7 Tesla Magnetic Resonance Imaging to Detect Cortical Pathology in Multiple Sclerosis

Background

Neocortical lesions (NLs) are an important pathological component of multiple sclerosis (MS), but their visualization by magnetic resonance imaging (MRI) remains challenging.

Objectives

We aimed at assessing the sensitivity of multi echo gradient echo (ME-GRE) T₂ ^*-weighted MRI at 7.0 Tesla in depicting NLs compared to myelin and iron staining.

Methods

Samples from two MS patients were imaged post mortem using a whole body 7T MRI scanner with a 24-channel receive-only array. Isotropic 200 micron resolution images with varying T₂ ^* weighting were reconstructed from the ME-GRE data and converted into R₂ ^* maps. Immunohistochemical staining for myelin (proteolipid protein, PLP) and diaminobenzidine-enhanced Turnbull blue staining for iron were performed.

Results

Prospective and retrospective sensitivities of MRI for the detection of NLs were 48% and 67% respectively. We observed MRI maps detecting only a small portion of 20 subpial NLs extending over large cortical areas on PLP stainings. No MRI signal changes suggestive of iron accumulation in NLs were observed. Conversely, R₂ ^* maps indicated iron loss in NLs, which was confirmed by histological quantification.

Conclusions

High-resolution post mortem imaging using R₂ ^* and magnitude maps permits detection of focal NLs. However, disclosing extensive subpial demyelination with MRI remains challenging.     

Is R2* a New MRI Biomarker for the Progression of Parkinson’s Disease? A Longitudinal Follow-Up

Purpose

To study changes of iron content in basal ganglia in Parkinson’s disease (PD) through a three-year longitudinal follow-up of the effective transverse relaxation rate R₂*, a validated MRI marker of brain iron content which can be rapidly measured under clinical conditions.

Methods

Twenty-seven PD patients and 26 controls were investigated by a first MRI (t₀). Longitudinal analysis was conducted among the 18 controls and 14 PD patients who underwent a second MRI (t₁) 3 years after. The imaging protocol consisted in 6 gradient echo images obtained at different echo-times for mapping R₂*. Quantitative exploration of basal ganglia was performed by measuring the variation of R₂* [R₂*(t₁) – R₂*(t₀)] in several regions of interest.

Results

During the three-year evolution of PD, R₂* increased in Substantia nigra (SN) (by 10.2% in pars compacta, p = 0.001, and 8.1% in pars reticulata, p = 0.013) and in the caudal putamen (11.4%, p = 0.011), without significant change in controls. Furthermore, we showed a positive correlation between the variation of R₂* and the worsening of motor symptoms of PD (p = 0.028).

Conclusion

Significant variation of R₂* was longitudinally observed in the SN and caudal putamen of patients with PD evolving over a three-year period, emphasizing its interest as a biomarker of disease progression. Our results suggest that R₂* MRI follow-up could be an interesting tool for individual assessment of neurodegeneration due to PD, and also be useful for testing the efficiency of disease-modifying treatments.     

High Dynamic Range Processing for Magnetic Resonance Imaging

Purpose

To minimize feature loss in T₁- and T₂-weighted MRI by merging multiple MR images acquired at different T_R and T_E to generate an image with increased dynamic range.

Materials and Methods

High Dynamic Range (HDR) processing techniques from the field of photography were applied to a series of acquired MR images. Specifically, a method to parameterize the algorithm for MRI data was developed and tested. T₁- and T₂-weighted images of a number of contrast agent phantoms and a live mouse were acquired with varying T_R and T_E parameters. The images were computationally merged to produce HDR-MR images. All acquisitions were performed on a 7.05 T Bruker PharmaScan with a multi-echo spin echo pulse sequence.

Results

HDR-MRI delineated bright and dark features that were either saturated or indistinguishable from background in standard T₁- and T₂-weighted MRI. The increased dynamic range preserved intensity gradation over a larger range of T₁ and T₂ in phantoms and revealed more anatomical features in vivo.

Conclusions

We have developed and tested a method to apply HDR processing to MR images. The increased dynamic range of HDR-MR images as compared to standard T₁- and T₂-weighted images minimizes feature loss caused by magnetization recovery or low SNR.     

