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1.  Metabolic spectroscopy of inflammation in a bleomycin-induced lung injury model using hyperpolarized 1-13C pyruvate 
NMR in biomedicine  2014;27(8):939-947.
Metabolic activity in the lung is known to change in response to external insults, inflammation, and in cancer. We report measurements of metabolism in the isolated, perfused rat lung of healthy controls and in diseased lungs undergoing acute inflammation using hyperpolarized 1-13C-labeled pyruvate. The overall apparent activity of lactate dehydrogenase is shown to increase significantly (on average by a factor of 3.3) at the 7-day acute stage and to revert substantially to baseline at 21 days, while other markers indicating monocarboxylate uptake and transamination rate are unchanged. Elevated lung lactate signal levels correlate well with phosphodiester levels as determined with 31P spectroscopy and to the presence of neutrophils as determined by histology, consistent with a relationship between intracellular lactate pool labeling and the density and type of inflammatory cells present. We discuss several alternate hypotheses, and conclude that the most probable source of the observed signal increase is direct uptake and metabolism of pyruvate by inflammatory cells and primarily neutrophils. This signal is seen in high contrast to the low baseline activity of the lung.
PMCID: PMC4110199  PMID: 24865640
Lung; Inflammation; Hyperpolarized; Metabolism; Neutrophil; Macrophage
2.  Intraoperative hypertensive crisis due to a catecholamine-secreting esthesioneuroblastoma 
Head & neck  2015;37(6):E74-E80.
Although uncommon, esthesioneuroblastomas may produce clinically significant amounts of catecholamines.
We report a patient with a catecholamine-secreting esthesioneuroblastoma who developed intraoperative hypertensive crisis.
A patient with history of hypertension was referred to our skull base center for management of a residual esthesioneuroblastoma. A staged endonasal endoscopic approach was planned. At the conclusion of the first stage, a hypertensive crisis occurred. Work-up revealed elevated levels of serum and urinary catecholamines. The patient was treated with alpha adrenoceptor blockade prior to the second stage. Serum catecholamine levels following this second stage were normal. On immunohistochemical analysis, the tumor cells were found to be positive for tyrosine hydroxylase, the rate limiting enzyme in cathecholamine synthesis, and achaete-scute homologue 1, a transcription factor essential in the development of olfactory and sympathetic neurons.
Catecholamine production should be considered in the differential of unexpected extreme hypertension during surgical resection of esthesioneuroblastoma.
PMCID: PMC4412780  PMID: 25352487
catecholamine; expanded endonasal endoscopic approach; esthesioneuroblastoma; hypertensive crisis; tyrosine hydroxylase
3.  Transplanted Bone Marrow–Derived Circulating PDGFRα+ Cells Restore Type VII Collagen in Recessive Dystrophic Epidermolysis Bullosa Mouse Skin Graft 
Recessive dystrophic epidermolysis bullosa (RDEB) is an intractable genetic blistering skin disease in which the epithelial structure easily separates from the underlying dermis because of genetic loss of functional type VII collagen (Col7) in the cutaneous basement membrane zone. Recent studies have demonstrated that allogeneic bone marrow transplantation (BMT) ameliorates the skin blistering phenotype of RDEB patients by restoring Col7. However, the exact therapeutic mechanism of BMT in RDEB remains unclear. In this study, we investigated the roles of transplanted bone marrow–derived circulating mesenchymal cells in RDEB (Col7-null) mice. In wild-type mice with prior GFP-BMT after lethal irradiation, lineage-negative/GFP-positive (Lin−/GFP+) cells, including platelet-derived growth factor receptor α-positive (PDGFRα+) mesenchymal cells, specifically migrated to skin grafts from RDEB mice and expressed Col7. Vascular endothelial cells and follicular keratinocytes in the deep dermis of the skin grafts expressed SDF-1α, and the bone marrow–derived PDGFRα+ cells expressed CXCR4 on their surface. Systemic administration of the CXCR4 antagonist AMD3100 markedly decreased the migration of bone marrow–derived PDGFRα+ cells into the skin graft, resulting in persistent epidermal detachment with massive necrosis and inflammation in the skin graft of RDEB mice; without AMD3100 administration, Col7 was significantly supplemented to ameliorate the pathogenic blistering phenotype. Collectively, these data suggest that the SDF1α/CXCR4 signaling axis induces transplanted bone marrow–derived circulating PDGFRα+ mesenchymal cells to migrate and supply functional Col7 to regenerate RDEB skin.
