Search

PDF file, 82K.

PDF file, 77K.

PDF file, 157K, Ex vivo serum rescue assay.

PDF file, 304K, Effect of copper on virus therapy for glioma cells.

Purpose: Copper in serum supports angiogenesis and inhibits replication of wild-type HSV-1. Copper chelation is currently being investigated as an antiangiogenic and antineoplastic agent in patients diagnosed with cancer. Herpes simplex virus–derived oncolytic viruses (oHSV) are being evaluated for safety and efficacy in patients, but several host barriers limit their efficacy. Here, we tested whether copper inhibits oHSV infection and replication and whether copper chelation would augment therapeutic efficacy of oHSV.Experimental Design: Subcutaneous and intracranial tumor-bearing mice were treated with oHSV ± ATN-224 to evaluate tumor burden and survival. Virus replication and cell killing was measured in the presence or absence of the copper chelating agent ATN-224 and in the presence or absence of copper in vitro. Microvessel density and changes in perfusion were evaluated by immunohistochemistry and dynamic contrast enhanced MRI (DCE-MRI). Serum stability of oHSV was measured in mice fed with ATN-224. Tumor-bearing mice were injected intravenously with oHSV; tumor burden and amount of virus in tumor tissue were evaluated.Results: Combination of systemic ATN-224 and oHSV significantly reduced tumor growth and prolonged animal survival. Immunohistochemistry and DCE-MRI imaging confirmed that ATN-224 reduced oHSV-induced blood vessel density and vascular leakage. Copper at physiologically relevant concentrations inhibited oHSV replication and glioma cell killing, and this effect was rescued by ATN-224. ATN-224 increased serum stability of oHSV and enhanced the efficacy of systemic delivery.Conclusion: This study shows that combining ATN-224 with oHSV significantly increased serum stability of oHSV and greatly enhanced its replication and antitumor efficacy. Clin Cancer Res; 18(18); 4931–41. ©2012 AACR.

PDF file, 65K, ATN-224 by itself does not affect glioma cell viability or oHSV efficacy in the absence of copper.

PDF file, 46K, Quantification of color coded Ktrans and ve parametric images following DCE-MRI.

Supplementary Methods, Table 1, Figures 1-8 from Loss of Metallothionein Predisposes Mice to Diethylnitrosamine-Induced Hepatocarcinogenesis by Activating NF-κB Target Genes

Aortic aneurism open repair surgery can cause spinal cord (SC) injury with 5-15% of patients developing paraparesis or paraplegia. Using a mouse model of transient aortic cross-clamping (ACC), we have previously found that the expression of proinflammatory microRNA

Experience-dependent white matter plasticity offers new potential for rehabilitation-induced recovery after neurotrauma. This first-in-human translational experiment combined myelin water imaging in humans and genetic fate-mapping of oligodendrocyte lineage cells in mice to investigate whether downhill locomotor rehabilitation that emphasizes eccentric muscle actions promotes white matter plasticity and recovery in chronic, incomplete spinal cord injury (SCI). In humans, of 20 individuals with SCI that enrolled, four passed the imaging screen and had myelin water imaging before and after a 12-week (3 times/week) downhill locomotor treadmill training program (SCI+DH). One individual was excluded for imaging artifacts. Uninjured control participants (n = 7) had two myelin water imaging sessions within the same day. Changes in myelin water fraction (MWF), a histopathologically-validated myelin biomarker, were analyzed in a priori motor learning and non-motor learning brain regions and the cervical spinal cord using statistical approaches appropriate for small sample sizes. PDGFRα-CreER(T2):mT/mG mice, that express green fluorescent protein on oligodendrocyte precursor cells and subsequent newly-differentiated oligodendrocytes upon tamoxifen-induced recombination, were either naive (n = 6) or received a moderate (75 kilodyne), contusive SCI at T9 and were randomized to downhill training (n = 6) or unexercised groups (n = 6). We initiated recombination 29 days post-injury, seven days prior to downhill training. Mice underwent two weeks of daily downhill training on the same 10% decline grade used in humans. Between-group comparison of functional (motor and sensory) and histological (oligodendrogenesis, oligodendroglial/axon interaction, paranodal structure) outcomes occurred post-training. In humans with SCI, downhill training increased MWF in brain motor learning regions (postcentral, precuneus) and mixed motor and sensory tracts of the ventral cervical spinal cord compared to control participants (P < 0.05). In mice with thoracic SCI, downhill training induced oligodendrogenesis in cervical dorsal and lateral white matter, increased axon-oligodendroglial interactions, and normalized paranodal structure in dorsal column sensory tracts (P < 0.05). Downhill training improved sensorimotor recovery in mice by normalizing hip and knee motor control and reducing hyperalgesia, both of which were associated with new oligodendrocytes in the cervical dorsal columns (P < 0.05). Our findings indicate that eccentric-focused, downhill rehabilitation promotes white matter plasticity and improved function in chronic SCI, likely via oligodendrogenesis in nervous system regions activated by the training paradigm. Together, these data reveal an exciting role for eccentric training in white matter plasticity and sensorimotor recovery after SCI.

