Development of a clinical assay to measure chlorinated tyrosine in hair and tissue samples using a mouse chlorine inhalation exposure model.

Chlorine is a toxic industrial chemical with a history of use as a chemical weapon. Chlorine is also produced, stored, and transported in bulk making it a high-priority pulmonary threat in the USA. Due to the high reactivity of chlorine, few biomarkers exist to identify exposure in clinical and environmental samples. Our laboratory evaluates acute chlorine exposure in clinical samples by measuring 3-chlorotyrosine (Cl-Tyr) and 3,5-dichlorotyrosine (Cl2-Tyr) using liquid chromatography tandem mass spectrometry (LC-MS/MS). Individuals can have elevated biomarker levels due to their environment and chronic health conditions, but levels are significantly lower in individuals exposed to chlorine. Historically these biomarkers have been evaluated in serum, plasma, blood, and bronchoalveolar lavage (BAL) fluid. We report the expansion into hair and lung tissue samples using our newly developed tissue homogenization protocol which fits seamlessly with our current chlorinated tyrosine quantitative assay. Furthermore, we have updated the chlorinated tyrosine assay to improve throughput and ruggedness and reduce sample volume requirements. The improved assay was used to measure chlorinated tyrosine levels in 198 mice exposed to either chlorine gas or air. From this animal study, we compared Cl-Tyr and Cl2-Tyr levels among three matrices (i.e., lung, hair, and blood) and found that hair had the most abundant chlorine exposure biomarkers. Furthermore, we captured the first timeline of each analyte in the lung, hair, and blood samples. In mice exposed to chlorine gas, both Cl-Tyr and Cl2-Tyr were present in blood and lung samples up to 24 h and up to 30 days in hair samples.

Impact of oviductal versus ovarian epithelial cell of origin on ovarian endometrioid carcinoma phenotype in the mouse.

Endometrioid carcinoma (EC) is a relatively indolent ovarian carcinoma subtype that is nonetheless deadly if detected late. Existing genetically engineered mouse models (GEMMs) of the disease, based on transformation of the ovarian surface epithelium (OSE), take advantage of known ovarian EC driver gene lesions, but do not fully recapitulate the disease features seen in patients. An EC model in which the Apc and Pten tumour suppressor genes are conditionally deleted in murine OSE yields tumours that are biologically more aggressive and significantly less differentiated than human ECs. Importantly, OSE is not currently thought to be the tissue of origin of most ovarian cancers, including ECs, suggesting that tumour initiation in Müllerian epithelium may produce tumours that more closely resemble their human tumour counterparts. We have developed Ovgp1-iCreERT2 mice in which the Ovgp1 promoter controls expression of tamoxifen (TAM)-regulated Cre recombinase in oviductal epithelium - the murine equivalent of human Fallopian tube epithelium. Ovgp1-iCreERT2 ;Apcfl/fl ;Ptenfl/fl mice treated with TAM or injected with adenovirus expressing Cre into the ovarian bursa uniformly develop oviductal or ovarian ECs, respectively. On the basis of their morphology and global gene expression profiles, the oviduct-derived tumours more closely resemble human ovarian ECs than do OSE-derived tumours. Furthermore, mice with oviductal tumours survive much longer than their counterparts with ovarian tumours. The slow progression and late metastasis of oviductal tumours resembles the relatively indolent behaviour characteristic of so-called Type I ovarian carcinomas in humans, for which EC is a prototype. Our studies demonstrate the utility of Ovgp1-iCreERT2 mice for manipulating genes of interest specifically in the oviductal epithelium, and establish that the cell of origin is an important consideration in mouse ovarian cancer GEMMs. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Type I to type II ovarian carcinoma progression: mutant Trp53 or Pik3ca confers a more aggressive tumor phenotype in a mouse model of ovarian cancer.