PMCID: PMC4319308  PMID: 25601922
4.  Regional Alveolar Partial Pressure of Oxygen Measurement with Parallel Accelerated Hyperpolarized Gas MRI 
Academic radiology  2013;20(10):10.1016/j.acra.2013.07.002.
PMCID: PMC3856723  PMID: 24029054
5.  Evaluation of a System for High-Accuracy 3D Image-Based Registration of Endoscopic Video to C-Arm Cone-Beam CT for Image-Guided Skull Base Surgery 
IEEE transactions on medical imaging  2013;32(7):1215-1226.
The safety of endoscopic skull base surgery can be enhanced by accurate navigation in preoperative computed tomography (CT) or, more recently, intraoperative cone-beam CT (CBCT). The ability to register real-time endoscopic video with CBCT offers an additional advantage by rendering information directly within the visual scene to account for intraoperative anatomical change. However, tracker localization error (~ 1–2 mm) limits the accuracy with which video and tomographic images can be registered. This paper reports the first implementation of image-based video-CBCT registration, conducts a detailed quantitation of the dependence of registration accuracy on system parameters, and demonstrates improvement in registration accuracy achieved by the image-based approach. Performance was evaluated as a function of parameters intrinsic to the image-based approach, including system geometry, CBCT image quality, and computational runtime. Overall system performance was evaluated in a cadaver study simulating transsphenoidal skull base tumor excision. Results demonstrated significant improvement (p < 0.001)in registration accuracy with a mean reprojection distance error of 1.28 mm for the image-based approach versus 1.82 mm for the conventional tracker-based method. Image-based registration was highly robust against CBCT image quality factors of noise and resolution, permitting integration with low-dose intraoperative CBCT.
PMCID: PMC4118820  PMID: 23372078
Cone-beam computed tomography (CBCT); endoscopy; image-guided treatment; registration; surgical guidance/navigation
6.  Dynamic Visualization of Dendritic Cell-Antigen Interactions in the Skin Following Transcutaneous Immunization 
PLoS ONE  2014;9(2):e89503.
Delivery of vaccines into the skin provides many advantages over traditional parenteral vaccination and is a promising approach due to the abundance of antigen presenting cells (APC) residing in the skin including Langerhans cells (LC) and dermal dendritic cells (DDC). However, the main obstacle for transcutaneous immunization (TCI) is the effective delivery of the vaccine through the stratum corneum (SC) barrier to the APC in the deeper skin layers. This study therefore utilized microneedles (MN) and a lipid-based colloidal delivery system (cubosomes) as a synergistic approach for the delivery of vaccines to APC in the skin. The process of vaccine uptake and recruitment by specific types of skin APC was investigated in real-time over 4 hours in B6.Cg-Tg (Itgax-EYFP) 1 Mnz/J mice by two-photon microscopy. Incorporation of the vaccine into a particulate delivery system and the use of MN preferentially increased vaccine antigen uptake by a highly motile subpopulation of skin APC known as CD207+ DC. No uptake of antigen or any response to immunisation by LC could be detected.
PMCID: PMC3933627  PMID: 24586830
7.  Imaging the Interaction of Atelectasis and Overdistention in Surfactant Depleted Lungs 
Critical care medicine  2013;41(2):527-535.
Atelectasis and surfactant depletion may contribute to greater distension – and thereby injury – of aerated lung regions; recruitment of atelectatic lung may protect these regions by attenuating such overdistension. However, the effects of atelectasis (and recruitment) on aerated airspaces remain elusive. We tested the hypothesis that during mechanical ventilation, surfactant depletion increases the dimensions of aerated airspaces and that lung recruitment reverses these changes.
Prospective imaging study in an animal model.
Research imaging facility
27 healthy Sprague Dawley rats
Surfactant depletion was obtained by saline lavage in anesthetized, ventilated rats. Alveolar recruitment was accomplished using positive end-expiratory pressure (PEEP) and exogenous surfactant administration.