Introduction: Spontaneously Hypertensive Rats- Stroke Prone (SHRSP) are a relevant model for human cerebral small vessel disease (cSVD). However, data are still lacking regarding the neuroimaging correlates of cSVD lesions in SHRSP and their temporal evolution in relationship to histological findings. Methods: 40 SHRSP and 40 Wistar Kyoto control (WKY) male rats were divided into 4 groups (10 WKY and 10 SHRSP) per group. Systolic blood pressure (SBP) was measured using tail-cuff device, weekly. Select animals of each group had brain MRI at 9.4T machine at 7, 16, 24, and 32 weeks of age. Volumetric analysis was completed using manual segmentation of the total brain, ventricles and bilateral hippocampi. Following MRI, brain histology was completed at the same time points. Results: At 6 weeks, SBP was similar (WKY 123.8±0.1 vs SHRSP 130.7±3.2, P=0.09). SHRSP developed hypertension between 9-11 weeks of age and maintained it throughout the experiment (SBP at 31 weeks: WKY 138.1±6 vs SHRSP 169.1±6.7, p=0.0006). At 7 weeks, brain MRI was normal in SHRSP and WKY. Histology was largely unremarkable except for few areas of red blood cell extravasation in couple of SHRSP. At 16 weeks, MRI was normal in WKY and it showed small subcortical hyperintensity on T2 sequences in one SHRSP while histology showed microbleeds (MBs) in 85% of SHRSP. At 24 weeks, brain MRI consistently identified subcortical hyperintensities in SHRSP and H&E showed MBs in all SHRSP in addition to hemosiderin deposition and arteriosclerosis. LFB staining showed areas of demyelination in the corpus callosum. At 32 weeks, SHRSP had hydrocephalus and H&E showed widespread hemosiderin deposition. Volumetric analysis showed larger ventricles in SHRSP (SHRSP 42.3±18.1 ml vs WKY 28.4±7.2 ml, p=0.013) and ventricle size significantly increased with age in SHRSP. Hippocampal and brain volumes were similar in both groups (hippocampal volume: WKY 84.5±8.1 ml vs SHRSP 81.6±2.6 ml, p=0.39; brain volume WKY 2103.4±100.4 ml vs SHRSP 2169.6 ±164.2 ml, p=0.2). Conclusions: SHRSP develop cSVD histological changes early in life and brain MRI showed consistent abnormalities at a later time point. These results have implications in defining cSVD phenotypes in SHRSP in future mechanistic studies.

The relationship between adiposity and metabolic health is well established. However, very little is known about the fat depot, known as paracardial fat (pCF), located superior to and surrounding the heart. Here, we show that pCF remodels with aging and a high-fat diet and that the size and function of this depot are controlled by alcohol dehydrogenase 1 (ADH1), an enzyme that oxidizes retinol into retinaldehyde. Elderly individuals and individuals with obesity have low ADH1 expression in pCF, and in mice, genetic ablation of Adh1 is sufficient to drive pCF accumulation, dysfunction, and global impairments in metabolic flexibility. Metabolomics analysis revealed that pCF controlled the levels of circulating metabolites affecting fatty acid biosynthesis. Also, surgical removal of the pCF depot was sufficient to rescue the impairments in cardiometabolic flexibility and fitness observed in Adh1-deficient mice. Furthermore, treatment with retinaldehyde prevented pCF remodeling in these animals. Mechanistically, we found that the ADH1/retinaldehyde pathway works by driving PGC-1α nuclear translocation and promoting mitochondrial fusion and biogenesis in the pCF depot. Together, these data demonstrate that pCF is a critical regulator of cardiometabolic fitness and that retinaldehyde and its generating enzyme ADH1 act as critical regulators of adipocyte remodeling in the pCF depot.

Objective Since enhanced cardiac magnetic resonance imaging (cMRI) signals have been associated with lupus disease activity in humans prior to renal failure and novel, cardiac-focused therapeutic strategies could be investigated with an associated animal model, autoimmune myocarditis was characterized in murine lupus nephritis (NZM2410). Methods Weekly blood urea nitrogen (BUN) levels and weights were recorded. Cardiac function was assessed by echocardiogram. Myocardial edema was measured with quantitative T2 cMRI mapping. Endpoint serum and cardiac tissue were collected for histopathological analysis and cytokine measurements. Results Despite showing no signs of significant renal disease, mice displayed evidence of myocarditis and fibrosis histologically at 30–35 weeks. Moreover, T2 cMRI mapping displayed robust signals and analysis of sagittal heart sections showed significant myocardium thickening. Cytokine expression levels of IL-2, IL-10, TNF-α, CXCL1, and IL-6 were significantly enhanced in serum. Echocardiograms demonstrated significantly increased ventricular diameters and reduced ejection fractions, while immunohistochemical staining identified CD4+ and CD8+ T cells, and IL-17 in cardiac infiltrates. Human lupus cardiac tissue showed similar histopathology with enhanced infiltrates by H&E, fibrosis, and CD4+ detection. Conclusions Histopathology, functional abnormalities, and enhanced cMRI signals indicative of myocarditis are detected in NZM2410 mice without glomerulonephritis, which supports the primary pathological role of autoimmune-mediated, cardiac-targeted inflammation in lupus.

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are particularly devastating, difficult to treat, and confer a poor prognosis. While the precise incidence of brain metastases in the United States remains hard to estimate, it is likely to increase as extracranial therapies continue to become more efficacious in treating cancer. Thus, it is necessary to identify and develop novel therapeutic approaches to treat metastasis at this site. To this end, intracranial injection of cancer cells has become a well-established method in which to model brain metastasis. Previously, the inability to directly measure tumor growth has been a technical hindrance to this model; however, increasing availability and quality of small animal imaging modalities, such as magnetic resonance imaging (MRI), are vastly improving the ability to monitor tumor growth over time and infer changes within the brain during the experimental period. Herein, intracranial injection of murine mammary tumor cells into immunocompetent mice followed by MRI is demonstrated. The presented injection approach utilizes isoflurane anesthesia and a stereotactic setup with a digitally controlled, automated drill and needle injection to enhance precision, and reduce technical error. MRI is measured over time using a 9.4 Tesla instrument in The Ohio State University James Comprehensive Cancer Center Small Animal Imaging Shared Resource. Tumor volume measurements are demonstrated at each time point through use of ImageJ. Overall, this intracranial injection approach allows for precise injection, day-to-day monitoring, and accurate tumor volume measurements, which combined greatly enhance the utility of this model system to test novel hypotheses on the drivers of brain metastases.

