Laura Calderan

ADIPOSE-DERIVED MESENCHYMAL STEM CELLS AMELIORATE CHRONIC EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS

Mesenchymal stem cells (MSCs) represent a promising therapeutic approach for neurological autoimmune diseases; previous studies have shown that treatment with bone marrow‐derived MSCs induces immune modulation and reduces disease severity in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Here we show that intravenous administration of adipose‐derived MSCs (ASCs) before disease onset significantly reduces the severity of EAE by immune modulation and decreases spinal cord inflammation and demyelination. ASCs preferentially home into lymphoid organs but also migrates inside the central nervous system (CNS). Most importantly, administration of ASCs in chronic established EAE significantly ameliorates the disease course and reduces both demyelination and axonal loss, and induces a Th2‐type cytokine shift in T cells. Interestingly, a relevant subset of ASCs expresses activated α4 integrins and adheres to inflamed brain venules in intravital microscopy experiments. Bioluminescence imaging shows that α4 integrins control ASC accumulation in inflamed CNS. Importantly, we found that ASC cultures produce basic fibroblast growth factor, brain‐derived growth factor, and platelet‐derived growth factor‐AB. Moreover, ASC infiltration within demyelinated areas is accompanied by increased number of endogenous oligodendrocyte progenitors. In conclusion, we show that ASCs have clear therapeutic potential by a bimodal mechanism, by suppressing the autoimmune response in early phases of disease as well as by inducing local neuroregeneration by endogenous progenitors in animals with established disease. Overall, our data suggest that ASCs represent a valuable tool for stem cell–based therapy in chronic inflammatory diseases of the CNS. STEM CELLS 2009;27:2624–2635

Cerenkov radiation allows in vivo optical imaging of positron emitting radiotracers.

In this paper, we showed that Cerenkov radiation (CR) escaping from the surface of small living animals injected with (18)F-FDG can be detected with optical imaging techniques. (18)F decays by emitting positrons with a maximum energy of 0.635 MeV; such positrons, when travelling into tissues faster than the speed of light in the same medium, are responsible of CR emission. A detailed model of the CR spectrum considering the positron energy spectrum was developed in order to quantify the amount of light emission. The results presented in this work were obtained using a commercial optical imager equipped with charged coupled detectors (CCD). Our data open the door to optical imaging (OI) in vivo of the glucose metabolism, at least in pre-clinical research. We found that the heart and bladder can be clearly identified in the animal body reflecting the accumulation of the (18)F-FDG. Moreover, we describe two different methods based on the spectral analysis of the CR that can be used to estimate the depth of the source inside the animal. We conclude that (18)F-FDG can be employed as it is as a bimodal tracer for positron emission tomography (PET) and OI techniques. Our results are encouraging, suggesting that it could be possible to apply the proposed approach not only to beta(+) but also to pure beta(-) emitters.

Early antiangiogenic activity of SU11248 evaluated in vivo by dynamic contrast-enhanced magnetic resonance imaging in an experimental model of colon carcinoma.

Purpose: To compare two dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) techniques in terms of their ability in assessing the early antiangiogenic effect of SU11248, a novel selective multitargeted tyrosine kinase inhibitor, that exhibits direct antitumor and antiangiogenic activity via inhibition of the receptor tyrosine kinases platelet-derived growth factor receptor, vascular endothelial growth factor receptor, KIT, and FLT3. Experimental Design: A s.c. tumor model of HT29 human colon carcinoma in athymic mice was used. Two DCE-MRI techniques were used based, respectively, on macromolecular [Gd-diethylenetriaminepentaacetic acid (DTPA)-albumin] and low molecular weight (Gd-DTPA) contrast agents. The first technique provided a quantitative measurement of transendothelial permeability and fractional plasma volume, accepted surrogate markers of tumor angiogenesis. With the second technique, we quantified the initial area under the concentration-time curve, which gives information related to tumor perfusion and vascular permeability. Experiments were done before and 24 hours after a single dose administration of SU11248. Results: The early antiangiogenic effect of SU11248 was detected by DCE-MRI with macromolecular contrast agent as a 42% decrease in vascular permeability measured in the tumor rim. The effect was also detected by DCE-MRI done with Gd-DTPA as a 31% decrease in the initial area under the concentration-time curve. Histologic slices showed a statistically significant difference in mean vessel density between the treated and control groups. Conclusions: The early antiangiogenic activity of SU11248 was detected in vivo by DCE-MRI techniques using either macromolecular or low molecular weight contrast agents. Because DCE-MRI techniques with low molecular weight contrast agents can be used in clinical studies, these results could be relevant for the design of clinical trials based on new paradigms.

