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1.  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.
doi:10.1371/journal.pone.0089503
PMCID: PMC3933627  PMID: 24586830
2.  Imaging the Interaction of Atelectasis and Overdistention in Surfactant Depleted Lungs 
Critical care medicine  2013;41(2):527-535.
Objective
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.
Design
Prospective imaging study in an animal model.
Setting
Research imaging facility
Subjects
27 healthy Sprague Dawley rats
Interventions
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).
Conclusions
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.
doi:10.1097/CCM.0b013e31826ab1f2
PMCID: PMC3557664  PMID: 23263577
Imaging/MRI; ARDS; Ventilator-Induced Lung Injury; Alveolar Recruitment; Artificial Respiration; Hyperpolarized Gas
3.  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.
doi:10.1002/mrm.24604
PMCID: PMC3834003  PMID: 23382040
4.  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.
doi:10.1371/journal.pone.0083629
PMCID: PMC3875446  PMID: 24386239
5.  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.
doi:10.1172/JCI71488
PMCID: PMC3871248  PMID: 24316976
6.  Accelerated Fractional Ventilation Imaging with Hyperpolarized Gas MRI 
PURPOSE
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.
METHODS
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.
RESULTS
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].
CONCLUSION
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.
doi:10.1002/mrm.24582
PMCID: PMC3833087  PMID: 23400938
Pulmonary ventilation; Quantitative lung imaging; Fractional ventilation; Hyperpolarized gas MRI; Mechanical ventilation; Parallel MRI; GRAPPA; Accelerated ventilation imaging
7.  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.
doi:10.1002/nbm.2763
PMCID: PMC3362674  PMID: 22290603
Pulmonary ventilation; Quantitative lung imaging; Fractional ventilation; Hyperpolarized gas MRI; Mechanical ventilation
8.  Establishment and characterization of a primary human chordoma xenograft model 
Journal of neurosurgery  2012;116(4):801-809.
Object
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.3171/2011.12.JNS111123
PMCID: PMC3715057  PMID: 22283186
animal model; brachyury; chordoma; xenograft; oncology
9.  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.
doi:10.3892/mco.2013.127
PMCID: PMC3915624  PMID: 24649241
Jumonji/Arid1B; esophageal cancer; gastrointestinal organs
10.  CognitionMaster: an object-based image analysis framework 
Diagnostic Pathology  2013;8:34.
Background
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.
Results
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.
Conclusions
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.
doi:10.1186/1746-1596-8-34
PMCID: PMC3626931  PMID: 23445542
Software; Open source; Image analysis; Object-based image analysis
11.  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.
doi:10.1172/JCI65054
PMCID: PMC3561830  PMID: 23321670
12.  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.
doi:10.1038/nprot.2009.129
PMCID: PMC3443679  PMID: 19696749
13.  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.
doi:10.1007/s10059-011-1010-x
PMCID: PMC3887611  PMID: 21360199
cell dynamics; chemokine; chemotaxis; lipid mediator; live imaging
14.  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.
doi:10.1172/JCI60746
PMCID: PMC3314466  PMID: 22406534
15.  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.
doi:10.1002/mrm.22186
PMCID: PMC3285263  PMID: 19877277
quantitative pulmonary imaging; regional lung ventilation; fractional ventilation; hyperpolarized gas MRI; mathematical models of lung
16.  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.
RATIONALE AND OBJECTIVES
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.
MATERIALS AND METHODS
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.
RESULTS
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).
CONCLUSION
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.
doi:10.1016/j.acra.2010.06.019
PMCID: PMC2953546  PMID: 20934126
bleomycin; interstitial lung disease; hyperpolarized 3He MRI; idiopathic pulmonary fibrosis; interstitial lung disease
17.  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.
doi:10.1016/j.jmr.2010.03.004
PMCID: PMC2885468  PMID: 20427216
18.  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.
doi:10.1084/jem.20101474
PMCID: PMC3005230  PMID: 21135136
19.  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.
doi:10.1002/mrm.21854
PMCID: PMC3056443  PMID: 19230017
Hyperpolarized 3He MRI; apparent diffusion coefficient; partial pressure of oxygen; multiple regression method
20.  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.
doi:10.1513/pats.200902-008AW
PMCID: PMC2731803  PMID: 19687215
hyperpolarized gas MRI; hyperpolarized 13C MRI; quantitative lung imaging
21.  Robust Motion Estimation and Structure Recovery from Endoscopic Image Sequences With an Adaptive Scale Kernel Consensus Estimator 
To correctly estimate the camera motion parameters and reconstruct the structure of the surrounding tissues from endoscopic image sequences, we need not only to deal with outliers (e.g., mismatches), which may involve more than 50% of the data, but also to accurately distinguish inliers (correct matches) from outliers. In this paper, we propose a new robust estimator, Adaptive Scale Kernel Consensus (ASKC), which can tolerate more than 50 percent outliers while automatically estimating the scale of inliers. With ASKC, we develop a reliable feature tracking algorithm. This, in turn, allows us to develop a complete system for estimating endoscopic camera motion and reconstructing anatomical structures from endoscopic image sequences. Preliminary experiments on endoscopic sinus imagery have achieved promising results.