Spinal cord paralysis is relatively common after surgical repair of thoraco-abdominal aortic aneurysm (TAAA) and its etiology is unknown. The present study was designed to examine the histopathology of the disease and investigate whether miR-155 ablation would reduce spinal cord ischemic damage and delayed hindlimb paralysis induced by aortic cross-clamping (ACC) in our mouse model. The loss of locomotor function in ACC-paralyzed mice correlated with the presence of extensive gray matter damage and central cord edema, with minimal white matter histopathology. qRTPCR and Western blotting showed that the spinal cords of wild-type ACC mice that escaped paralysis showed lower miR-155 expression and higher levels of transcripts encoding Mfsd2a, which is implicated in the maintenance of blood-brain barrier integrity. In situ based testing demonstrated that increased miR-155 detection in neurons was highly correlated with the gray matter damage and the loss of one of its targets, Mfsd2a, could serve as a good biomarker of the endothelial cell damage. In vitro, we demonstrated that miR-155 targeted Mfsd2a in endothelial cells and motoneurons and increased endothelial cell permeability. Finally, miR-155 ablation slowed the progression of central cord edema, and reduced the incidence of paralysis by 40%. In sum, the surgical pathology findings clearly indicated that the epicenter of the ischemic-induced paralysis was the gray matter and that endothelial cell damage correlated to Mfsd2a loss is a good biomarker of the disease. MiR-155 targeting therefore offers new therapeutic opportunity for edema caused by traumatic spinal cord injury and diagnostic pathologists, by using immunohistochemistry, can clarify if this mechanism also is important in other ischemic diseases of the CNS, including stroke.

In older adults, the loss of muscle strength (dynapenia) and the loss of muscle mass (sarcopenia) are important contributors to the loss of physical function. We sought to investigate dynapenia, sarcopenia, and the loss of motor unit function in aging mice. C57BL/6J mice were analyzed with cross-sectional (males: 3 vs. 27 months; males and females: 8 vs. 12 vs. 20 months) and longitudinal studies (males: 10–25 months) using in vivo electrophysiological measures of motor unit connectivity (triceps surae compound muscle action potential and motor unit number estimation), in vivo measures of plantar flexion torque, magnetic resonance imaging of hind limb muscle volume, and grip strength. Compound muscle action potential amplitude, motor unit number estimation, and plantar flexion torque were decreased at 20 months. In contrast, grip strength was reduced at 24 months. Motor unit number estimates correlated with muscle torque and hind limb muscle volume. Our results demonstrate that the loss of motor unit connectivity is an early finding in aging male and female mice and that muscle size and contractility are both associated with motor unit number.

Nanoparticles have been widely explored for combined therapeutic and diagnostic applications. For example, lipid-based nanoparticles have been used to encapsulate multiple types of agents and achieve multi-functions. Herein, we enabled a co-delivery of mRNA molecules and superparamagnetic iron oxide nanoparticles (SPIONs) by using an amino-ester lipid-like nanomaterial. An orthogonal experimental design was used to identify the optimal formulation. The optimal formulation, MPA-Ab-8 LLNs, not only showed high encapsulation of both mRNA and SPIONs, but also increased the r(2) relaxivity of SPIONs by more than 1.5-fold in vitro. MPA-Ab-8 LLNs effectively delivered mRNA and SPIONs into cells, and consequently induced high protein expression as well as strong MRI contrast. Consistent herewith, we observed both mRNA-mediated protein expression and an evident negative contrast enhancement of MRI signal in mice. In conclusion, amino-ester nanomaterials demonstrate great potential as delivery vehicles for theranostic applications.

People who suffer a traumatic spinal cord injury (SCI) are at increased risk for developing dermatological complications. These conditions increase cost of care, incidence of rehospitalization, and the risk for developing other infections. The consequences of dermatological complications after SCI are likely exacerbated further by post-injury deficits in neural-immune signaling. Indeed, a functional immune system is essential for optimal host defense and tissue repair. Here, we tested the hypothesis that SCI at high spinal levels, which causes systemic immune suppression, would suppress cutaneous inflammation below the level of injury. C57BL/6 mice received an SCI (T3 spinal level) or sham injury; then one day later complete Freund's adjuvant (CFA) was injected subcutaneously below the injury level. Inflammation was quantified by injecting mice with V-Sense, a perfluorocarbon (PFC) tracer that selectively labels macrophages, followed by in vivo imaging. The total radiant efficiency, which is proportional to the number of macrophages, was measured over a 4-day period at the site of CFA injection. Fluorescent in vivo imaging revealed that throughout the analysis period, the macrophage reaction in SCI mice was reduced ∼50% compared with sham-injured mice. Radiant efficiency data were confirmed using magnetic resonance imaging (MRI), and together the data indicate that SCI significantly impairs subcutaneous inflammation. Future studies should determine whether enhancing local inflammation or boosting systemic immune function can improve the rate or efficiency of cutaneous wound healing in individuals with SCI. Doing so also could limit wound infections or secondary complications of impaired healing after SCI.

Multi-functional nanomaterials possess unique properties, facilitating both therapeutic and diagnostic applications among others. Herein, we developed dual-functional lipid-like nanoparticles for simultaneous delivery of mRNA and magnetic resonance imaging (MRI) contrast agents in order to express functional proteins and provide real-time visualization. TT3-Gd18 LLNs were identified as a lead formulation, which was able to encapsulate 91% of mRNA and 74% of Gd. This formulation showed a comparable or a slightly higher delivery efficiency of mRNA compared to the initial TT3 LLNs. Moreover, a strong MRI signal was observed in the cell pellets treated with TT3-Gd18 LLNs. More importantly, TT3-Gd18 LLNs demonstrated an efficient delivery of mRNA and Gd contrast agents in vivo.