Measurements and Main Results
Airspace dimensions were estimated by measuring the apparent diffusion coefficient (ADC) of 3He, using diffusion-weighted hyperpolarized gas magnetic resonance imaging (MRI). Atelectasis was demonstrated using computerized tomography (CT) and by measuring oxygenation. Saline lavage increased atelectasis (increase in non-aerated tissue from 1.2 to 13.8% of imaged area, P<0.001), and produced a concomitant increase in mean ADC (~33%, P<0.001) vs. baseline; the heterogeneity of the CT signal and the variance of ADC were also increased. Application of PEEP and surfactant reduced the mean ADC (~23%, P<0.001), and its variance, in parallel to alveolar recruitment (i.e. less CT densities and heterogeneity, increased oxygenation).
Overdistension of aerated lung occurs during atelectasis, is detectable using clinically relevant MRI technology, and could be a key factor in the generation of lung injury during mechanical ventilation. Lung recruitment by higher PEEP and surfactant administration reduces airspace distension.
PMCID: PMC3557664  PMID: 23263577
Imaging/MRI; ARDS; Ventilator-Induced Lung Injury; Alveolar Recruitment; Artificial Respiration; Hyperpolarized Gas
8.  A Variability Study of Regional Alveolar Oxygen Tension Measurement in Humans Using Hyperpolarized 3He MRI 
To presents the first systematic reproducibility measurement of alveolar partial pressure of oxygen (pAO2) in the human lung, regional variability is defined in terms of an intraclass correlation coefficient (ICC) between co-localized, same-subject measurements separated by one-week or couple of minutes (short-term). In addition, the repeatability of the average lung pAO2 is compared to that of the standard pulmonary function tests (PFT).
PFT and pAO2 imaging were performed on eight subjects: 4 nonsmokers (1 man, 3 women; 56 ± 1.7 years), 4 smokers (1 woman, 3 men; 52 ± 7.5 years) in three visits during two weeks. Regional variability was assessed based on a mixed-effects model and an ICC. The coefficient of variation (CV) of mean and standard deviation of pAO2 in three days was also compared to CV of PFT results.
Short-term regional reproducibility based on ICC was 0.71 and 0.63 for nonsmokers and smokers; respectively. The one-week variability was lower (ICC=0.59 and 0.47; respectively). The CV of whole-lung average pAO2 was significantly higher than that of FVC(forced vital capacity; P=0.02) but not from DLCO (diffusing capacity).
The smoker group shows more variability in pAO2 measurements both between experiments and in each individual pAO2 maps. pAO2 had a similar repeatability to DLCO.
PMCID: PMC3834003  PMID: 23382040
9.  Cell Cycle-Dependent Rho GTPase Activity Dynamically Regulates Cancer Cell Motility and Invasion In Vivo 
PLoS ONE  2013;8(12):e83629.
The mechanism behind the spatiotemporal control of cancer cell dynamics and its possible association with cell proliferation has not been well established. By exploiting the intravital imaging technique, we found that cancer cell motility and invasive properties were closely associated with the cell cycle. In vivo inoculation of human colon cancer cells bearing fluorescence ubiquitination-based cell cycle indicator (Fucci) demonstrated an unexpected phenomenon: S/G2/M cells were more motile and invasive than G1 cells. Microarray analyses showed that Arhgap11a, an uncharacterized Rho GTPase-activating protein (RhoGAP), was expressed in a cell-cycle-dependent fashion. Expression of ARHGAP11A in cancer cells suppressed RhoA-dependent mechanisms, such as stress fiber formation and focal adhesion, which made the cells more prone to migrate. We also demonstrated that RhoA suppression by ARHGAP11A induced augmentation of relative Rac1 activity, leading to an increase in the invasive properties. RNAi-based inhibition of Arhgap11a reduced the invasion and in vivo expansion of cancers. Additionally, analysis of human specimens showed the significant up-regulation of Arhgap11a in colon cancers, which was correlated with clinical invasion status. The present study suggests that ARHGAP11A, a cell cycle-dependent RhoGAP, is a critical regulator of cancer cell mobility and is thus a promising therapeutic target in invasive cancers.