In Vivo Assessment of Antiangiogenic Activity of SU6668 in an Experimental Colon Carcinoma Model

Purpose: The purpose of this research was to assess in vivo by dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) the antiangiogenic effect of SU6668, an oral, small molecule inhibitor of the angiogenic receptor tyrosine kinases vascular endothelial growth factor receptor 2 (Flk-1/KDR), platelet-derived growth factor receptor, and fibroblast growth factor receptor 1. Experimental Design: A s.c. tumor model of HT29 human colon carcinoma in athymic mice was used. DCE-MRI with a macromolecular contrast agent was used to measure transendothelial permeability and fractional plasma volume, accepted surrogate markers of tumor angiogenesis. CD31 immunohistochemical staining was used for assessing microvessels density and vessels area. Experiments were performed after 24 h, and 3, 7, and 14 days of treatment. Results: DCE-MRI clearly detected the early effect (after 24 h of treatment) of SU6668 on tumor vasculature as a 51% and 26% decrease in the average vessel permeability measured in the tumor rim and core (respectively). A substantial decrease was also observed in average fractional plasma volume in the rim (59%) and core (35%) of the tumor. Histological results confirmed magnetic resonance imaging findings. After 3, 7, and 14 days of treatment, postcontrast magnetic resonant images presented a thin strip of strongly enhanced tissue at the tumor periphery; histology examination showed that this hyperenhanced ring corresponded to strongly vascularized tissue adjacent but external to the tumor. Histology also revealed a strong decrease in the thickness of peripheral viable tissue, with a greatly reduced vessel count. SU6668 greatly inhibited tumor growth, with 60% inhibition at 14 days of treatment. Conclusions: DCE-MRI detected in vivo the antiangiogenic efficacy of SU6668.

A novel near-infrared indocyanine dye-polyethylenimine conjugate allows DNA delivery imaging in vivo

Near-infrared (NIR) fluorescence light has been applied to monitor several biological events in vivo since it penetrates tissues more efficiently than visible light. Dyes exhibiting NIR fluorescence and having large Stokes shift are key elements for this promising optical imaging technology. Here, we report the synthesis of a novel conjugate between a near-infrared indocyanine dye and an organic polyamine polymer (polyethylenimine, PEI) (IR820-PEI) with high chemical stability and good optical properties. IR820-PEI absorbs at 665 nm, emits at 780 nm, and displays a large Stokes shift (115 nm). Moreover, the reported conjugate is able to bind DNA, and the delivery process can be monitored in vivo with noninvasive optical imaging techniques. These characteristics make IR820-PEI one of the most effective and versatile indocyanine dye polymeric-conjugate reported so far.

In Vivo Phenotyping of the ob/ob Mouse by Magnetic Resonance Imaging and 1H-Magnetic Resonance Spectroscopy

Objective: We studied ob/ob and wild‐type (WT) mice to characterize the adipose tissues depots and other visceral organs and to establish an experimental paradigm for in vivo phenotyping.

In vivo mapping of Fractional Plasma Volume (FPV) and endothelial transfer coefficient (KPS) in solid tumors using a macromolecular contrast agent: Correlation with histology and ultrastructure

Contrast‐enhanced MRI, immunostaining and electron microscopy were used to detect areas of intense angiogenesis in experimental tumors. This work was also aimed at evaluating the possible effect of the surrounding tissues on tumor microvasculature and at studying the penetration of macromolecules in avascular areas. Human colon carcinoma cells were implanted in subcutaneous tissue of nude mice. Dynamic T1‐weigthed 3D pulse sequences were acquired before and after administration of Gd‐DTPA‐albumin to obtain parametric maps of fractional plasma volume (fpv) and transendothelial permeability (Kps). The maps suggested that tumor can be subdivided into 4 zones located in the peripheral rim (zones I–II) or in the core (zones III–IV) of the tumor itself. Significant differences (p<0.001) were found in the values of Kps and fpv of zones I–II with respect to zones III–IV. In the peripheral rim, permeability was significantly higher (p<0.01) in the muscle‐peripheral region (zone I) with respect to the skin‐peripheral region (zone II). In areas with high Kps, histological and ultrastructural examination revealed clusters of newly formed vessels and signs of intense permeability. Numerous vascular vesicular organs were visible in these areas. In the tumoral core, analysis of the microcirculatory parameters revealed regions with mild permeability (zone III) and regions with negligible permeability (zone IV). These 2 zones were discriminated by the average value of Kps (p<0.05), while their fpv was not significantly different. Upon histological examination, the tumoral core exhibited necrotic areas; CD31 immunocytochemistry exhibited that it was diffusely hypovascularized with large avascular areas. Upon ultrastructural examination, capillaries were rarely visible and exhibited signs of endothelial cell damage. The results suggest that segmentation based on microvascular parameters detects in vivo zones characterized by immunocytochemical and ultrastructural aspects of intense angiogenesis. The finding that a certain amount of contrast agent penetrates in the tumoral core suggests that high oncotic and hydrostatic pressure only partially hinders the penetration of macromolecules.