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized clinically by cognitive decline and memory loss. The pathological features are amyloid-β peptide (Aβ) plaques and intracellular neurofibrillary tangles. Many studies have suggested that oxidative damage induced by reactive oxygen species (ROS) is an important mechanism for AD progression. Our recent study demonstrated that oxidative stress could further impair mitochondrial function. In the present study, we adopted a transgenic mouse model of AD (mAPP, overexpressing AβPP/Aβ in neurons) and performed redox measurements using in vivo electron paramagnetic resonance (EPR) imaging with methoxycarbamyl-proxyl (MCP) as a redox-sensitive probe for studying oxidative stress in an early stage of pathology in a transgenic AD mouse model. Through assessing oxidative stress, mitochondrial function and cognitive behaviors of mAPP mice at the age of 8-9 months, we found that oxidative stress and mitochondrial dysfunction appeared in the early onset of AD. Increased ROS levels were associated with defects of mitochondrial and cognitive dysfunction. Notably, the in vivo EPR method offers a unique way of assessing tissue oxidative stress in living animals under noninvasive conditions, and thus holds a potential for early diagnosis and monitoring the progression of AD.

BACKGROUND: Clinical multi-electrode mapping of atrial fibrillation (AF) drivers suffers from variable contact, signal processing, and structural complexity within the 3D human atrial wall, raising questions on the validity of such drivers. OBJECTIVES: To improve AF driver identification by integrating clinical multi-electrode mapping with driver fingerprints defined by high-resolution ex-vivo 3D functional and structural imaging. METHODS: Sustained AF was mapped in coronary-perfused explanted human hearts (n=11) with transmural near-infrared optical mapping (NIOM, ~0.3mm(2) resolution). Simultaneously, custom FIRMap catheters (~9×9mm(2) resolution) mapped endocardial and epicardial surfaces, which were analyzed by Focal Impulse and Rotor Mapping (FIRM) activation and Rotational Activity Profile (RAP). Functional maps were integrated with contrast-enhanced MRI (CE-MRI, ~0.1mm(3) resolution) analysis of 3D fibrosis architecture. RESULTS: During sustained AF, NIOM identified 1-2 intramural, spatially stable reentrant AF drivers per heart. Driver targeted ablation affecting 2.2±1.1% of the atrial surface terminated and prevented AF. Driver regions had significantly higher phase singularity density, and dominant frequency versus neighboring non-driver regions. FIRM had 80% sensitivity to NIOM-defined driver locations (16/20), and matched 14/20 driver visualizations: 10/14 reentries seen with RAP, and 4/6 breakthrough/focal patterns. FIRM detected 1.1±0.9 false-positive RAP per recording, but these regions had lower intramural CE-MRI fibrosis than driver regions (14.9±7.9% vs 23.2±10.5%, p

Aims The complex architecture of the human atria may create physical substrates for sustained re-entry to drive atrial fibrillation (AF). The existence of sustained, anatomically defined AF drivers in humans has been challenged partly due to the lack of simultaneous endocardial–epicardial (Endo–Epi) mapping coupled with high-resolution 3D structural imaging. Methods and results Coronary-perfused human right atria from explanted diseased hearts ( n = 8, 43–72 years old) were optically mapped simultaneously by three high-resolution CMOS cameras (two aligned Endo–Epi views (330 µm2 resolution) and one panoramic view). 3D gadolinium-enhanced magnetic resonance imaging (GE-MRI, 80 µm3 resolution) revealed the atrial wall structure varied in thickness (1.0 ± 0.7–6.8 ± 2.4 mm), transmural fiber angle differences, and interstitial fibrosis causing transmural activation delay from 23 ± 11 to 43 ± 22 ms at increased pacing rates. Sustained AF (>90 min) was induced by burst pacing during pinacidil (30–100 µM) perfusion. Dual-sided sub-Endo–sub-Epi optical mapping revealed that AF was driven by spatially and temporally stable intramural re-entry with 107 ± 50 ms cycle length and transmural activation delay of 67 ± 31 ms. Intramural re-entrant drivers were captured primarily by sub-Endo mapping, while sub-Epi mapping visualized re-entry or ‘breakthrough’ patterns. Re-entrant drivers were anchored on 3D micro-anatomic tracks (15.4 ± 2.2 × 6.0 ± 2.3 mm2, 2.9 ± 0.9 mm depth) formed by atrial musculature characterized by increased transmural fiber angle differences and interstitial fibrosis. Targeted radiofrequency ablation of the tracks verified these re-entries as drivers of AF. Conclusions Integrated 3D structural–functional mapping of diseased human right atria ex vivo revealed that the complex atrial microstructure caused significant differences between Endo vs. Epi activation during pacing and sustained AF driven by intramural re-entry anchored to fibrosis-insulated atrial bundles.

Background Structural remodeling of human atria plays a key role in sustaining atrial fibrillation ( AF ), but insufficient quantitative analysis of human atrial structure impedes the treatment of AF . We aimed to develop a novel 3‐dimensional (3D) structural and computational simulation analysis tool that could reveal the structural contributors to human reentrant AF drivers. Methods and Results High‐resolution panoramic epicardial optical mapping of the coronary‐perfused explanted intact human atria (63‐year‐old woman, chronic hypertension, heart weight 608 g) was conducted during sinus rhythm and sustained AF maintained by spatially stable reentrant AF drivers in the left and right atrium. The whole atria (107×61×85 mm 3 ) were then imaged with contrast‐enhancement MRI (9.4 T, 180×180×360‐μm 3 resolution). The entire 3D human atria were analyzed for wall thickness (0.4–11.7 mm), myofiber orientations, and transmural fibrosis (36.9% subendocardium; 14.2% midwall; 3.4% subepicardium). The 3D computational analysis revealed that a specific combination of wall thickness and fibrosis ranges were primarily present in the optically defined AF driver regions versus nondriver tissue. Finally, a 3D human heart–specific atrial computer model was developed by integrating 3D structural and functional mapping data to test AF induction, maintenance, and ablation strategies. This 3D model reproduced the optically defined reentrant AF drivers, which were uninducible when fibrosis and myofiber anisotropy were removed from the model. Conclusions Our novel 3D computational high‐resolution framework may be used to quantitatively analyze structural substrates, such as wall thickness, myofiber orientation, and fibrosis, underlying localized AF drivers, and aid the development of new patient‐specific treatments.