PMCID: PMC3875446  PMID: 24386239
10.  LRIG1 inhibits STAT3-dependent inflammation to maintain corneal homeostasis 
Corneal integrity and transparency are indispensable for good vision. Cornea homeostasis is entirely dependent upon corneal stem cells, which are required for complex wound-healing processes that restore corneal integrity following epithelial damage. Here, we found that leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) is highly expressed in the human holoclone-type corneal epithelial stem cell population and sporadically expressed in the basal cells of ocular-surface epithelium. In murine models, LRIG1 regulated corneal epithelial cell fate during wound repair. Deletion of Lrig1 resulted in impaired stem cell recruitment following injury and promoted a cell-fate switch from transparent epithelium to keratinized skin-like epidermis, which led to corneal blindness. In addition, we determined that LRIG1 is a negative regulator of the STAT3-dependent inflammatory pathway. Inhibition of STAT3 in corneas of Lrig1–/– mice rescued pathological phenotypes and prevented corneal opacity. Additionally, transgenic mice that expressed a constitutively active form of STAT3 in the corneal epithelium had abnormal features, including corneal plaques and neovascularization similar to that found in Lrig1–/– mice. Bone marrow chimera experiments indicated that LRIG1 also coordinates the function of bone marrow–derived inflammatory cells. Together, our data indicate that LRIG1 orchestrates corneal-tissue transparency and cell fate during repair, and identify LRIG1 as a key regulator of tissue homeostasis.
PMCID: PMC3871248  PMID: 24316976
11.  Accelerated Fractional Ventilation Imaging with Hyperpolarized Gas MRI 
To investigate the utility of accelerated imaging to enhance multi-breath fractional ventilation (r) measurement accuracy using HP gas MRI. Undersampling shortens the breath-hold time, thereby reducing the O2-induced signal decay and allows subjects to maintain a more physiologically relevant breathing pattern. Additionally it may improve r estimation accuracy by reducing RF destruction of HP gas.
Image acceleration was achieved by using an 8-channel phased array coil. Undersampled image acquisition was simulated in a series of ventilation images and images were reconstructed for various matrix sizes (48–128) using GRAPPA. Parallel accelerated r imaging was also performed on five mechanically ventilated pigs.
Optimal acceleration factor was fairly invariable (2.0–2.2×) over the range of simulated resolutions. Estimation accuracy progressively improved with higher resolutions (39–51% error reduction). In vivo r values were not significantly different between the two methods: 0.27±0.09, 0.35±0.06, 0.40±0.04 (standard) versus 0.23±0.05, 0.34±0.03, 0.37±0.02 (accelerated); for anterior, medial and posterior slices, respectively, whereas the corresponding vertical r gradients were significant (P < 0.001): 0.021±0.007 (standard) versus 0.019±0.005 (accelerated) [cm−1].
Quadruple phased array coil simulations resulted in an optimal acceleration factor of ~2× independent of imaging resolution. Results advocate undersampled image acceleration to improve accuracy of fractional ventilation measurement with HP gas MRI.
PMCID: PMC3833087  PMID: 23400938
Pulmonary ventilation; Quantitative lung imaging; Fractional ventilation; Hyperpolarized gas MRI; Mechanical ventilation; Parallel MRI; GRAPPA; Accelerated ventilation imaging
12.  Multi-slice Fractional Ventilation Imaging in Large Animals with Hyperpolarized Gas MRI 
NMR in biomedicine  2012;25(9):1015-1025.
Noninvasive assessment of regional lung ventilation is of critical importance in quantifying the severity of disease and evaluating response to therapy in many pulmonary diseases. This work presents for the first time the implementation of a hyperpolarized (HP) gas MRI technique for measuring whole-lung regional fractional ventilation (r) in Yorkshire pigs (n = 5) through the use of a gas mixing and delivery device in supine position. The proposed technique utilizes a series of back-to-back HP gas breaths with images acquired during short end-inspiratory breath-holds. In order to decouple the RF pulse decay effect from ventilatory signal build-up in the airways, regional distribution of flip angle (α) was estimated in the imaged slices by acquiring a series of back-to-back images with no inter-scan time delay during a breath-hold at the tail-end of the ventilation sequence. Analysis was performed to assess the multi-slice ventilation model sensitivity to noise, oxygen and number of flip angle images. The optimal α value was determined based on minimizing the error in r estimation; αopt = 5–6° for the set of acquisition parameters in pigs. The mean r values for the group of pigs were 0.27±0.09, 0.35±0.06, 0.40±0.04 for ventral, middle and dorsal slices, respectively, (excluding conductive airways r > 0.9). A positive gravitational (ventral-dorsal) ventilation gradient effect was present in all animals. The trachea and major conductive airways showed a uniform near-unity r value, with progressively smaller values corresponding to smaller diameter airways, and ultimately leading to lung parenchyma. Results demonstrate the feasibility of measurements of fractional ventilation in large species, and provides a platform to address technical challenges associated with long breathing time scales through the optimization of acquisition parameters in species with a pulmonary physiology very similar to that of human beings.