A dualistic pathway model of ovarian carcinoma (OvCA) pathogenesis has been proposed: type I OvCAs are low grade, genetically stable, and relatively more indolent than type II OvCAs, most of which are high-grade serous carcinomas. Endometrioid OvCA (EOC) is a prototypical type I tumor, often harboring mutations that affect the Wnt and phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin signaling pathways. Molecular and histopathologic analyses indicate type I and II OvCAs share overlapping features, and a subset of EOCs may undergo type I→type II progression accompanied by acquisition of somatic TP53 or PIK3CA mutations. We used a murine model of EOC initiated by conditional inactivation of the Apc and Pten tumor suppressor genes to investigate mutant Trp53 or Pik3ca alleles as key drivers of type I→type II OvCA progression. In the mouse EOC model, the presence of somatic Trp53 or Pik3ca mutations resulted in shortened survival and more widespread metastasis. Activation of mutant Pik3ca alone had no demonstrable effect on the ovarian surface epithelium but resulted in papillary hyperplasia when coupled with Pten inactivation. Our findings indicate that the adverse prognosis associated with TP53 and PIK3CA mutations in human cancers can be functionally replicated in mouse models of type I→type II OvCA progression. Moreover, the models should represent a robust platform for assessment of the contributions of Trp53 or Pik3ca defects in the response of EOCs to conventional and targeted drugs.

Mitigating the risk of radiation-induced cancers: limitations and paradigms in drug development.

The United States radiation medical countermeasures (MCM) programme for radiological and nuclear incidents has been focusing on developing mitigators for the acute radiation syndrome (ARS) and delayed effects of acute radiation exposure (DEARE), and biodosimetry technologies to provide radiation dose assessments for guiding treatment. Because a nuclear accident or terrorist incident could potentially expose a large number of people to low to moderate doses of ionising radiation, and thus increase their excess lifetime cancer risk, there is an interest in developing mitigators for this purpose. This article discusses the current status, issues, and challenges regarding development of mitigators against radiation-induced cancers. The challenges of developing mitigators for ARS include: the long latency between exposure and cancer manifestation, limitations of animal models, potential side effects of the mitigator itself, potential need for long-term use, the complexity of human trials to demonstrate effectiveness, and statistical power constraints for measuring health risks (and reduction of health risks after mitigation) following relatively low radiation doses (<0.75 Gy). Nevertheless, progress in the understanding of the molecular mechanisms resulting in radiation injury, along with parallel progress in dose assessment technologies, make this an opportune, if not critical, time to invest in research strategies that result in the development of agents to lower the risk of radiation-induced cancers for populations that survive a significant radiation exposure incident.

Granulocyte Colony-Stimulating Factor (Neupogen®; Filgrastim) Accelerates Neutrophil Recovery in a Rodent Model of Sulfur Mustard-Induced Hematologic Toxicity.

OBJECTIVE: Evidence of myelosuppression has been negatively correlated with patient outcomes following cases of high dose sulfur mustard (SM) exposure. These hematologic complications can negatively impact overall immune function and increase the risk of infection and life-threatening septicemia. Currently, there are no approved medical treatments for the myelosuppressive effects of SM exposure. METHODS: Leveraging a recently developed rodent model of SM-induced hematologic toxicity, post-exposure efficacy testing of the granulocyte colony-stimulating factor drug Neupogen® was performed in rats intravenously challenged with SM. Before efficacy testing, pharmacokinetic/pharmacodynamic analyses were performed in naïve rats to identify the apparent human equivalent dose of Neupogen® for efficacy evaluation. RESULTS: When administered 1 d after SM-exposure, daily subcutaneous Neupogen® treatment did not prevent the delayed onset of hematologic toxicity but significantly accelerated recovery from neutropenia. Compared with SM controls, Neupogen®-treated animals recovered body weight faster, resolved toxic clinical signs more rapidly, and did not display transient febrility at time points generally concurrent with marked pancytopenia. CONCLUSIONS: Collectively, this work corroborates the results of a previous pilot large animal study, validates the utility of a rodent screening model, and provides further evidence for the potential clinical utility of Neupogen® as an adjunct treatment following SM exposure.