Autonomic dysreflexia (AD), a potentially dangerous complication of high-level spinal cord injury (SCI) characterized by exaggerated activation of spinal autonomic (sympathetic) reflexes, can cause pulmonary embolism, stroke, and, in severe cases, death. People with high-level SCI also are immune compromised, rendering them more susceptible to infectious morbidity and mortality. The mechanisms underlying postinjury immune suppression are not known. Data presented herein indicate that AD causes immune suppression. Using in vivo telemetry, we show that AD develops spontaneously in SCI mice with the frequency of dysreflexic episodes increasing as a function of time postinjury. As the frequency of AD increases, there is a corresponding increase in splenic leucopenia and immune suppression. Experimental activation of spinal sympathetic reflexes in SCI mice (e.g., via colorectal distension) elicits AD and exacerbates immune suppression via a mechanism that involves aberrant accumulation of norepinephrine and glucocorticoids. Reversal of postinjury immune suppression in SCI mice can be achieved by pharmacological inhibition of receptors for norepinephrine and glucocorticoids during the onset and progression of AD. In a human subject with C5 SCI, stimulating the micturition reflex caused AD with exaggerated catecholamine release and impaired immune function, thus confirming the relevance of the mouse data. These data implicate AD as a cause of secondary immune deficiency after SCI and reveal novel therapeutic targets for overcoming infectious complications that arise due to deficits in immune function.

Aims The adult human sinoatrial node (SAN) has a specialized fibrotic intramural structure (35–55% fibrotic tissue) that provides mechanical and electrical protection from the surrounding atria. We hypothesize that late gadolinium-enhanced cardiovascular magnetic resonance (LGE-CMR) can be applied to define the fibrotic human SAN structure in vivo . Methods and results LGE-CMR atrial scans of healthy volunteers ( n olu, 23–52 y.o.) using a 3 Tesla magnetic resonance imaging system with a spatial resolution of 1.0 mm3 or 0.625 × 0.625 × 1.25 mm3 were obtained and analysed. Percent fibrosis of total connective and cardiomyocyte tissue area in segmented atrial regions were measured based on signal intensity differences of fibrotic vs. non-fibrotic cardiomyocyte tissue. A distinct ellipsoidal fibrotic region (length: 23.6 ± 1.9 mm; width: 7.2 ± 0.9 mm; depth: 2.9 ± 0.4 mm) in all hearts was observed along the posterior junction of the crista terminalis and superior vena cava extending towards the interatrial septum, corresponding to the anatomical location of the human SAN. The SAN fibrotic region consisted of 41.9 ± 5.4% of LGE voxels above an average threshold of 2.7 SD (range 2–3 SD) from the non-fibrotic right atrial free wall tissue. Fibrosis quantification and SAN identification by in vivo LGE-CMR were validated in optically mapped explanted donor hearts ex vivo ( n ivo, 19–65 y.o.) by contrast-enhanced CMR (9.4 Tesla; up to 90 µm3 resolution) correlated with serial histological sections of the SAN. Conclusion This is the first study to visualize the 3D human SAN fibrotic structure in vivo using LGE-CMR. Identification of the 3D SAN location and its high fibrotic content by LGE-CMR may provide a new tool to avoid or target SAN structure during ablation.

Acute respiratory disease is associated with significant morbidity and mortality in influenza. Because antiviral drugs are only effective early in infection, new agents are needed to treat nonvaccinated patients presenting with late-stage disease, particularly those who develop acute respiratory distress syndrome. We found previously that the de novo pyrimidine synthesis inhibitor A77-1726 reversed the influenza-induced impairment of alveolar fluid clearance. Patients with acute respiratory distress syndrome and intact alveolar fluid clearance demonstrate lower mortality than those with compromised fluid clearance. We therefore investigated the effects of treatment with nebulized A77-1726 (67.5 mg/kg) on indices of cardiopulmonary function relevant to the diagnosis of acute respiratory distress syndrome. BALB/cAnNCr mice (8-12 wk old) were inoculated intranasally with 10,000 plaque-forming units/mouse influenza A/WSN/33 (H1N1). Pulse oximetry was performed daily. Alveolar fluid clearance, lung water, and lung mechanics were measured at 2 and 6 days after inoculation in live, ventilated mice by BSA instillation, magnetic resonance imaging, and forced-oscillation techniques, respectively. A77-1726 treatment at 1 day after inoculation delayed mortality. Treatment on Days 1 or 5 reduced viral replication on Day 6, and improved alveolar fluid clearance, peripheral oxygenation, and cardiac function. Nebulized A77-1726 also reversed influenza-induced increases in lung water content and volume, improved pulmonary mechanics, reduced bronchoalveolar lavage fluid ATP and neutrophil content, and increased IL-6 concentrations. The ability of A77-1726 to improve cardiopulmonary function in influenza-infected mice and to reduce the severity of ongoing acute respiratory distress syndrome late in infection suggests that pyrimidine synthesis inhibitors are promising therapeutic candidates for the management of severe influenza.

Here, we describe the construction and testing of a novel herpes simplex virus type 1 (HSV-1) derived oncolytic virus (OV): 34.5ENVE (viral ICP34.5 Expressed by Nestin promotor and Vstat120 Expressing), for the treatment of cancer. This virus showed significant glioma-specific killing and antiangiogenic effects in vitro and in vivo. Treatment of subcutaneous and intracranial glioma-bearing mice with 34.5ENVE showed a significant increase in median survival of mice in four different glioma models. Histology and dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) revealed reduced microvessel density (MVD) and increased tumoral necrosis in 34.5ENVE-treated tumor tissue compared to control OV-treated tumor tissue. Collectively, these results describe the construction, efficacy, and impact on tumor microenvironment of a transcriptionally driven OV armed with Vstat120 gene expression. These preclinical results will facilitate future clinical testing of 34.5ENVE.