PMCID: PMC3362674  PMID: 22290603
Pulmonary ventilation; Quantitative lung imaging; Fractional ventilation; Hyperpolarized gas MRI; Mechanical ventilation
13.  Establishment and characterization of a primary human chordoma xenograft model 
Journal of neurosurgery  2012;116(4):801-809.
Chordomas are rare tumors arising from remnants of the notochord. Because of the challenges in achieving a complete resection, the radioresistant nature of these tumors, and the lack of effective chemotherapeutics, the median survival for patients with chordomas is approximately 6 years. Reproducible preclinical model systems that closely mimic the original patient’s tumor are essential for the development and evaluation of effective therapeutics. Currently, there are only a few established chordoma cell lines and no primary xenograft model. In this study, the authors aimed to develop a primary chordoma xenograft model.
The authors implanted independent tumor samples from 2 patients into athymic nude mice. The resulting xenograft line was characterized by histopathological analysis and immunohistochemical staining. The patient’s tumor and serial passages of the xenograft were genomically analyzed using a 660,000 single-nucleotide polymorphism array.
A serially transplantable xenograft was established from one of the 2 patient samples. Histopathological analysis and immunohistochemical staining for S100 protein, epithelial membrane antigen, and cytokeratin AE1/AE3 of the primary patient sample and the xenografts confirmed that the xenografts were identical to the original chordoma obtained from the patient. Immunohistochemical staining and western blot analysis confirmed the presence of brachyury, a recently described marker of chordomas, in the tumor from the patient and each of the xenografts. Genome-wide variation was assessed between the patient’s tumor and the xenografts and was found to be more than 99.9% concordant.
To the best of their knowledge, the authors have established the first primary chordoma xenograft that will provide a useful preclinical model for this disease and a platform for therapeutic development.
PMCID: PMC3715057  PMID: 22283186
animal model; brachyury; chordoma; xenograft; oncology
14.  Jumonji/Arid1b (Jarid1b) protein modulates human esophageal cancer cell growth 
Molecular and Clinical Oncology  2013;1(4):753-757.
Although esophageal cancer is highly heterogeneous and the involvement of epigenetic regulation of cancer stem cells is highly suspected, the biological significance of epigenetically modified molecules that regulate different subpopulations remains to be firmly established. Using esophageal cancer cells, we investigated the functional roles of the H3K4 demethylase Jumonji/Arid1b (Jarid1b) (Kdm5b/Plu-1/Rbp2-h1), an epigenetic factor that is required for continuous cell growth in melanoma. JARID1B knockdown resulted in the suppression of esophageal cancer cell growth, sphere formation and invasion ability and was associated with loss of epithelial marker expression. However, these inhibitory effects observed on tumor formation were reverted subsequent to subcutaneous inoculation of these cells into immune-deficient mice. These results indicated that JARID1B plays a role in maintaining cancer stem cells in the esophagus and justifies the rationale for studying the effects of continuous inhibition of this epigenetic factor in esophageal cancer.
PMCID: PMC3915624  PMID: 24649241
Jumonji/Arid1B; esophageal cancer; gastrointestinal organs
15.  CognitionMaster: an object-based image analysis framework 
Diagnostic Pathology  2013;8:34.
Automated image analysis methods are becoming more and more important to extract and quantify image features in microscopy-based biomedical studies and several commercial or open-source tools are available. However, most of the approaches rely on pixel-wise operations, a concept that has limitations when high-level object features and relationships between objects are studied and if user-interactivity on the object-level is desired.
In this paper we present an open-source software that facilitates the analysis of content features and object relationships by using objects as basic processing unit instead of individual pixels. Our approach enables also users without programming knowledge to compose “analysis pipelines“ that exploit the object-level approach. We demonstrate the design and use of example pipelines for the immunohistochemistry-based cell proliferation quantification in breast cancer and two-photon fluorescence microscopy data about bone-osteoclast interaction, which underline the advantages of the object-based concept.
We introduce an open source software system that offers object-based image analysis. The object-based concept allows for a straight-forward development of object-related interactive or fully automated image analysis solutions. The presented software may therefore serve as a basis for various applications in the field of digital image analysis.