Overlapping Science in Radiation and Sulfur Mustard Exposures of Skin and Lung: Consideration of Models, Mechanisms, Organ Systems, and Medical Countermeasures: Overlapping science in radiation and sulfur mustard injuries to lung and skin.

PURPOSE: To summarize presentations and discussions from the 2022 trans-agency workshop titled "Overlapping science in radiation and sulfur mustard (SM) exposures of skin and lung: Consideration of models, mechanisms, organ systems, and medical countermeasures." METHODS: Summary on topics includes: (1) an overview of the radiation and chemical countermeasure development programs and missions; (2) regulatory and industry perspectives for drugs and devices; 3) pathophysiology of skin and lung following radiation or SM exposure; 4) mechanisms of action/targets, biomarkers of injury; and 5) animal models that simulate anticipated clinical responses. RESULTS: There are striking similarities between injuries caused by radiation and SM exposures. Primary outcomes from both types of exposure include acute injuries, while late complications comprise chronic inflammation, oxidative stress, and vascular dysfunction, which can culminate in fibrosis in both skin and lung organ systems. This workshop brought together academic and industrial researchers, medical practitioners, US Government program officials, and regulators to discuss lung-, and skin- specific animal models and biomarkers, novel pathways of injury and recovery, and paths to licensure for products to address radiation or SM injuries. CONCLUSIONS: Regular communications between the radiological and chemical injury research communities can enhance the state-of-the-science, provide a unique perspective on novel therapeutic strategies, and improve overall US Government emergency preparedness.

Assessment of cell infiltration in myocardial infarction: a dose-dependent study using micrometer-sized iron oxide particles.

Myocardial infarction (MI) is a leading cause of death and disabilities. Inflammatory cells play a vital role in the process of postinfarction remodeling and repair. Inflammatory cell infiltration into the infarct site can be monitored using T 2-weighted MRI following an intravenous administration of iron oxide particles. In this study, various doses of micrometer-sized iron oxide particles (1.1-14.5 µg Fe/g body weight) were injected into the mouse blood stream before a surgical induction of MI. Cardiac MRIs were performed at 3, 7, 14, and 21 days postinfarction to monitor the signal attenuation at the infarct site. A dose-dependent phenomenon of signal attenuation was observed at the infarct site, with a higher dose leading to a darker signal. The study suggests an optimal temporal window for monitoring iron oxide particles-labeled inflammatory cell infiltration to the infarct site using MRI. The dose-dependent signal attenuation also indicates an optimal iron oxide dose of approximately 9.1-14.5 µg Fe/g body weight. A lower dose cannot differentiate the signal attenuation, whereas a higher dose would cause significant artifacts. This iron oxide-enhanced MRI technique can potentially be used to monitor cell migration and infiltration at the pathological site or to confirm any cellular response following some specific treatment strategies.

Indirectly probing Ca(2+) handling alterations following myocardial infarction in a murine model using T(1)-mapping manganese-enhanced magnetic resonance imaging.

Prolonged ischemia causes cellular necrosis and myocardial infarction (MI) via intracellular calcium (Ca(2+)) overload. Manganese-enhanced MRI indirectly assesses Ca(2+) influx movement in vivo as manganese (Mn(2+)) is a Ca(2+) analog. To characterize myocardial Mn(2+) efflux properties, T(1)-mapping manganese-enhanced MRI studies were performed on adult male C57Bl/6 mice in which Ca(2+) efflux was altered using pharmacological intervention agents or MI-inducing surgery. Results showed that (1) Mn(2+) efflux rate increased exponentially with increasing Mn(2+) doses; (2) SEA0400 (a sodium-calcium exchanger inhibitor) decreased the rate of Mn(2+) efflux; and (3) dobutamine (a positive inotropic agent) increased the Mn(2+) efflux rate. A novel analysis technique also delineated regional features in the MI mice, which showed an increased Mn(2+) efflux rate in the necrosed and peri-infarcted tissue zones. The T(1)-mapping manganese-enhanced MRI technique characterized alterations in myocardial Mn(2+) efflux rates following both pharmacologic intervention and an acute MI. The Mn(2+) efflux results were consistent with those in ex vivo studies showing an increased Ca(2+) concentration under similar conditions. Thus, T(1)-mapping manganese-enhanced MRI has the potential to indirectly identify and quantify intracellular Ca(2+) handling in the peri-infarcted tissue zones, which may reveal salvageable tissue in the post-MI myocardium.

