and matrix metalloproteinases (MMPs). IL-13 is a key TH2 cytokine that directs many features of airway remodeling through TGF-β1 and MMPs.
Objectives: We hypothesized that, in human asthma, IL-13 stimulates increased airway fibroblast invasiveness via TGF-β1 and MMPs in asthma compared with normal controls.
Methods: Fibroblasts were cultured from endobronchial biopsies in 20 subjects with mild asthma (FEV1: 90 ± 3.6% pred) and 17 normal control subjects (FEV1: 102 ± 2.9% pred) who underwent bronchoscopy. Airway fibroblast invasiveness was investigated using Matrigel chambers. IL-13 or IL-13 with TGF-β1 neutralizing antibody or pan-MMP inhibitor (GM6001) was added to the lower chamber as a chemoattractant. Flow cytometry and immunohistochemistry were performed in a subset of subjects to evaluate IL-13 receptor levels.
Measurements and Main Results: IL-13 significantly stimulated invasion in asthmatic airway fibroblasts, compared with normal control subjects. Inhibitors of both TGF-β1 and MMPs blocked IL-13–induced invasion in asthma, but had no effect in normal control subjects. At baseline, in airway tissue, IL-13 receptors were expressed in significantly higher levels in asthma, compared with normal control subjects. In airway fibroblasts, baseline IL-13Rα2 was reduced in asthma compared with normal control subjects.
Conclusions: IL-13 potentiates airway fibroblast invasion through a mechanism involving TGF-β1 and MMPs. IL-13 receptor subunits are differentially expressed in asthma. These effects may result in IL-13–directed airway remodeling in asthma.
PMCID: PMC3136991  PMID: 21471104
airway remodeling; interleukin-13; transforming growth factor-β; matrix metalloproteinase
7.  c-Kit Is Essential for Alveolar Maintenance and Protection from Emphysema-like Disease in Mice 
Rationale: Previously, we demonstrated a candidate region for susceptibility to airspace enlargement on mouse chromosome 5. However, the specific candidate genes within this region accounting for emphysema-like changes remain unrecognized. c-Kit is a receptor tyrosine kinase within this candidate gene region that has previously been recognized to contribute to the survival, proliferation, and differentiation of hematopoietic stem cells. Increases in the percentage of cells expressing c-Kit have previously been associated with protection against injury-induced emphysema.
Objectives: Determine whether genetic variants of c-Kit are associated with spontaneous airspace enlargement.
Methods: Perform single-nucleotide polymorphism association studies in the mouse strains at the extremes of airspace enlargement phenotype for variants in c-Kit tyrosine kinase. Characterize mice bearing functional variants of c-Kit compared with wild-type controls for the development of spontaneous airspace enlargement. Epithelial cell proliferation was measured in culture.
Measurements and Main Results: Upstream regulatory single-nucleotide polymorphisms in the divergent mouse strains were associated with the lung compliance difference observed between the extreme strains. c-Kit mutant mice (KitW-sh/W-sh), when compared with genetic controls, developed altered lung histology, increased total lung capacity, increased residual volume, and increased lung compliance that persist into adulthood. c-Kit inhibition with imatinib attenuated in vitro proliferation of cells expressing epithelial cell adhesion molecule.
Conclusions: Our findings indicate that c-Kit sustains and/or maintains normal alveolar architecture in the lungs of mice. In vitro data suggest that c-Kit can regulate epithelial cell clonal expansion. The precise mechanisms that c-Kit contributes to the development of airspace enlargement and increased lung compliance remain unclear and warrants further investigation.
PMCID: PMC3136992  PMID: 21471107
genetic; tyrosine kinase; SASH; chronic obstructive pulmonary disease; aging
8.  Centrifugal Migration of Mesenchymal Cells in Embryonic Lung 
Developmental Dynamics  2008;237(3):750-757.