Human tumor cell proliferation evaluated using manganese-enhanced MRI

Background

Tumor cell proliferation can depend on calcium entry across the cell membrane. As a first step toward the development of a non-invasive test of the extent of tumor cell proliferation in vivo, we tested the hypothesis that tumor cell uptake of a calcium surrogate, Mn²⁺ [measured with manganese-enhanced MRI (MEMRI)], is linked to proliferation rate in vitro.

Methodology/Principal Findings

Proliferation rates were determined in vitro in three different human tumor cell lines: C918 and OCM-1 human uveal melanomas and PC-3 prostate carcinoma. Cells growing at different average proliferation rates were exposed to 1 mM MnCl₂ for one hour and then thoroughly washed. MEMRI R₁ values (longitudinal relaxation rates), which have a positive linear relationship with Mn²⁺ concentration, were then determined from cell pellets. Cell cycle distributions were determined using propidium iodide staining and flow cytometry. All three lines showed Mn²⁺-induced increases in R₁ compared to cells not exposed to Mn²⁺. C918 and PC-3 cells each showed a significant, positive correlation between MEMRI R₁ values and proliferation rate (p≤0.005), while OCM-1 cells showed no significant correlation. Preliminary, general modeling of these positive relationships suggested that pellet R₁ for the PC-3 cells, but not for the C918 cells, could be adequately described by simply accounting for changes in the distribution of the cell cycle-dependent subpopulations in the pellet.

Conclusions/Significance

These data clearly demonstrate the tumor-cell dependent nature of the relationship between proliferation and calcium influx, and underscore the usefulness of MEMRI as a non-invasive method for investigating this link. MEMRI is applicable to study tumors in vivo, and the present results raise the possibility of evaluating proliferation parameters of some tumor types in vivo using MEMRI.     

Determining Duration of HER2-Targeted Therapy Using Stem Cell Extinction Models

Introduction

Trastuzumab dramatically improves survival in breast cancer patients whose tumor overexpresses HER2. A subpopulation of cells in human breast tumors has been identified with characteristics of cancer stem cells. These breast cancer stem-like cells (BCSCs) rely on HER2 signaling for self-renewal, suggesting that HER2-targeted therapy targets BCSCs even when the bulk of the tumor does not overexpress HER2. In order to guide clinical trials examining HER2-targeted therapy in the adjuvant setting, we propose a mathematical model to examine BCSC population dynamics and predict optimal duration of therapy.

Methods

Varying the susceptibility of BCSCs to HER2-targeted therapy, we quantify the average time to extinction of BCSCs. We expand our model using stochastic simulation to include the partially differentiated tumor cells (TCs) that represent bulk tumor population and examine effects of plasticity on required duration of therapy.

Results

Lower susceptibility of BCSCs and increased rates of dedifferentiation entail longer extinction times, indicating a need for prolonged administration of HER2-targeted therapy. We predict that even when therapy does not appreciably reduce tumor size in the advanced cancer setting, it will eventually eradicate the tumor in the adjuvant setting as long as there is at least a modest effect on BCSCs.

Conclusions

We anticipate that our results will inform clinical trials of targeted therapies in planning the duration of therapy needed to eradicate BCSCs. Our predictions also address safety, as longer duration of therapy entails a greater potential impact on normal stem cells that may also be susceptible to stem cell-targeted therapies.     

In vivo Tracking of Dendritic Cell using MRI Reporter Gene,Ferritin

The noninvasive imaging of dendritic cells (DCs) migrated into lymph nodes (LNs) can provide helpful information on designing DCs-based immunotherapeutic strategies. This study is to investigate the influence of transduction of human ferritin heavy chain (FTH) and green fluorescence protein (GFP) genes on inherent properties of DCs, and the feasibility of FTH as a magnetic resonance imaging (MRI) reporter gene to track DCs migration into LNs. FTH-DCs were established by the introduction of FTH and GFP genes into the DC cell line (DC2.4) using lentivirus. The changes in the rate of MRI signal decay (R₂*) resulting from FTH transduction were analyzed in cell phantoms as well as popliteal LN of mice after subcutaneous injection of those cells into hind limb foot pad by using a multiple gradient echo sequence on a 9.4 T MR scanner. The transduction of FTH and GFP did not influence the proliferation and migration abilities of DCs. The expression of co-stimulatory molecules (CD40, CD80 and CD86) in FTH-DCs was similar to that of DCs. FTH-DCs exhibited increased iron storage capacity, and displayed a significantly higher transverse relaxation rate (R₂*) as compared to DCs in phantom. LNs with FTH-DCs exhibited negative contrast, leading to a high R₂* in both in vivo and ex vivo T₂*-weighted images compared to DCs. On histological analysis FTH-DCs migrated to the subcapsular sinus and the T cell zone of LN, where they highly expressed CD25 to bind and stimulate T cells. Our study addresses the feasibility of FTH as an MRI reporter gene to track DCs migration into LNs without alteration of their inherent properties. This study suggests that FTH-based MRI could be a useful technique to longitudinally monitor DCs and evaluate the therapeutic efficacy of DC-based vaccines.     