PMCID: PMC3626931  PMID: 23445542
Software; Open source; Image analysis; Object-based image analysis
16.  Dynamic visualization of RANKL and Th17-mediated osteoclast function 
Osteoclasts are bone resorbing, multinucleate cells that differentiate from mononuclear macrophage/monocyte-lineage hematopoietic precursor cells. Although previous studies have revealed important molecular signals, how the bone resorptive functions of such cells are controlled in vivo remains less well characterized. Here, we visualized fluorescently labeled mature osteoclasts in intact mouse bone tissues using intravital multiphoton microscopy. Within this mature population, we observed cells with distinct motility behaviors and function, with the relative proportion of static – bone resorptive (R) to moving – nonresorptive (N) varying in accordance with the pathophysiological conditions of the bone. We also found that rapid application of the osteoclast-activation factor RANKL converted many N osteoclasts to R, suggesting a novel point of action in RANKL-mediated control of mature osteoclast function. Furthermore, we showed that Th17 cells, a subset of RANKL-expressing CD4+ T cells, could induce rapid N-to-R conversion of mature osteoclasts via cell-cell contact. These findings provide new insights into the activities of mature osteoclasts in situ and identify actions of RANKL-expressing Th17 cells in inflammatory bone destruction.
PMCID: PMC3561830  PMID: 23321670
17.  Quantifying cellular interaction dynamics in 3-D fluorescence microscopy data 
Nature protocols  2009;4(9):1305-1311.
The wealth of information available from advanced fluorescence imaging techniques used to analyze biological processes with high spatial and temporal resolution calls for high-throughput image analysis methods. Here, we describe a fully automated approach to analyzing cellular interaction behavior in 3-D fluorescence microscopy images. As example application we present the analysis of drug-induced and S1P1-knock-out-related changes in bone-osteoclast interactions. Moreover, we apply our approach to images showing the spatial association of dendritic cells with the fibroblastic reticular cell network within lymph nodes and to microscopy data about T-B lymphocyte synapse formation. Such analyses that yield important information about the molecular mechanisms determining cellular interaction behavior would be very difficult to perform with approaches that rely on manual/semi-automated analyses. This protocol integrates adaptive threshold segmentation, object detection, adaptive color channel merging and neighborhood analysis and permits rapid, standardized, quantitative analysis and comparison of the relevant features in large data sets.
PMCID: PMC3443679  PMID: 19696749
18.  The Role of Sphingosine 1-Phosphate in Migration of Osteoclast Precursors; an Application of Intravital Two-Photon Microscopy 
Molecules and Cells  2011;31(5):399-403.
Sphingosine-1-phosphate (S1P), a biologically active lysophospholipid that is enriched in blood, controls the trafficking of osteoclast precursors between the circulation and bone marrow cavities via G protein-coupled receptors, S1PRs. While S1PR1 mediates chemoattraction toward S1P in bone marrow, where S1P concentration is low, S1PR2 mediates chemorepulsion in blood, where the S1P concentration is high. The regulation of precursor recruitment may represent a novel therapeutic strategy for controlling osteoclast-dependent bone remodeling. Through intravital multiphoton imaging of bone tissues, we reveal that the bidirectional function of S1P temporospatially regulates the migration of osteoclast precursors within intact bone tissues. Imaging technologies have enabled in situ visualization of the behaviors of several players in intact tissues. In addition, intravital microscopy has the potential to be more widely applied to functional analysis and intervention.
PMCID: PMC3887611  PMID: 21360199
cell dynamics; chemokine; chemotaxis; lipid mediator; live imaging
19.  The sphingosine-1-phosphate transporter Spns2 expressed on endothelial cells regulates lymphocyte trafficking in mice 
The Journal of Clinical Investigation  2012;122(4):1416-1426.
The bioactive lysophospholipid mediator sphingosine-1-phosphate (S1P) promotes the egress of newly formed T cells from the thymus and the release of immature B cells from the bone marrow. It has remained unclear, however, where and how S1P is released. Here, we show that in mice, the S1P transporter spinster homolog 2 (Spns2) is responsible for the egress of mature T cells and immature B cells from the thymus and bone marrow, respectively. Global Spns2-KO mice exhibited marked accumulation of mature T cells in thymi and decreased numbers of peripheral T cells in blood and secondary lymphoid organs. Mature recirculating B cells were reduced in frequency in the bone marrow as well as in blood and secondary lymphoid organs. Bone marrow reconstitution studies revealed that Spns2 was not involved in S1P release from blood cells and suggested a role for Spns2 in other cells. Consistent with these data, endothelia-specific deletion of Spns2 resulted in defects of lymphocyte egress similar to those observed in the global Spns2-KO mice. These data suggest that Spns2 functions in ECs to establish the S1P gradient required for T and B cells to egress from their respective primary lymphoid organs. Furthermore, Spns2 could be a therapeutic target for a broad array of inflammatory and autoimmune diseases.