Hypoxia, which is commonly observed in many solid tumors, is a major impediment to chemo- or radiation therapy. Hypoxia is also known to overexpress/activate signal transducer and activator of transcription 3 (STAT3) leading to tumor progression as well as drug resistance. We hypothesized that increased oxygenation of the hypoxic tumor may have an inhibitory effect on STAT3 activation and hence tumor-growth inhibition. Mice containing human ovarian cancer xenograft tumor were exposed to hyperbaric oxygen (HBO; 100% oxygen; 2 atm; 90-min duration) daily, for up to 21 days. Mice exposed to HBO showed a significant reduction in tumor volume, with no effect on body weight. STAT3 (Tyr 705) activation and cyclin-D1 protein/mRNA levels were significantly decreased up on HBO exposure. Interestingly, HBO exposure, in combination with weekly administration of cisplatin, also significantly reduced the tumor volume; however, this group of mice had drastically reduced body weight when compared to other groups. While conventional wisdom might suggest that increased oxygenation of tumors would promote tumor growth, the results of the present study indicated otherwise. Hyperoxia appears to inhibit STAT3 activation, which is a key step in the ovarian tumor progression. The study may have important implications for the treatment of ovarian cancer in the clinic.

Signal transducer and activator of transcription 3 (STAT3) is activated in a variety of human cancers, including ovarian cancer. The molecular mechanism by which the STAT3 is activated in cancer cells is poorly understood. We observed that human ovarian xenograft tumors (A2780) in mice were severely hypoxic (pO(2) approximately 2 mmHg). We further observed that hypoxic exposure significantly increased the phosphorylation of STAT3 (pSTAT3) at the Tyr705 residue in A2780 cell line. The pSTAT3 (Tyr705) level was highly dependent on cellular oxygenation levels, with a significant increase at2% O(2), and without any change in the pSTAT3 (Ser727) or total STAT3 levels. The pSTAT3 (Tyr705) elevation following hypoxic exposure could be reversed within 12 hr after returning the cells to normoxia. The increased level of pSTAT3 was partly mediated by increased levels of reactive oxygen species generation in the hypoxic cancer cells. Conventional chemotherapeutic drugs cisplatin and taxol were far less effective in eliminating the hypoxic ovarian cancer cells suggesting a role for pSTAT3 in cellular resistance to chemotherapy. Inhibition of STAT3 by AG490 followed by treatment with cisplatin or taxol resulted in a significant increase in apoptosis suggesting that hypoxia-induced STAT3 activation is responsible for chemoresistance. The results have important clinical implications for the treatment of hypoxic ovarian tumors using STAT3-specific inhibitors.

The use of oxygen-sensing water-insoluble paramagnetic probes, such as lithium octa-n-butoxynaphthalocyanine (LiNc-BuO), enables repeated measurements of pO(2) from the same location in tissue by electron paramagnetic resonance (EPR) spectroscopy. In order to facilitate direct in vivo application, and hence eventual clinical applicability, of LiNc-BuO, we encapsulated LiNc-BuO microcrystals in polydimethylsiloxane (PDMS), an oxygen-permeable and bioinert polymer, and developed an implantable chip. In vitro evaluation of the chip, performed under conditions of sterilization, high-energy irradiation, and exposure to cultured cells, revealed that it is biostable and biocompatible. Implantation of the chip in the gastrocnemius muscle tissue of mice showed that it is capable of repeated and real-time measurements of tissue oxygenation for an extended period. Functional evaluation using a murine tumor model established the suitability and applicability of the chip for monitoring tumor oxygenation. This study establishes PDMS-encapsulated LiNc-BuO as a promising choice of probe for clinical EPR oximetry.

Lithium octa-n-butoxynaphthalocyanine (LiNc-BuO) is a promising probe for biological electron paramagnetic resonance (EPR) oximetry and is being developed for clinical use. However, clinical applicability of LiNc-BuO may be hindered by potential limitations associated with biocompatibility, biodegradation, and migration of individual crystals in tissue. To overcome these limitations, we have encapsulated LiNc-BuO crystals in polydimethyl siloxane (PDMS), an oxygen-permeable and bioinert polymer, to fabricate conveniently implantable and retrievable oxygen-sensing chips. Encapsulation was performed by a simple cast-molding process, giving appreciable control over size, shape, thickness and spin density of chips. The in vitro oxygen response of the chip was linear, reproducible, and not significantly different from that of unencapsulated crystals. Cast-molding of the structurally-flexible PDMS enabled the fabrication of chips with tailored spin densities, and ensured non-exposure of embedded LiNc-BuO, mitigating potential biocompatibility/toxicological concerns. Our results establish PDMS-encapsulated LiNc-BuO as a promising candidate for further biological evaluation and potential clinical application.

Under normal conditions, cells meticulously maintain redox homeostasis. However, under abnormal conditions, such as stress, the redox equilibrium is disturbed, leading to a threat to the cellular wellbeing. Techniques that are capable of measurement of the redox status might prove useful in the detection and treatment of adverse conditions that change the cellular redox equilibrium. Electron paramagnetic resonance (EPR) imaging is a unique non-invasive imaging technique that is capable of redox measurements in vivo. EPR utilizes redox-sensitive spin labels, nitroxides. These EPR-active probes are reduced by cellular reducing agents to EPR-inactive species (hydroxylamines). The rate of reduction, among various factors, depends on the redox status of the cell, thus providing information about the redox environment of the region of interest. This review focuses on the principle, method, and application of redox mapping to biological systems.