A Novel Cre Recombinase Imaging System for Tracking Lymphotropic Virus Infection In Vivo

Background

Detection, isolation, and identification of individual virus infected cells during long term infection are critical to advance our understanding of mechanisms of pathogenesis for latent/persistent viruses. However, current approaches to study these viruses in vivo have been hampered by low sensitivity and effects of cell-type on expression of viral encoded reporter genes. We have designed a novel Cre recombinase (Cre)-based murine system to overcome these problems, and thereby enable tracking and isolation of individual in vivo infected cells.

Methodology/Principal findings

Murine gammaherpesvirus 68 (MHV-68) was used as a prototypic persistent model virus. A Cre expressing recombinant virus was constructed and characterised. The virus is attenuated both in lytic virus replication, producing ten-fold lower lung virus titres than wild type virus, and in the establishment of latency. However, despite this limitation, when the sEGFP7 mouse line containing a Cre-activated enhanced green fluorescent protein (EGFP) was infected with the Cre expressing virus, sites of latent and persistent virus infection could be identified within B cells and macrophages of the lymphoid system on the basis of EGFP expression. Importantly, the use of the sEGFP7 mouse line which expresses high levels of EGFP allowed individual virus positive cells to be purified by FACSorting. Virus gene expression could be detected in these cells. Low numbers of EGFP positive cells could also be detected in the bone marrow.

Conclusions/Significance

The use of this novel Cre-based virus/mouse system allowed identification of individual latently infected cells in vivo and may be useful for the study and long-term monitoring of other latent/persistent virus infections.     

Pathological Mechanism for Delayed Hyperenhancement of Chronic Scarred Myocardium in Contrast Agent Enhanced Magnetic Resonance Imaging

Objectives

To evaluate possible mechanism for delayed hyperenhancement of scarred myocardium by investigating the relationship of contrast agent (CA) first pass and delayed enhancement patterns with histopathological changes.

Materials and Methods

Eighteen pigs underwent 4 weeks ligation of 1 or 2 diagonal coronary arteries to induce chronic infarction. The hearts were then removed and perfused in a Langendorff apparatus. The hearts firstly experienced phosphorus 31 MR spectroscopy. The hearts in group I (n = 9) and II (n = 9) then received the bolus injection of Gadolinium diethylenetriamine pentaacetic acid (0.05 mmol/kg) and gadolinium-based macromolecular agent (P792, 15 µmol/kg), respectively. First pass T₂ ^* MRI was acquired using a gradient echo sequence. Delayed enhanced T₁ MRI was acquired with an inversion recovery sequence. Masson''s trichrome and anti- von Willebrand Factor (vWF) staining were performed for infarct characterization.

Results

Wash-in of both kinds of CA caused the sharp and dramatic T₂ ^* signal decrease of scarred myocardium similar to that of normal myocardium. Myocardial blood flow and microvessel density were significantly recovered in 4-week-old scar tissue. Steady state distribution volume (ΔR₁ relaxation rate) of Gd-DTPA was markedly higher in scarred myocardium than in normal myocardium, whereas ΔR₁ relaxation rate of P792 did not differ significantly between scarred and normal myocardium. The ratio of extracellular volume to the total water volume was significantly greater in scarred myocardium than in normal myocardium. Scarred myocardium contained massive residual capillaries and dilated vessels. Histological stains indicated the extensively discrete matrix deposition and lack of cellular structure in scarred myocardium.