PMCID: PMC3314466  PMID: 22406534
20.  Improved Technique for Measurement of Regional Fractional Ventilation by Hyperpolarized 3He MRI 
Magnetic Resonance in Medicine  2010;63(1):137-150.
Quantitative measurement of regional lung ventilation is of great significance in assessment of lung function in many obstructive and restrictive pulmonary diseases. A new technique for regional measurement of fractional ventilation using hyperpolarized 3He MRI is proposed, addressing the shortcomings of an earlier approach that limited its use to small animals. The new approach allows for the acquisition of similar quantitative maps over a shortened period and requires substantially less 3He gas. This technique is therefore a better platform for implementation in large species, including humans. The measurements using the two approaches were comparable to a great degree, as verified in a healthy rat lung, and are very reproducible. Preliminary validation is performed in a lung phantom system. Volume dependency of measurements was assessed both in vivo and in vitro. A scheme for selecting an optimum flip angle is proposed. In addition, a dead space modeling approach is proposed to yield more accurate measurements of regional fractional ventilation using either method. Finally, sensitivity of the new technique to model parameters, noise, and number of included images were assessed numerically. As a prelude to application in humans, the technique was implemented in a large animal study successfully.
PMCID: PMC3285263  PMID: 19877277
quantitative pulmonary imaging; regional lung ventilation; fractional ventilation; hyperpolarized gas MRI; mathematical models of lung
21.  Quantitative Assessment of Lung Ventilation and Microstructure in an Animal Model of Idiopathic Pulmonary Fibrosis Using Hyperpolarized Gas MRI 
Academic radiology  2010;17(11):1433-1443.
The use of hyperpolarized 3He MRI as a quantitative lung imaging tool has progressed rapidly in the past decade, mostly in assessment of the airways diseases COPD and asthma. This technique has shown potential to assess both structural and functional information in healthy and diseased lungs. In this study, we apply the regional measurements of structure and function to a bleomycin rat model of interstitial lung disease.
Male Sprague Dawley rats (300–350 g) were administered intra-tracheal bleomycin. After 3 weeks, apparent diffusion coefficient and fractional ventilation were measured by 3He MRI and pulmonary function testing using a rodent-specific plethysmography chamber. Sensitized and healthy animals were then compared using threshold analysis to assess the potential sensitivity of these techniques to pulmonary abnormalities.
No significant changes were observed in total lung volume and compliance between the two groups. Airway resistance elevated and forced expiratory volume significantly declined in the 3-wk bleomycin rats, and fractional ventilation was significantly decreased compared to control animals (p < 4×10−4). Apparent diffusion coefficient of 3He showed a smaller change, but still a significant decrease in 3-wk bleomycin animals (p < 0.05).
Preliminary results suggest that quantitative 3He MRI can be a sensitive and non-invasive tool to assess changes in an animal interstitial lung disease model. This technique may be useful for longitudinal animal studies and also in the investigation of human interstitial lung diseases.
PMCID: PMC2953546  PMID: 20934126
bleomycin; interstitial lung disease; hyperpolarized 3He MRI; idiopathic pulmonary fibrosis; interstitial lung disease
22.  Optimal transfer of spin-order between a singlet nuclear pair and a heteronucleus 
Intramolecular spin-order transfer is a useful technique for signal enhancement of insensitive and low-concentration molecular species. We present a closed-form, optimized pulse sequence which maximizes the efficiency of transfer between a singlet (para) nuclear pair and a vicinal heteronucleus. Neglecting the decay of coherences while the nuclei are in the transverse plane, the scheme is unity efficient for all combinations of internuclear scalar couplings. Efficiency loss due to T2-like decay is also minimized by keeping the sequence as short as possible. We expect this result to be useful for hyperpolarization experiments in which the spin-order originates in parahydrogen, as well as studies of singlet state decay aimed at longer-term storage of spin-order in hyperpolarized Magnetic Resonance Imaging.
PMCID: PMC2885468  PMID: 20427216
23.  Chemorepulsion by blood S1P regulates osteoclast precursor mobilization and bone remodeling in vivo 
The Journal of Experimental Medicine  2010;207(13):2793-2798.