Monitoring bone marrow-originated mesenchymal stem cell traffic to myocardial infarction sites using magnetic resonance imaging.

How stem cells promote myocardial repair in myocardial infarction (MI) is not well understood. The purpose of this study was to noninvasively monitor and quantify mesenchymal stem cells (MSC) from bone marrow to MI sites using magnetic resonance imaging (MRI). MSC were dual-labeled with an enhanced green fluorescent protein and micrometer-sized iron oxide particles prior to intra-bone marrow transplantation into the tibial medullary space of C57Bl/6 mice. Micrometer-sized iron oxide particles labeling caused signal attenuation in T(2)*-weighted MRI and thus allowed noninvasive cell tracking. Longitudinal MRI demonstrated MSC infiltration into MI sites over time. Fluorescence from both micrometer-sized iron oxide particles and enhanced green fluorescent protein in histology validated the presence of dual-labeled cells at MI sites. This study demonstrated that MSC traffic to MI sites can be noninvasively monitored in MRI by labeling cells with micrometer-sized iron oxide particles. The dual-labeled MSC at MI sites maintained their capability of proliferation and differentiation. The dual-labeling, intra-bone marrow transplantation, and MRI cell tracking provided a unique approach for investigating stem cells' roles in the post-MI healing process. This technique can potentially be applied to monitor possible effects on stem cell mobilization caused by given treatment strategies.

Relationship between blood and myocardium manganese levels during manganese-enhanced MRI (MEMRI) with T1 mapping in rats.

Manganese ions (Mn(2+) ) enter viable myocardial cells via voltage-gated calcium channels. Because of its shortening of T(1) and its relatively long half-life in cells, Mn(2+) can serve as an intracellular molecular contrast agent to study indirect calcium influx into the myocardium. One major concern in using Mn(2+) is its sensitivity over a limited range of concentrations employing T(1)-weighted images for visualization, which limits its potential in quantitative techniques. Therefore, this study assessed the implementation of a T(1) mapping method for cardiac manganese-enhanced MRI to enable a quantitative estimate of the influx of Mn(2+) over a wide range of concentrations in male Sprague-Dawley rats. This MRI method was used to compare the relationship between T(1) changes in the heart as a function of myocardium and blood Mn(2+) levels. Results showed a biphasic relationship between ΔR(1) and the total Mn(2+) infusion dose. Nonlinear relationships were observed between the total Mn(2+) infusion dose versus blood levels and left ventricular free wall ΔR(1) . At low blood levels of Mn(2+) , there was proportionally less cardiac enhancement seen than at higher levels of blood Mn(2+) . We hypothesize that Mn(2+) blood levels increase as a result of rate-limiting excretion by the liver and kidneys at these higher Mn(2+) doses.

Temporal and noninvasive monitoring of inflammatory-cell infiltration to myocardial infarction sites using micrometer-sized iron oxide particles.