Disulfide nitroxide biradicals, DNB, have been used for glutathione, GSH, measurements by X-band electron paramagnetic resonance, EPR, in various cells and tissues. In the present paper, the postulated potential use of DNB for EPR detection of GSH in vivo was explored. Isotopic substitution in the structure of the DNB was performed for the enhancement of its EPR spectral properties. 15 N substitution in the NO fragment of the DNB decreased the number of EPR spectral lines and resulted in an approximately two-fold increase in the signal-to-noise ratio, SNR. An additional two-fold increase in the SNR was achieved by substitution of the hydrogen atoms with deuterium resulting in narrowing the EPR lines from 1.35 G to 0.95 G. The spectral changes of DNB upon reaction with GSH and cysteine were studied in vitro in a wide range of pHs at room temperature and “body” temperature, 37°C, and the corresponding bimolecular rate constants were calculated. In in vivo experiments the kinetics of the L-band EPR spectral changes after injection of DNB into ovarian xenograft tumors grown in nude mice were measured by L-band EPR spectroscopy, and analyzed in terms of the two main contributing reactions, splitting of the disulfide bond and reduction of the NO fragment. The initial exponential increase of the “monoradical” peak intensity has been used for the calculation of the GSH concentration using the value of the observed rate constant for the reaction of DNB with GSH, k obs (pH 7.1, 37°C) = 2.6 M - 1 s - 1 . The concentrations of GSH in cisplatin-resistant and cisplatin-sensitive tumors were found to be 3.3 mM and 1.8 mM, respectively, in quantitative agreement with the in vitro data.

Methods currently available for the measurement of oxygen concentrations (oximetry) in viable tissues differ widely from each other in their methodological basis and applicability. The goal of this study was to compare two novel methods, particulate-based electron paramagnetic resonance (EPR) and OxyLite oximetry, in an experimental tumor model. EPR oximetry uses implantable paramagnetic particulates, whereas OxyLite uses fluorescent probes affixed on a fiber-optic cable. C3H mice were transplanted with radiation-induced fibrosarcoma (RIF-1) tumors in their hind limbs. Lithium phthalocyanine (LiPc) microcrystals were used as EPR probes. The pO(2) measurements were taken from random locations at a depth of approximately 3 mm within the tumor either immediately or 48 h after implantation of LiPc. Both methods revealed significant hypoxia in the tumor. However, there were striking differences between the EPR and OxyLite readings. The differences were attributed to the volume of tissue under examination and the effect of needle invasion at the site of measurement. This study recognizes the unique benefits of EPR oximetry in terms of robustness, repeatability and minimal invasiveness.

High-power ultrasound (20 kHz) was used to encapsulate a solution of perchlorotriphenylmethyl triester (PTM-TE, a stable organic free radical) dissolved in hexamethyldisiloxane (HMDS) into a polymerized shell of bovine serum albumin (BSA). The size distribution of the microspheres was between 0.5 and 3 microm with a maximum at approximately 1.2 microm. The electron paramagnetic resonance spectrum of PTM-TE consists of a single, sharp line which is sensitive to the surrounding concentration of oxygen. It was found that the technique of encapsulating a solution of PTM-TE dissolved in HMDS into the BSA microspheres resulted in an overall loss of EPR signal intensity from the washed suspension of microspheres. However, the encapsulated PTM-TE/HMDS solution remained sensitive to the partial pressure of oxygen in the surrounding environment. The microspheres were found to be useful for determining the partial pressure of oxygen in the muscle and tumor tissue of mice.

A novel procedure for in vivo imaging of the oxygen partial pressure (pO2) in implanted tumors is reported. The procedure uses electron paramagnetic resonance imaging (EPRI) of oxygen-sensing nanoprobes embedded in the tumor cells. Unlike existing methods of pO2 quantification, wherein the probes are physically inserted at the time of measurement, the new approach uses cells that are preinternalized (labeled) with the oxygen-sensing probes, which become permanently embedded in the developed tumor. Radiation-induced fibrosarcoma (RIF-1) cells, internalized with nanoprobes of lithium octa-n-butoxy-naphthalocyanine (LiNc-BuO), were allowed to grow as a solid tumor. In vivo imaging of the growing tumor showed a heterogeneous distribution of the spin probe, as well as oxygenation in the tumor volume. The pO2 images obtained after the tumors were exposed to a single dose of 30-Gy X-radiation showed marked redistribution as well as an overall increase in tissue oxygenation, with a maximum increase 6 hr after irradiation. However, larger tumors with a poorly perfused core showed no significant changes in oxygenation. In summary, the use of in vivo EPR technology with embedded oxygen-sensitive nanoprobes enabled noninvasive visualization of dynamic changes in the intracellular pO2 of growing and irradiated tumors.