Murine lung development begins at embryonic day (E) 9.5. Normal lung structure and function depend on the patterns of localization of differentiated cells. Pulmonary mesenchymal cell lineages have been relatively unexplored. Importantly, there has been no prior evidence of clonality of any lung cells. Herein we use a definitive genetic approach to demonstrate a common origin for proximal and distal pulmonary mesenchymal cells. A retroviral library with 3,400 unique inserts was microinjected into the airway lumen of E11.5 lung buds. After 7–11 days of culture, buds were stained for placental alkaline phosphatase (PLAP). Most PLAP+ cells are peribronchial smooth muscle cells, initially localized laterally near the hilum, then migrating down airways to the subpleural region. Laser-capture microdissection and polymerase chain reaction confirm the clonal identities of PLAP+ cells proximally and distally. Our observation of this fundamental process during lung development opens new avenues for investigation of maladaptive mesenchymal responses in lung diseases.
PMCID: PMC3340126  PMID: 18297731
retrovirus; microinjection; alkaline phosphatase; smooth muscle; clonal analysis
9.  Nitric Oxide Mediates Relative Airway Hyporesponsiveness to Lipopolysaccharide in Surfactant Protein A–Deficient Mice 
Surfactant protein A (SP-A) mediates innate immune cell responses to LPS, a cell wall component of gram-negative bacteria that is found ubiquitously in the environment and is associated with adverse health effects. Inhaled LPS induces lung inflammation and increases airway responsiveness (AR). However, the role of SP-A in mediating LPS-induced AR is not well-defined. Nitric oxide (NO) is described as a potent bronchodilator, and previous studies showed that SP-A modulates the LPS-induced production of NO. Hence, we tested the hypothesis that increased AR, observed in response to aerosolized LPS exposure, would be significantly reduced in an SP-A–deficient condition. Wild-type (WT) and SP-A null (SP-A−/−) mice were challenged with aerosolized LPS. Results indicate that despite similar inflammatory indices, LPS-treated SP-A−/− mice had attenuated AR after methacholine challenge, compared with WT mice. The attenuated AR could not be attributed to inherent differences in SP-D concentrations or airway smooth muscle contractile and relaxation properties, because these measures were similar between WT and SP-A−/− mice. LPS-treated SP-A−/− mice, however, had elevated nitrite concentrations, inducible nitric oxide synthase (iNOS) expression, and NOS activity in their lungs. Moreover, the administration of the iNOS-specific inhibitor 1400W completely abrogated the attenuated AR. Thus, when exposed to aerosolized LPS, SP-A−/− mice demonstrate a relative airway hyporesponsiveness that appears to be mediated at least partly via an iNOS-dependent mechanism. These findings may have clinical significance, because recent studies reported associations between surfactant protein polymorphisms and a variety of lung diseases.
PMCID: PMC3049231  PMID: 20348208
surfactant protein A; lipopolysaccharide; airway responsiveness
10.  In vivo and ex vivo epi-mode pump-probe imaging of melanin and microvasculature 
Biomedical Optics Express  2011;2(6):1576-1583.
We performed epi-mode pump-probe imaging of melanin in excised human pigmented lesions and both hemoglobin and melanin in live xenograft mouse melanoma models to depths greater than 100 µm. Eumelanin and pheomelanin images, which have been previously demonstrated to differentiate melanoma from benign lesions, were acquired at the dermal-epidermal junction with cellular resolution and modest optical powers (down to 15 mW). We imaged dermal microvasculature with the same wavelengths, allowing simultaneous acquisition of melanin, hemoglobin and multiphoton autofluorescence images. Molecular pump-probe imaging of melanocytes, skin structure and microvessels allows comprehensive, non-invasive characterization of pigmented lesions.