Micrometer-sized iron oxide particles (MPIO) are a more sensitive MRI contrast agent for tracking cell migration compared to ultrasmall iron oxide particles. This study investigated the temporal relationship between inflammation and tissue remodeling due to myocardial infarction (MI) using MPIO-enhanced MRI. C57Bl/6 mice received an intravenous MPIO injection for cell labeling, followed by a surgically induced MI seven days later (n=7). For controls, two groups underwent either sham-operated surgery without inducing an MI post-MPIO injection (n=7) or MI surgery without MPIO injection (n=6). The MRIs performed post-MI showed significant signal attenuation around the MI site for the mice that received an intravenous MPIO injection for cell labeling, followed by a surgically induced MI seven days later, compared to the two control groups (P<0.01). The findings suggested that the prelabeled inflammatory cells mobilized and infiltrated into the MI site. Furthermore, the linear regression of contrast-to-noise ratio at the MI site and left ventricular ejection function suggested a positive correlation between the labeled inflammatory cell infiltration and cardiac function attenuation during post-MI remodeling (r2=0.98). In conclusion, this study demonstrated an MRI technique for noninvasively and temporally monitoring inflammatory cell migration into the myocardium while potentially providing additional insight concerning the pathologic progression of a myocardial infarction.

Porous-wall hollow glass microspheres as novel potential nanocarriers for biomedical applications.

Porous-wall hollow glass microspheres (PW-HGMs) are a novel form of glass material consisting of a 10- to 100-microm-diameter hollow central cavity surrounded by a 1-microm-thick silica shell. A tortuous network of nanometer-scale channels completely penetrates the shell. We show here that these channels promote size-dependent uptake and controlled release of biological molecules in the 3- to 8-nm range, including antibodies and a modified single-chain antibody variable fragment. In addition, a 6-nm (70-kDa) dextran can be used to gate the porous walls, facilitating controlled release of an internalized short interfering RNA. PW-HGMs remained in place after mouse intratumoral injection, suggesting a possible application for the delivery of anticancer drugs. The combination of a hollow central cavity that can carry soluble therapeutic agents with mesoporous walls for controlled release is a unique characteristic that distinguishes PW-HGMs from other glass materials for biomedical applications. FROM THE CLINICAL EDITOR: Porous-wall hollow glass microspheres (PW-HGMs) are a novel form of glass microparticles with a tortuous network of nanometer-scale channels. These channels allow size-dependent uptake and controlled release of biological molecules including antibodies and single-chain antibody fragments. PW-HGMs remained in place after mouse intratumoral injection, suggesting a possible application for the delivery of anti-cancer drugs.

Assessing manganese efflux using SEA0400 and cardiac T1-mapping manganese-enhanced MRI in a murine model.

The sodium-calcium exchanger (NCX) is one of the transporters contributing to the control of intracellular calcium (Ca(2+)) concentration by normally mediating net Ca(2+) efflux. However, the reverse mode of the NCX can cause intracellular Ca(2+) concentration overload, which exacerbates the myocardial tissue injury resulting from ischemia. Although the NCX inhibitor SEA0400 has been shown to therapeutically reduce myocardial injury, no in vivo technique exists to monitor intracellular Ca(2+) fluctuations produced by this drug. Cardiac manganese-enhanced MRI (MEMRI) may indirectly assess Ca(2+) efflux by estimating changes in manganese (Mn(2+)) content in vivo, since Mn(2+) has been suggested as a surrogate marker for Ca(2+). This study used the MEMRI technique to examine the temporal features of cardiac Mn(2+) efflux by implementing a T(1)-mapping method and inhibiting the NCX with SEA0400. The change in (1)H(2)O longitudinal relaxation rate, Delta R(1), in the left ventricular free wall, was calculated at different time points following infusion of 190 nmol/g manganese chloride (MnCl(2)) in healthy adult male mice. The results showed 50% MEMRI signal attenuation at 3.4 +/- 0.6 h post-MnCl(2) infusion without drug intervention. Furthermore, treatment with 50 +/- 0.2 mg/kg of SEA0400 significantly reduced the rate of decrease in Delta R(1). At 4.9-5.9 h post-MnCl(2) infusion, the average Delta R(1) values for the two groups treated with SEA0400 were 2.46 +/- 0.29 and 1.72 +/- 0.24 s(-1) for 50 and 20 mg/kg doses, respectively, as compared to the value of 1.27 +/- 0.28 s(-1) for the control group. When this in vivo data were compared to ex vivo absolute manganese content data, the MEMRI T(1)-mapping technique was shown to effectively quantify Mn(2+) efflux rates in the myocardium. Therefore, combining an NCX inhibitor with MEMRI may be a useful technique for assessing Mn(2+) transport mechanisms and rates in vivo, which may reflect changes in Ca(2+) transport.