Effect of tamoxifen in an experimental model of breast tumor studied by dynamic contrast-enhanced magnetic resonance imaging and different contrast agents

Objectives:The aim of this study was to compare the efficacy of gadoteridol, B22956/1 (a new protein binding blood pool contrast agent), and (Gd-DTPA)37-albumin in detecting, by dynamic contrast-enhanced magnetic resonance imaging (MRI), the effect in vivo of tamoxifen in an experimental model of breast tumor implanted in rats. Materials and Methods:Tumors were induced by subcutaneous injection of 106 mammary adenocarcinoma cells (13762 MAT B III). Treatment with tamoxifen (or vehicle) started on day 4 after implantation. On day 10 after implantation, animals were observed by MRI using B22956/1 (or gadoteridol) and, 24 hours later, using (Gd-DTPA)37–albumin. Results:Dynamic contrast-enhanced magnetic resonance imaging data showed that tamoxifen treatment decreased vascular permeability to B22956/1, whereas no difference was detectable in permeability to gadoteridol or to (Gd-DTPA)37–albumin. No effect on fractional plasma volume was detected with either of contrast agents. Conclusions:B22956/1 is superior to both small Gd chelates and macromolecular contrast agents in the assessment of the effect of tamoxifen treatment on tumor vasculature.

Combined optical and single photon emission imaging: preliminary results.

In vivo optical imaging instruments are generally devoted to the acquisition of light coming from fluorescence or bioluminescence processes. Recently, an instrument was conceived with radioisotopic detection capabilities (Kodak in Vivo Multispectral System F) based on the conversion of x-rays from the phosphorus screen. The goal of this work is to demonstrate that an optical imager (IVIS 200, Xenogen Corp., Alameda, USA), designed for in vivo acquisitions of small animals in bioluminescent and fluorescent modalities, can even be employed to detect signals due to radioactive tracers. Our system is based on scintillator crystals for the conversion of high-energy rays and a collimator. No hardware modifications are required. Crystals alone permit the acquisition of photons coming from an in vivo 20 g nude mouse injected with a solution of methyl diphosphonate technetium 99 metastable (Tc99m-MDP). With scintillator crystals and collimators, a set of measurements aimed to fully characterize the system resolution was carried out. More precisely, system point spread function and modulation transfer function were measured at different source depths. Results show that system resolution is always better than 1.3 mm when the source depth is less than 10 mm. The resolution of the images obtained with radioactive tracers is comparable with the resolution achievable with dedicated techniques. Moreover, it is possible to detect both optical and nuclear tracers or bi-modal tracers with only one instrument.

Cerebral cortex three‐dimensional profiling in human fetuses by magnetic resonance imaging

Seven human fetuses of crown/rump length corresponding to gestational ages ranging from the 12th to the 16th week were studied using a paradigm based on three‐dimensional reconstruction of the brain obtained by magnetic resonance imaging (MRI). The aim of the study was to evaluate brain morphology in situ and to describe developmental dynamics during an important period of fetal morphogenesis. Three‐dimensional MRI showed the increasing degree of maturation of the brains; fronto‐occipital distance, bitemporal distance and occipital angle were examined in all the fetuses. The data were interpreted by correlation with the internal structure as visualized using high‐spatial‐resolution MRI, acquired using a 4.7‐T field intensity magnet with a gradient power of 20 G cm−1. The spatial resolution was sufficient for a detailed detection of five layers, and the contrast was optimized using sequences with different degrees of T1 and T2 weighting. Using the latter, it was possible to visualize the subplate and marginal zones. The cortical thickness was mapped on to the hemispheric surface, describing the thickness gradient from the insular cortex to the periphery of the hemispheres. The study demonstrates the utility of MRI for studying brain development. The method provides a quantitative profiling of the brain, which allows the calculation of important morphological parameters, and it provides informative regarding transient features of the developing brain.