Conclusions

Collateral circulation formation and residual vessel effectively delivered CA into scarred myocardium. However, residual vessel without abnormal hyperpermeability allowed Gd-DTPA rather than P792 to penetrate into extravascular compartment. Discrete collagen fiber meshwork and loss of cellularity enlarged extracellular space accessible to Gd-DTPA, resulting in the delayed hyper-enhanced scar.     

A Low Cost Metal-Free Vascular Access Mini-Port for Artifact Free Imaging and Repeated Injections in Mice

Purpose

Small injection ports for mice are increasingly used for drug testing or when administering contrast agents. Commercially available mini-ports are expensive single-use items that cause imaging-artifacts. We developed and tested an artifact-free, low-cost, vascular access mini-port (VAMP) for mice.

Procedures

Leakage testing of the VAMP was conducted with high speed bolus injections of different contrast agents. VAMP-induced artifacts were assessed using a micro-CT and a small animal MRI (9.4T) scanner ex vivo. Repeated contrast administration was performed in vivo.

Results

With the VAMP there was no evidence of leakage with repeated punctures, high speed bolus contrast injections, and drawing of blood samples. In contrast to the tested commercially available ports, the VAMP did not cause artifacts with MRI or CT imaging.

Conclusions

The VAMP is an alternative to commercially available mini-ports and has useful applications in animal research involving imaging procedures and contrast agent testing.     

Magnetic resonance imaging of tumors colonized with bacterial ferritin-expressing Escherichia coli

Background

Recent studies have shown that human ferritin can be used as a reporter of gene expression for magnetic resonance imaging (MRI). Bacteria also encode three classes of ferritin-type molecules with iron accumulation properties.

Methods and Findings

Here, we investigated whether these bacterial ferritins can also be used as MRI reporter genes and which of the bacterial ferritins is the most suitable reporter. Bacterial ferritins were overexpressed in probiotic E. coli Nissle 1917. Cultures of these bacteria were analyzed and those generating highest MRI contrast were further investigated in tumor bearing mice. Among members of three classes of bacterial ferritin tested, bacterioferritin showed the most promise as a reporter gene. Although all three proteins accumulated similar amounts of iron when overexpressed individually, bacterioferritin showed the highest contrast change. By site-directed mutagenesis we also show that the heme iron, a unique part of the bacterioferritin molecule, is not critical for MRI contrast change. Tumor-specific induction of bacterioferritin-expression in colonized tumors resulted in contrast changes within the bacteria-colonized tumors.

Conclusions

Our data suggest that colonization and gene expression by live vectors expressing bacterioferritin can be monitored by MRI due to contrast changes.     

Comparative Study of Nanosecond Electric Fields In Vitro and In Vivo on Hepatocellular Carcinoma Indicate Macrophage Infiltration Contribute to Tumor Ablation In Vivo

Background and Aim

Recurrence and metastasis are associated with poor prognosis in hepatocellular carcinoma even in the patients who have undergone radical resection. Therefore, effective treatment is urgently needed for improvement of patients'' survival. Previously, we reported that nanosecond pulse electric fields (nsPEFs) can ablate melanoma by induction of apoptosis and inhibition of angiogenesis. This study aims to investigate the in vivo ablation strategy by comparing the dose effect of nanosecond electric fields in vitro and in vivo on hepatocellular carcinoma.

Materials and Methods

Four hepatocellular carcinoma cell lines HepG2, SMMC7721, Hep1-6, and HCCLM3 were pulsed to test the anti-proliferation and anti-migration ability of 100 ns nsPEFs in vitro. The animal model of human subdermal xenograft HCCLM3 cells into BALB/c nude mouse was used to test the anti-tumor growth and macrophage infiltration in vivo.

Results

In vitro assays showed anti-tumor effect of nsPEFs is dose-dependant. But the in vivo study showed the strategy of low dose and multiple treatments is superior to high dose single treatment. The macrophages infiltration significantly increased in the tumors which were treated by multiple low dose nsPEFs.

Conclusion

The low dose multiple nsPEFs application is more efficient than high dose single treatment in inhibiting the tumor volume in vivo, which is quite different from the dose-effect relationship in vitro. Beside the electric field strength, the macrophage involvement must be considered to account for effect variability and toxicology in vivo.     