The use of novel gradient directions with DTI to synthesize data with complicated diffusion behavior.

This study demonstrates a new technique for synthesizing diffusion tensor imaging (DTI) data sets that exhibit complex diffusion characteristics by performing operations on acquired DTI data of simple structures with anisotropic diffusive properties. The motivation behind this technique is to characterize the behavior of noise in complicated data using a phantom. Compared to simulations, an advantage to this approach is that the acquired data contain noise characteristic of the scanner and protocol. Using this technique, a simple capillary phantom is employed to infer the quality of data for more clinically realistic tissue structures (e.g., crossing fiber tracts). A water-filled phantom containing capillary arrays was constructed to demonstrate this technique, which uses a DTI protocol with typical clinical parameters. Eigenvalues and fractional anisotropy were calculated for the initial prolate data. Data were adjusted to synthesize different apparent diffusion coefficient (ADC) spatial distributions, which were compared to theoretical and analytical models. RMS differences and volumetric overlap between expected and measured ADC distributions were quantified for all synthesized distributions. Differences between synthesized and actual distributions were discussed.

An empirical characterization of the quality of DTI data and the efficacy of dyadic sorting.

Metrics calculated from images acquired using the diffusion tensor imaging (DTI) technique possess a systematic bias that depends on signal-to-noise ratio (SNR). Dyadic sorting provides a simple method for remediating some of this bias within a region(s) of interest (ROI). Although this bias and its removal using dyadic sorting have been studied previously within a theoretical framework, one can employ precise geometric knowledge of microstructures to perform an empirical comparison between expected DTI results and those measured with a scanner. In this project, the biasing effect of low SNR (approximately 1-10) on DTI eigenvalues was measured directly using water-filled capillary structures of two different sizes, and the magnitude of the corrective effect of dyadically sorting eigenvector-eigenvalue pairs was characterized. Multiple DTI series were acquired for determining DTI metrics at eight unique SNR values, using T(R) to vary signal intensity via T(1) contrast. Differences between the second and third eigenvalues, which should be equal for prolate geometry, ranged from approximately 23% to 45% and from 19% to 41% for large and small inner diameter capillaries after sorting eigenvalues by magnitude, and ranged from approximately 1% to 18% and from 1% to 4% after dyadic sorting. A high-resolution DTI series was used to observe the effect of ROI size on dyadic sorting. For restriction of diffusion on the scale of the small capillary at SNR approximately 18, an ROI with > or =50 pixels is adequate to determine fractional anisotropy to 99% accuracy, while larger ROI are required to resolve the two smaller eigenvalues to the same accuracy ( approximately 330-390 pixels). At low values of SNR, the iteration of dyadic sorting is suggested to achieve good accuracy. A method for the incorporation of empirical measurements into a bias-correction map, which would be useful for characterizing uncertainty and for reducing systematic bias in DTI data, is introduced.

Troponin I protein kinase C phosphorylation sites and ventricular function.