doi:10.1109/CVPR.2008.4587687
PMCID: PMC2895276  PMID: 20607128
22.  Toward Video-Based Navigation for Endoscopic Endonasal Skull Base Surgery 
Endoscopic endonasal skull base surgery (ESBS) requires high accuracy to ensure safe navigation of the critical anatomy at the anterior skull base. Current navigation systems provide approximately 2mm accuracy. This level of registration error is due in part from the indirect nature of tracking used. We propose a method to directly track the position of the endoscope using video data. Our method first reconstructs image feature points from video in 3D, and then registers the reconstructed point cloud to pre-operative data (e.g. CT/MRI). After the initial registration, the system tracks image features and maintains the 2D–3D correspondence of image features and 3D locations. These data are then used to update the current camera pose. We present registration results within 1mm, which matches the accuracy of our validation framework.
doi:10.1007/978-3-642-04268-3_12
PMCID: PMC2850286  PMID: 20376205
23.  Anatomical reconstruction from endoscopic images: Toward quantitative endoscopy 
American journal of rhinology  2008;22(1):47-51.
Background
Recent advances in computational image processing have made it possible to reconstruct camera motion and scene geometry from a series of monocular images. By applying these methods to endoscopic image sequences, it is possible to create detailed, quantitative anatomic reconstructions. Such anatomic reconstructions have many potential clinical uses. Our objectives in this study are to (1) develop a process flow for reconstruction from endoscopic image sequences and (2) present results supporting the hypothesis that such reconstructions can be computed.
Methods
We first outline the overall process flow for endoscopic reconstruction. Then, we present an instantiation of this process flow using recently developed methods in computational vision. We apply these methods to cadaverous specimens for which ground truth endoscopic motion is known.
Results
We are able to produce consistent estimates of endoscopic motion and dense reconstructions of the surrounding anatomy for >65% of 1373 image pairs.
Conclusion
Our study indicates that processing endoscopic images to produce anatomic structure is feasible. Such reconstructions have high potential clinical value for intraoperative navigation, diagnosis, and treatment planning.
doi:10.2500/ajr.2008.22.3129
PMCID: PMC2850107  PMID: 18284859
Anatomic reconstruction; computer-integrated surgery; computer vision; endoscopy; image processing; navigation; robust statistics; surgery; visual modeling
24.  Sphingosine-1-phosphate mobilizes osteoclast precursors and regulates bone homeostasis 
Nature  2009;458(7237):524-528.
Osteoclasts (OCs) are bone-resorbing multinuclear giant cells that differentiate from mononuclear macrophage/monocyte-lineage hematopoietic precursors. Although many molecules are known to contribute to OC differentiation, RANKL chief among them, the mechanisms controlling the recruitment and homing of OC precursors (OPs) to the bone surface have not been elucidated. Here we report that sphingosine-1-phosphate (S1P) controls the movement of OPs between the blood and their site of differentiation. Cells with the properties of OPs express functional S1P1 receptors and exhibit positive chemotaxis along an S1P gradient in vitro. Intravital two-photon imaging of bone tissues revealed that a potent S1P1 agonist, SEW2871, stimulated motility of OP-containing monocytoid populations in vivo. OC/monocyte (CD11b) lineage-specific conditional S1P1 knockout mice showed osteoporotic changes due to increased OC attachment to bone surface, suggesting a crucial role of the S1P-S1P1 system in recirculation of OPs to blood where S1P levels are high. Furthermore, treatment with the S1P1 agonist FTY720 relieved ovariectomy-induced osteoporosis in mice by facilitating recirculation of OP-containing cell populations and reducing the number of mature OCs attached to the bone surface. This study provides evidence that S1P controls the migratory behavior of OPs, dynamically regulating bone mineral homeostasis, and identifies a critical control point in osteoclastogenesis that may be promising as a therapeutic target.
doi:10.1038/nature07713
PMCID: PMC2785034  PMID: 19204730
25.  Visualization and Identification of IL-7 Producing Cells in Reporter Mice 
PLoS ONE  2009;4(11):e7637.
Interleukin-7 (IL-7) is required for lymphocyte development and homeostasis although the actual sites of IL-7 production have never been clearly identified. We produced a bacterial artificial chromosome (BAC) transgenic mouse expressing ECFP in the Il7 locus. The construct lacked a signal peptide and ECFP (enhanced cyan fluorescent protein ) accumulated inside IL-7-producing stromal cells in thoracic thymus, cervical thymus and bone marrow. In thymus, an extensive reticular network of IL-7-containing processes extended from cortical and medullary epithelial cells, closely contacting thymocytes. Central memory CD8 T cells, which require IL-7 and home to bone marrow, physically associated with IL-7-producing cells as we demonstrate by intravital imaging.
doi:10.1371/journal.pone.0007637
PMCID: PMC2770321  PMID: 19907640

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