PMCID: PMC3114225  PMID: 21698020
(170.3880) Medical and biological imaging; (180.5810) Scanning microscopy; (320.7150) Ultrafast spectroscopy
11.  NPAS1 Regulates Branching Morphogenesis in Embryonic Lung 
Drosophila trachealess (Trl), master regulator of tracheogenesis, has no known functional mammalian homolog. We hypothesized that genes similar to trachealess regulate lung development. Quantitative (Q)RT-PCR and immunostaining were used to determine spatial and temporal patterns of npas1 gene expression in developing murine lung. Immunostaining for α-smooth muscle actin demonstrated myofibroblasts, and protein gene product (PGP)9.5 identified neuroendocrine cells. Branching morphogenesis of embryonic lung buds was analyzed in the presence of antisense or sense oligodeoxynucleotides (ODN). Microarray analyses were performed to screen for changes in gene expression in antisense-treated lungs. QRT-PCR was used to validate the altered expression of key genes identified on the microarrays. We demonstrate that npas1 is expressed in murine embryonic lung. npas1 mRNA peaks early at Embryonic Day (E)10.5–E11.5, then drops to low levels. Sequencing verifies the identity of npas1 transcripts in embryonic lung. NPAS1 immunostaining occurs in nuclei of parabronchial mesenchymal cells, especially at the tracheal bifurcation. Arnt, the murine homolog of Tango (the heterodimerization partner for Trl) is also expressed in developing lung but at constant levels. npas1- or arnt-antisense ODN inhibit lung branching morphogenesis, with altered myofibroblast development and increased pulmonary neuroendocrine cells. On microarrays, we identify > 50 known genes down-regulated by npas1-antisense, including multiple genes regulating cell migration and cell differentiation. QRT-PCR confirms significantly decreased expression of the neurogenic genes RBP-Jk and Tle, and three genes involved in muscle development: β−ig-h3, claudin-11, and myocardin. Npas1 can regulate myofibroblast distribution, branching morphogenesis, and neuroendocrine cell differentiation in murine embryonic lung.
PMCID: PMC1899329  PMID: 17110583
branching morphogenesis; myofibroblasts; smooth muscle actin; cell migration; neuroendocrine cells
12.  Accelerated Thymic Maturation and Autoreactive T Cells in Bronchopulmonary Dysplasia 
Rationale: Bronchopulmonary dysplasia (BPD), a chronic lung disease of newborns triggered by oxygen and barotrauma, is characterized by arrested alveolarization. Increased levels of bombesin-like peptides shortly after birth mediate lung injury: anti-bombesin antibody 2A11 protects against BPD in two baboon models. The role of adaptive immunity in BPD has not been explored previously.
Objectives: Our goal was to test the hypothesis that thymic architecture and/or T-cell function is altered with BPD, leading to autoimmunity and immunodeficiency.
Methods: Thymic structure was analyzed by histopathology of thymic architecture and immunohistochemistry for thymic maturation markers (terminal deoxynucleotidyl transferase, proliferating cell nuclear antigen, CD4, and CD8). Thymic cortical epithelial cells (nurse cells) were studied using HLA-DR and protein gene product 9.5 as markers. Functional analysis was performed with “mixed lymphocyte reaction” of thymocyte or splenocyte responder cells with autologous lung cells as the stimulators.
Measurements and Main Results: 2A11 treatment attenuates thymic cortical involution in BPD animals, sustaining terminal deoxynucleotidyl transferase–positive prothymocytes and thymocyte proliferation. BPD animals have increased CD4+ cells in thymic cortex and lung interstitium, which are reduced by 2A11. Conversely, cortical protein gene product 9.5/HLA-DR–positive thymic nurse cells are depleted in BPD animals, but are preserved by 2A11-treatment. Whereas fetal thymocytes and splenocytes respond to phythemagglutinin/ionomycin and to a lesser extent, to autologous lung, BPD thymocytes and splenocytes are phythemagglutinin/ionomycin-unresponsive, and yet react strongly to autologous lung. The 2A11 normalizes these responses.
Conclusions: These observations suggest that bombesin-like peptides mediate premature thymic maturation and thymic nurse-cell depletion, leading to autoreactive T cells that could contribute to lung injury.
PMCID: PMC2662921  PMID: 16574933
animal model; bombesin; immunohistochemistry; mixed lymphocyte reaction; thymic nurse cells

Results 1-12 (12)