OBJECTIVE: Cardiac Troponin I (cTnI) phosphorylation by protein kinase C (PKC) results in a reduction of maximal actomyosin ATPase activity, an effect that is more marked at higher levels of calcium (Ca2+) and is likely to reduce active force development. We postulated that there would be greater Ca2+-dependent changes in ventricular function in hearts of cTnI transgenic (TG) mice expressing mutant troponin I lacking PKC sites compared to wild-type (WT). METHODS: We studied left ventricular function in isolated perfused hearts over a wide range of left ventricular volumes (Frank-Starling relationships) and mechanical restitution at three levels of perfusate Ca2+ (1.5, 2.5, and 3.5 mM). Manganese-enhanced magnetic resonance imaging (MRI) was used to study in-vivo sarcolemmal Ca2+ influx. The phosphorylation status of cTnI was examined by western blot analysis. RESULTS: Systolic contractile function in TG mice was altered in a calcium-dependent manner such that ventricular contractility was significantly greater in TG mice only at 3.5 mM perfusate Ca2+. The relaxation process and passive mechanical properties were unaltered in TG mice. Mechanical restitution parameters were abnormal in TG mice only at 1.5 mM perfusate Ca2+. In-vivo MRI data demonstrated up to 48% reduction in Mn2+-induced contrast enhancement, indicating reduced sarcolemmal Ca2+ influx. Western blot analysis indicated increased cTnI phosphorylation in TG mice. CONCLUSIONS: (1) TG mice exhibit calcium-dependent positive inotropy without slowed relaxation and this phenotype is mitigated by concomitant (compensatory) changes of reduced intracellular Ca2+ and increased phosphorylation of remaining cTnI sites. (2) The contractile phenotype in TG mice can be interpreted as an amplification of the normal response to changes in cellular Ca2+ observed in WT mice. Thus, PKC phosphorylation sites on cTnI play a role in attenuating contractile responses to changes in intracellular Ca2+.

Preclinical testing of PI3K/AKT/mTOR signaling inhibitors in a mouse model of ovarian endometrioid adenocarcinoma.

PURPOSE: Genetically engineered mouse (GEM) models of ovarian cancer that closely recapitulate their human tumor counterparts may be invaluable tools for preclinical testing of novel therapeutics. We studied murine ovarian endometrioid adenocarcinomas (OEA) arising from conditional dysregulation of canonical WNT and PI3K/AKT/mTOR pathway signaling to investigate their response to conventional chemotherapeutic drugs and mTOR or AKT inhibitors. EXPERIMENTAL DESIGN: OEAs were induced by injection of adenovirus expressing Cre recombinase (AdCre) into the ovarian bursae of Apc(flox/flox); Pten(flox/flox) mice. Tumor-bearing mice or murine OEA-derived cell lines were treated with cisplatin and paclitaxel, mTOR inhibitor rapamycin, or AKT inhibitors API-2 or perifosine. Treatment effects were monitored in vivo by tumor volume and bioluminescence imaging, in vitro by WST-1 proliferation assays, and in OEA tissues and cells by immunoblotting and immunostaining for levels and phosphorylation status of PI3K/AKT/mTOR signaling pathway components. RESULTS: Murine OEAs developed within 3 weeks of AdCre injection and were not preceded by endometriosis. OEAs responded to cisplatin + paclitaxel, rapamycin, and AKT inhibitors in vivo. In vitro studies showed that response to mTOR and AKT inhibitors, but not conventional cytotoxic drugs, was dependent on the status of PI3K/AKT/mTOR signaling. AKT inhibition in APC(-)/Pten(-) tumor cells resulted in compensatory upregulation of ERK signaling. CONCLUSIONS: The studies show the utility of this GEM model of ovarian cancer for preclinical testing of novel PI3K/AKT/mTOR signaling inhibitors and provide evidence for compensatory signaling, suggesting that multiple rather than single agent targeted therapy will be more efficacious for treating ovarian cancers with activated PI3K/AKT/mTOR signaling.

Feridex preloading permits tracking of CNS-resident macrophages after transient middle cerebral artery occlusion.