Insulin-Like Growth Factor 1 Receptor and Response to Anti-IGF1R Antibody Therapy in Osteosarcoma

Background

Survival outcomes for patients with osteosarcoma (OS) have remained stagnant over the past three decades. Insulin-like growth factor 1 receptor (IGF1R) is over-expressed in a number of malignancies, and anti-IGF1R antibodies have and are currently being studied in clinical trials. Understanding the molecular aberrations which result in increased tumor response to anti-IGF1R therapy could allow for the selection of patients most likely to benefit from IGF1R targeted therapy.

Methods

IGF1R mRNA expression was assessed by RT PCR in OS patient primary tumors, cell lines, and xenograft tumors. IGF1R copy number was assessed by 3 approaches: PCR, FISH, and dot blot analysis. Exons 1–20 of IGF1R were sequenced in xenograft tumors and 87 primary OS tumors, and surface expression of IGF1R was assessed by flow cytometry. Levels of mRNA and protein expression, copy number, and mutation status were compared with tumor response to anti-IGF1R antibody therapy in 4 OS xenograft models.

Results

IGF1R mRNA is expressed in OS. Primary patient samples and xenograft samples had higher mRNA expression and copy number compared with corresponding cell lines. IGF1R mRNA expression, cell surface expression, copy number, and mutation status were not associated with tumor responsiveness to anti-IGF1R antibody therapy.

Conclusions

IGF1R is expressed in OS, however, no clear molecular markers predict response to IGF1R antibody-mediated therapy. Additional pre-clinical studies assessing potential predictive biomarkers and investigating targetable molecular pathways critical to the proliferation of OS cells are needed.     

A Polymeric Prodrug of 5-Fluorouracil-1-Acetic Acid Using a Multi-Hydroxyl Polyethylene Glycol Derivative as the Drug Carrier

Purpose

Macromolecular prodrugs obtained by covalently conjugating small molecular drugs with polymeric carriers were proven to accomplish controlled and sustained release of the therapeutic agents in vitro and in vivo. Polyethylene glycol (PEG) has been extensively used due to its low toxicity, low immunogenicity and high biocompatibility. However, for linear PEG macromolecules, the number of available hydroxyl groups for drug coupling does not change with the length of polymeric chain, which limits the application of PEG for drug conjugation purposes. To increase the drug loading and prolong the retention time of 5-fluorouracil (5-Fu), a macromolecular prodrug of 5-Fu, 5-fluorouracil-1 acid-PAE derivative (5-FA-PAE) was synthesized and tested for the antitumor activity in vivo.

Methods

PEG with a molecular weight of 38 kDa was selected to synthesize the multi-hydroxyl polyethylene glycol derivative (PAE) through an addition reaction. 5-fluorouracil-1 acetic acid (5-FA), a 5-Fu derivative was coupled with PEG derivatives via ester bond to form a macromolecular prodrug, 5-FA-PAE. The in vitro drug release, pharmacokinetics, in vivo distribution and antitumor effect of the prodrug were investigated, respectively.

Results

The PEG-based prodrug obtained in this study possessed an exceedingly high 5-FA loading efficiency of 10.58%, much higher than the maximum drug loading efficiency of unmodified PEG with the same molecular weight, which was 0.98% theoretically. Furthermore, 5-FA-PAE exhibited suitable sustained release in tumors.

Conclusion

This study provides a new approach for the development of the delivery to tumors of anticancer agents with PEG derivatives.     

A 16-Channel Receive,Forced Current Excitation Dual-Transmit Coil for Breast Imaging at 7T

Purpose

To enable high spatial and temporal breast imaging resolution via combined use of high field MRI, array coils, and forced current excitation (FCE) multi channel transmit.

Materials and Methods

A unilateral 16-channel receive array insert was designed for use in a transmit volume coil optimized for quadrature operation with dual-transmit RF shimming at 7T. Signal-to-noise ratio (SNR) maps, g-factor maps, and high spatial and temporal resolution in vivo images were acquired to demonstrate the utility of the coil architecture.