Chemotaxis and chemorepulsion of osteoclast precursors depends on S1P concentrations and expression of the receptors S1PR1 and S1PR2, which act to regulate osteoclast precursor localization.
Sphingosine-1-phosphate (S1P), a lipid mediator enriched in blood, controls the dynamic migration of osteoclast (OC) precursors (OPs) between the blood and bone, in part via the S1P receptor 1 (S1PR1) which directs positive chemotaxis toward S1P. We show that OPs also express S1PR2, an S1P receptor which mediates negative chemotaxis (or chemorepulsion). OP-positive chemotaxis is prominent in gradients with low maximal concentrations of S1P, whereas such behavior is minimal in fields with high maximal S1P concentrations. This reverse-directional behavior is caused by S1PR2-mediated chemorepulsion acting to override S1PR1 upgradient motion. S1PR2-deficient mice exhibit moderate osteopetrosis as a result of a decrease in osteoclastic bone resorption, suggesting that S1PR2 contributes to OP localization on the bones mediated by chemorepulsion away from the blood where S1P levels are high. Inhibition of S1PR2 function by the antagonist JTE013 changed the migratory behavior of monocytoid cells, including OPs, and relieved osteoporosis in a mouse model by limiting OP localization and reducing the number of mature OCs attached to the bone surface. Thus, reciprocal regulation of S1P-dependent chemotaxis controls bone remodeling by finely regulating OP localization. This regulatory axis may be promising as a therapeutic target in diseases affecting OC-dependent bone remodeling.
PMCID: PMC3005230  PMID: 21135136
24.  Simultaneous measurement of pulmonary partial pressure of oxygen and apparent diffusion coefficient by hyperpolarized 3He MRI 
Hyperpolarized 3He (HP 3He) MRI shows promise to assess structural and functional pulmonary parameters in a sensitive, regional and non-invasive way. Structural HP 3He MRI has applied the apparent diffusion coefficient (ADC) for the detection of disease-induced lung microstructure changes at the alveolar level, and HP 3He pulmonary partial pressure of oxygen (pO2) imaging measures the oxygen transfer efficiency between the lung and blood stream. Although both parameters are affected in chronic obstructive pulmonary disease (COPD), a quantitative assessment of the regional correlation of the two parameters has not been reported in the literature. In this work, a single acquisition technique for the simultaneous measurement of ADC and pO2 is presented. This technique is based on the multiple regression method, in which a general linear estimator is used to retrieve the values of ADC and pO2 from a series of measurements. The measurement uncertainties are also analytically derived and used to find an optimal measurement scheme. The technique was first tested on a phantom model, and then on an in-vivo normal pig experiment. A case study was performed on a COPD patient, which showed that in a region-of-interest ADC was 29% higher while oxygen depletion rate was 61% lower than the corresponding global average values.
PMCID: PMC3056443  PMID: 19230017
Hyperpolarized 3He MRI; apparent diffusion coefficient; partial pressure of oxygen; multiple regression method
25.  Quantitative Assessment of Lung Using Hyperpolarized Magnetic Resonance Imaging 
Improvements in the quantitative assessment of structure, function, and metabolic activity in the lung, combined with improvements in the spatial resolution of those assessments, enhance the diagnosis and evaluation of pulmonary disorders. Radiologic methods are among the most attractive techniques for the comprehensive assessment of the lung, as they allow quantitative assessment of this organ through measurements of a number of structural, functional, and metabolic parameters. Hyperpolarized nuclei magnetic resonance imaging (MRI) has opened up new territories for the quantitative assessment of lung function and structure with an unprecedented spatial resolution and sensitivity. This review article presents a survey of recent developments in the field of pulmonary imaging using hyperpolarized nuclei MRI for quantitative imaging of different aspects of the lung, as well as preclinical applications of these techniques to diagnose and evaluate specific pulmonary diseases. After presenting a brief overview of various hyperpolarization techniques, this survey divides the research activities of the field into four broad areas: lung microstructure, ventilation, oxygenation, and perfusion. Finally, it discusses the challenges currently faced by researchers in this field to translate this rich body of methodology into wider-scale clinical applications.
PMCID: PMC2731803  PMID: 19687215
hyperpolarized gas MRI; hyperpolarized 13C MRI; quantitative lung imaging

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