At this time, the pathophysiology of macrophage involvement and their role in stroke progression are poorly understood. Recently, T2- and T2*-weighted magnetic resonance imaging (MRI), after intravenous administration of iron-oxide particles, have been used to understand the inflammatory cascade. Earlier studies report that image enhancement after stroke is from iron-laden macrophages; however, they do not account for potential blood-brain barrier disruption and nonspecific contrast enhancement. In this study, spontaneously hypertensive rats were preloaded with Feridex 7 days before stroke, permitting the labeling of bone-marrow-derived macrophages. Three-dimensional gradient-echo imaging showed average signal decreases of 13% to 23% in preloaded animals, concentrated on the lesion periphery and reaching a maximum on days 2 to 4 after stroke. Immunohistochemistry showed ED-2+, PB+, MHC-II+ and TNF-alpha+ perivascular macrophages (PVM), meningeal macrophages (MM), and choroid plexus macrophages (CPM). ED-1+ and IBA+ tissue macrophages and/or activated microglia were located throughout the lesion, but were PB-. These findings indicate the following: (1) Feridex preloading permits tracking of the central nervous system (CNS)-resident macrophages (PVM, MM, and CPM) and (2) CNS-resident macrophages likely play an integral role in the inflammatory cascade through antigen presentation and expression of proinflammatory cytokines. Further refinement of this method should permit noninvasive monitoring of inflammation and potential evaluation of antiinflammatory therapies in preclinical models of stroke.

Non-uniform gradient prescription for precise angular measurements using DTI.

Diffusion Tensor Imaging (DTI) calculates a tensor for each voxel, representing the mean diffusive characteristics in volume-averaged tissue. Gradients that phase-encode spins according to the amount of their diffusion are usually applied uniformly over a sphere during a DTI procedure for minimal bias of tensor information. If prior knowledge of diffusion direction exists, the angular precision for determining the principle eigenvector of cylindrically-symmetric ("prolate") tensors can be improved by specifying gradients non-uniformly. Improvements in precision of 30-40% can be achieved using a restricted band of zenith angle values for gradient directions. Sensitivity to the a priori angular range of the principle eigenvector can be adjusted with the width of the band. Simulations and phantom data are in agreement; a preliminary validation is presented.

Monitoring dynamic alterations in calcium homeostasis by T (1)-weighted and T (1)-mapping cardiac manganese-enhanced MRI in a murine myocardial infarction model.

Manganese has been used as a T(1)-weighted MRI contrast agent in a variety of applications. Because manganese ions (Mn(2+)) enter viable myocardial cells via voltage-gated Ca(2+) channels, manganese-enhanced MRI is sensitive to the viability and inotropic state of the heart. In spite of the established importance of Ca(2+) regulation in the heart both before and after myocardial injury, monitoring strategies to assess Ca(2+) homeostasis in affected cardiac tissues are limited. This study implements a T(1)-mapping method to obtain quantitative information both dynamically and over a range of MnCl(2) infusion doses. To optimize the current Mn(2+) infusion protocols, we performed both dose-dependent and temporal washout studies. A non-linear relationship between infused MnCl(2) solution dose and increase in left ventricular wall relaxation rate (DeltaR(1)) was observed. Control mice also exhibited significant Mn(2+) clearance over time, with a decrease in DeltaR(1) of approximately 50% occurring in just 2.5 h. The complicated efflux time dependence possibly suggests multiple efflux mechanisms. With the use of the measured relationship between infused Mn(2+) dose, DeltaR(1), and inductively coupled plasma mass spectrometry data analysis provided a means of estimating the absolute heart Mn concentration in vivo. We show that this technique has the sensitivity to observe or monitor potential alterations in Ca(2+) handling in vivo because of the physiological remodeling after myocardial infarction. Left ventricular free wall DeltaR(1) values were significantly lower (P = 0.005) in the adjacent zone, surrounding the injured myocardial tissue, than in healthy tissue. This inferred reduction in Mn concentration can be used to estimate potentially salvageable myocardium in vivo for future treatment or evaluation of disease progression.