Results

The dual-transmit FCE coil provided homogeneous excitation and the array provided an increase in average SNR of 3.3 times (max 10.8, min 1.5) compared to the volume coil in transmit/receive mode. High resolution accelerated in vivo breast imaging demonstrated the ability to achieve isotropic spatial resolution of 0.5 mm within clinically relevant 90 s scan times, as well as the ability to perform 1.0 mm isotropic resolution imaging, 7 s per dynamics, with the use of bidirectional SENSE acceleration of up to R = 9.

Conclusion

The FCE design of the transmit coil easily accommodates the addition of a sixteen channel array coil. The improved spatial and temporal resolution provided by the high-field array coil with FCE dual-channel transmit will ultimately be beneficial in lesion detection and characterization.     

Non-invasive Imaging of Endothelial Progenitor Cells in Tumor Neovascularization Using a Novel Dual-modality Paramagnetic/Near-Infrared Fluorescence Probe

Objective

Bone-marrow derived endothelial progenitor cells (EPCs) play an important role in tumor neovasculature. Due to their tumor homing property, EPCs are regarded as promising targeted vectors for delivering therapeutic agents in cancer treatment. Consequently, non-invasive confirmation of targeted delivery via imaging is urgently needed. This study shows the development and application of a novel dual-modality probe for in vivo non-invasively tracking of the migration, homing and differentiation of EPCs.

Methods

The paramagnetic/near-infrared fluorescence probe Conjugate 1 labeled EPCs were systemically transplanted into mice bearing human breast MDA-MB-231 tumor xenografts. Magnetic resonance imaging (MRI) and near-infrared (NIR) fluorescence optical imaging were performed at different stages of tumor development. The homing of EPCs and the tumor neovascularization were further evaluated by immunofluorescence.

Results

Conjugate 1 labeled EPCs can be monitored in vivo by MRI and NIR fluorescence optical imaging without altering tumor growth for up to three weeks after the systemic transplantation. Histopathological examination confirmed that EPCs were recruited into the tumor bed and then incorporated into new vessels two weeks after the transplantation. Tumor size and microvessel density was not influenced by EPCs transplantation in the first three weeks.

Conclusions

This preclinical study shows the feasibility of using a MRI and NIR fluorescence optical imaging detectable probe to non-invasively monitor transplanted EPCs and also provides strong evidence that EPCs are involved in the development of endothelial cells during the tumor neovascularization.     

Optical and NMR dose response of N-isopropylacrylamide normoxic polymer gel for radiation therapy dosimetry

Background

Application of less toxic normoxic polymer gel of N-isopropyl acrylamide (NIPAM) for radiation therapy has been studied in recent years.

Aim

In the current study the optical and NMR properties of NIPAM were studied for radiation therapy dosimetry application.

Materials and methods

NIPAM normoxic polymer gel was prepared and irradiated by 9 MV photon beam of a medical linac. The optical absorbance was measured using a conventional laboratory spectrophotometer in different wavelengths ranging from 390 to 860 nm. R₂ measurements of NIPAM gels were performed using a 1.5 T scanner and R₂–dose curve was obtained.

Results

Our results showed R₂ dose sensitivity of 0.193 ± 0.01 s⁻¹ Gy⁻¹ for NIPAM gel. Both R₂ and optical absorbance showed a linear relationship with dose from 1.5 to 11 Gy for NIPAM gel dosimeter. Moreover, absorbance–dose response varied considerably with light wavelength and highest sensitivity was seen for the blue part of the spectrum.

Conclusion

Our results showed that both optical and NMR approaches have acceptable sensitivity and accuracy for dose determination with NIPAM gel. However, for optical reading of the gel, utilization of an optimum optical wavelength is recommended.     

In Vivo Detection of Activated Platelets Allows Characterizing Rupture of Atherosclerotic Plaques with Molecular Magnetic Resonance Imaging in Mice

Background

Early and non-invasive detection of platelets on micro atherothrombosis provides a means to identify unstable plaque and thereby allowing prophylactic treatment towards prevention of stroke or myocardial infarction. Molecular magnetic resonance imaging (mMRI) of activated platelets as early markers of plaque rupture using targeted contrast agents is a promising strategy. In this study, we aim to specifically image activated platelets in murine atherothrombosis by in vivo mMRI, using a dedicated animal model of plaque rupture.