MicroRNAs (miRNAs), single-stranded non-coding RNAs, influence myriad biological processes that can contribute to cancer. Although tumor-suppressive and oncogenic functions have been characterized for some miRNAs, the majority of microRNAs have not been investigated for their ability to promote and modulate tumorigenesis. Here, we established that the miR-191/425 cluster is transcriptionally dependent on the host gene, DALRD3, and that the hormone 17β-estradiol (estrogen or E2) controls expression of both miR-191/425 and DALRD3. MiR-191/425 locus characterization revealed that the recruitment of estrogen receptor α (ERα) to the regulatory region of the miR-191/425-DALRD3 unit resulted in the accumulation of miR-191 and miR-425 and subsequent decrease in DALRD3 expression levels. We demonstrated that miR-191 protects ERα positive breast cancer cells from hormone starvation-induced apoptosis through the suppression of tumor-suppressor EGR1. Furthermore, enforced expression of the miR-191/425 cluster in aggressive breast cancer cells altered global gene expression profiles and enabled us to identify important tumor promoting genes, including SATB1, CCND2, and FSCN1, as targets of miR-191 and miR-425. Finally, in vitro and in vivo experiments demonstrated that miR-191 and miR-425 reduced proliferation, impaired tumorigenesis and metastasis, and increased expression of epithelial markers in aggressive breast cancer cells. Our data provide compelling evidence for the transcriptional regulation of the miR-191/425 cluster and for its context-specific biological determinants in breast cancers. Importantly, we demonstrated that the miR-191/425 cluster, by reducing the expression of an extensive network of genes, has a fundamental impact on cancer initiation and progression of breast cancer cells., Author Summary MicroRNAs are small noncoding RNAs that act as posttranscriptional repressors of gene expression. A pivotal role for miRNAs in all the molecular processes driving initiation and progression of various malignancies, including breast cancer, has been described. Divergent miRNA expression between normal and neoplastic breast tissues has been demonstrated, as well as differential miRNA expression among the molecular subtypes of breast cancer. Over half of all breast cancers overexpress ERα, and several studies have shown that miRNA expression is controlled by ERα. We assessed the global change in microRNA expression after estrogen starvation and stimulation in breast cancer cells and identified that miR-191/425 and the host gene DALRD3 are positively associated to ERα-positive tumors. We demonstrated that ERα regulates the miR-191/425 cluster and verified the existence of a transcriptional network that allows a dual effect of estrogen on miR-191/425 and their host gene. We show that estrogen induction of miR-191/425 supports in vitro and in vivo the estrogen-dependent proliferation of ERα positive breast cancer cells. On the contrary, miR-191/425 cluster reprograms gene expression to impair tumorigenicity and metastatic potential of highly aggressive ERα negative breast cancer cells.

The potential value of the nitric oxide (NO) level as a prognostic marker in human brain diseases is investigated. Cerebrospinal fluid (CSF) collected from neurological patients was examined for NO content using electron paramagnetic resonance (EPR) spectroscopy. In adult patients with meningitis, the level of NO was higher than that in other groups of brain disorders, such as brain traumas and brain tumors. Very high levels of NO in the CSF appeared to be correlated with a high incidence of fatal outcomes. In children with meningitis, it was possible to differentiate between viral and bacterial origin of the disease as evidenced by the EPR analysis of the CSF. The results indicated that NO levels in the CSF can be a useful prognostic marker in neurological diseases.

Background: Medulloblastoma is the most common type of pediatric brain tumor. Although numerous factors influence patient survival rates, more than 30% of all cases will ultimately be refractory to conventional therapies. Current standards of care are also associated with significant morbidities, giving impetus for the development of new treatments. We have previously shown that oncolytic measles virotherapy is effective against medulloblastoma, leading to significant prolongation of survival and even cures in mouse xenograft models of localized and metastatic disease. Because medulloblastomas are known to be highly vascularized tumors, we reasoned that the addition of angiogenesis inhibitors could further enhance the efficacy of oncolytic measles virotherapy. Toward this end, we have engineered an oncolytic measles virus that express a fusion protein of endostatin and angiostatin, two endogenous and potent inhibitors of angiogenesis.Methods: Oncolytic measles viruses encoding human and mouse variants of a secretable endostatin/angiostatin fusion protein were designed and rescued according to established protocols. These viruses, known as MV-hE:A and MV-mE:A respectively, were then evaluated for their anti-angiogenic potential and efficacy against medulloblastoma cell lines and orthotopic mouse models of localized disease.Results: Medulloblastoma cells infected by MV-E:A readily secrete endostatin and angiostatin prior to lysis. The inclusion of the endostatin/angiostatin gene did not negatively impact the measles virus' cytotoxicity against medulloblastoma cells or alter its growth kinetics. Conditioned media obtained from these infected cells was capable of inhibiting multiple angiogenic factors in vitro, significantly reducing endothelial cell tube formation, viability and migration compared to conditioned media derived from cells infected by a control measles virus. Mice that were given a single intratumoral injection of MV-E:A likewise showed reduced numbers of tumor-associated blood vessels and a trend for increased survival compared to mice treated with the control virus.Conclusions: These data suggest that oncolytic measles viruses encoding anti-angiogenic proteins may have therapeutic benefit against medulloblastoma and support ongoing efforts to target angiogenesis in medulloblastoma. © 2014 Hutzen et al.; licensee BioMed Central Ltd.

Loss of miR-122 causes chronic steatohepatitis and spontaneous hepatocellular carcinoma. However, the consequence of miR-122 deficiency on genotoxic stress–induced liver pathogenesis is poorly understood. Here, we investigated the impact of miR-122 depletion on liver pathobiology by treating liver-specific miR-122 knockout (LKO) mice with the hepatocarcinogen diethylnitrosamine (DEN). At 25 weeks post-DEN injection, all LKO mice developed CK-19–positive hepatobiliary cysts, which correlated with DEN-induced transcriptional activation of Cdc25a mediated through E2f1. Additionally, LKO livers were more fibrotic and vascular, and developed larger microscopic tumors, possibly due to elevation of the Axl oncogene, a receptor tyrosine kinase as a novel target of miR-122, and several protumorigenic miR-122 targets. At 35 weeks following DEN exposure, LKO mice exhibited a higher incidence of macroscopic liver tumors (71%) and cysts (86%) compared to a 21.4% and 0% incidence of tumors and cysts, respectively, in control mice. The tumors in LKO mice were bigger (ninefold, P = 0.015) and predominantly hepatocellular carcinoma, whereas control mice mostly developed hepatocellular adenoma. DEN treatment also reduced survival of LKO mice compared to control mice (P = 0.03). Interestingly, induction of oxidative stress and proinflammatory cytokines in LKO liver shortly after DEN exposure indicates predisposition of a pro-tumorigenic microenvironment. Collectively, miR-122 depletion facilitates cystogenesis and hepatocarcinogenesis in mice on DEN challenge by up-regulating several genes involved in proliferation, growth factor signaling, neovascularization, and metastasis.