Methods

An antibody targeting ligand-induced binding sites (LIBS) on the glycoprotein IIb/IIIa-receptor of activated platelets was conjugated to microparticles of iron oxide (MPIO) to form the LIBS-MPIO contrast agent causing a signal-extinction in T2*-weighted MRI. ApoE^−/− mice (60 weeks-old) were fed a high fat diet for 5 weeks. Using a small needle, the surface of their carotid plaques was scratched under blood flow to induce atherothrombosis. In vivo 9.4 Tesla MRI was performed before and repetitively after intravenous injection of either LIBS-MPIO versus non-targeted-MPIO.

Results

LIBS-MPIO injected animals showed a significant signal extinction (p<0.05) in MRI, corresponding to the site of plaque rupture and atherothrombosis in histology. The signal attenuation was effective for atherothrombosis occupying ≥2% of the vascular lumen. Histology further confirmed significant binding of LIBS-MPIO compared to control-MPIO on the thrombus developing on the surface of ruptured plaques (p<0.01).

Conclusion

in vivo mMRI detected activated platelets on mechanically ruptured atherosclerotic plaques in ApoE^−/− mice with a high sensititvity. This imaging technology represents a unique opportunity for noninvasive detection of atherothrombosis and the identification of unstable atherosclerotic plaques with the ultimate promise to prevent strokes and myocardial infarctions.     

Very Bright Green Fluorescent Proteins from the Pontellid Copepod Pontella mimocerami

Background

Fluorescent proteins (FP) homologous to the green fluorescent protein (GFP) from the jellyfish Aequorea victoria have revolutionized biomedical research due to their usefulness as genetically encoded fluorescent labels. Fluorescent proteins from copepods are particularly promising due to their high brightness and rapid fluorescence development.

Results

Here we report two novel FPs from Pontella mimocerami (Copepoda, Calanoida, Pontellidae), which were identified via fluorescence screening of a bacterial cDNA expression library prepared from the whole-body total RNA of the animal. The proteins are very similar in sequence and spectroscopic properties. They possess high molar extinction coefficients (79,000 M⁻¹ cm⁻) and quantum yields (0.92), which make them more than two-fold brighter than the most common FP marker, EGFP. Both proteins form oligomers, which we were able to counteract to some extent by mutagenesis of the N-terminal region; however, this particular modification resulted in substantial drop in brightness.

Conclusions

The spectroscopic characteristics of the two P. mimocerami proteins place them among the brightest green FPs ever described. These proteins may therefore become valuable additions to the in vivo imaging toolkit.     

Investigation of a MMP-2 Activity-Dependent Anchoring Probe for Nuclear Imaging of Cancer

Purpose

Since matrix metalloproteinase-2 (MMP-2) is an important marker of tumor malignancy, we developed an original drug design strategy, MMP-2 activity dependent anchoring probes (MDAP), for use in MMP-2 activity imaging, and evaluated the usefulness of this probe in in vitro and in vivo experiments.

Methods

We designed and synthesized MDAP₁₀₀₀, MDAP₃₀₀₀, and MDAP₅₀₀₀, which consist of 4 independent moieties: RI unit (¹¹¹In hydrophilic chelate), MMP-2 substrate unit (short peptide), anchoring unit (alkyl chain), and anchoring inhibition unit (polyethylene glycol (PEGn; where n represents the approximate molecular weight, n = 1000, 3000, and 5000). Probe cleavage was evaluated by chromatography after MMP-2 treatment. Cellular uptake of the probes was then measured. Radioactivity accumulation in tumor xenografts was evaluated after intravenous injection of the probes, and probe cleavage was evaluated in tumor homogenates.

Results

MDAP₁₀₀₀, MDAP₃₀₀₀, and MDAP₅₀₀₀ were cleaved by MMP-2 in a concentration-dependent manner. MDAP₃₀₀₀ pretreated with MMP-2 showed higher accumulation in tumor cells, and was completely blocked by additional treatment with an MMP inhibitor. MDAP₃₀₀₀ exhibited rapid blood clearance and a high tumor accumulation after intravenous injection in a rodent model. Furthermore, pharmacokinetic analysis revealed that MDAP₃₀₀₀ exhibited a considerably slow washout rate from tumors to blood. A certain fraction of cleaved MDAP₃₀₀₀ existed in tumor xenografts in vivo.

Conclusions

The results indicate the possible usefulness of our MDAP strategy for tumor imaging.