Time-lapse imaging of the dynamics of CNS glial-axonal interactions in vitro and ex vivo.

PLoS One. 2012;7(1):e30775

Authors: Ioannidou K, Anderson KI, Strachan D, Edgar JM, Barnett SC

Abstract
BACKGROUND: Myelination is an exquisite and dynamic example of heterologous cell-cell interaction, which consists of the concentric wrapping of multiple layers of oligodendrocyte membrane around neuronal axons. Understanding the mechanism by which oligodendrocytes ensheath axons may bring us closer to designing strategies to promote remyelination in demyelinating diseases. The main aim of this study was to follow glial-axonal interactions over time both in vitro and ex vivo to visualize the various stages of myelination.
METHODOLOGY/PRINCIPAL FINDINGS: WE TOOK TWO APPROACHES TO FOLLOW MYELINATION OVER TIME: i) time-lapse imaging of mixed CNS myelinating cultures generated from mouse spinal cord to which exogenous GFP-labelled murine cells were added, and ii) ex vivo imaging of the spinal cord of shiverer (Mbp mutant) mice, transplanted with GFP-labelled murine neurospheres. We demonstrate that oligodendrocyte-axonal interactions are dynamic events with continuous retraction and extension of oligodendroglial processes. Using cytoplasmic and membrane-GFP labelled cells to examine different components of the myelin-like sheath, we provide evidence from time-lapse fluorescence microscopy and confocal microscopy that the oligodendrocytes' cytoplasm-filled processes initially spiral around the axon in a corkscrew-like manner. This is followed subsequently by focal expansion of the corkscrew process to form short cuffs, which then extend longitudinally along the axons. We predict from this model that these spiral cuffs must extend over each other first before extending to form internodes of myelin.
CONCLUSION: These experiments show the feasibility of visualizing the dynamics of glial-axonal interaction during myelination over time. Moreover, these approaches complement each other with the in vitro approach allowing visualization of an entire internodal length of myelin and the ex vivo approach validating the in vitro data.

PMID: 22303455 [PubMed - in process]

Stratification of Nucleoside Analog Chemotherapy Using 1-(2'-Deoxy-2'-18F-Fluoro-β-D-Arabinofuranosyl)Cytosine and 1-(2'-Deoxy-2'-18F-Fluoro-β-L-Arabinofuranosyl)-5-Methylcytosine PET.

J Nucl Med. 2012 Feb;53(2):275-80

Authors: Lee JT, Campbell DO, Satyamurthy N, Czernin J, Radu CG

Abstract
UNLABELLED: The ability to measure tumor determinants of response to nucleoside analog (NA) chemotherapy agents such as gemcitabine and related compounds could significantly affect the management of several types of cancer. Previously we showed that the accumulation in tumors of the new PET tracer 1-(2'-deoxy-2'-(18)F-fluoro-β-d-arabinofuranosyl)cytosine ((18)F-FAC) is predictive of responses to gemcitabine. (18)F-FAC retention in cells requires deoxycytidine kinase (dCK), a rate-limiting enzyme in the deoxyribonucleoside salvage metabolism and in gemcitabine conversion from an inactive prodrug to a cytotoxic compound. The objectives of the current study were to determine whether (18)F-FAC tumor uptake is also influenced by cytidine deaminase (CDA), a determinant of resistance to gemcitabine; to develop a new PET assay using (18)F-FAC and the related probe 1-(2'-deoxy-2'-(18)F-fluoro-β-l-arabinofuranosyl)-5-methylcytosine (l-(18)F-FMAC) to profile tumor lesions for both dCK and CDA enzymatic activities; and to determine whether this PET assay can identify the most effective NA chemotherapy against tumors with differential expression of dCK and CDA.
METHODS: Isogenic murine leukemic cell lines with defined dCK and CDA activities were generated by retroviral transduction. A cell viability assay was used to determine the sensitivity of the isogenic cell lines to the dCK-dependent NA prodrugs gemcitabine and clofarabine. In vitro enzymatic and cell-based tracer uptake assays and in vivo PET with (18)F-FAC and l-(18)F-FMAC were used to predict tumor responses to gemcitabine and clofarabine.
RESULTS: dCK and CDA activities measured by kinase and tracer uptake assays correlated with the sensitivity of isogenic cell lines to gemcitabine and clofarabine. Coexpression of CDA decreased the sensitivity of dCK-positive cells to gemcitabine treatment in vitro by 15-fold but did not affect responses to clofarabine. Coexpression of CDA decreased (18)F-FAC but not l-(18)F-FMAC, phosphorylation, and uptake by dCK-positive cells. (18)F-FAC and l-(18)F-FMAC PET estimates of the enzymatic activities of dCK and CDA in tumor implants in mice were predictive of responses to gemcitabine and clofarabine treatment in vivo.
CONCLUSION: These findings support the utility of PET-based phenotyping of tumor nucleoside metabolism for guiding the selection of NA prodrugs.

PMID: 22302964 [PubMed - in process]

Synthesis and Preliminary Evaluation of 18F-Labeled Pyridaben Analogues for Myocardial Perfusion Imaging with PET.

J Nucl Med. 2012 Feb 2;

Authors: Mou T, Zhao Z, Fang W, Peng C, Guo F, Liu B, Ma Y, Zhang X

Abstract
In this study the (18)F-labeled pyridaben analogs 2-tertbutyl-4-chloro-5-(4-(2-(18)F-fluoroethoxy))benzyloxy-2H-pyridazin-3-one ((18)F-FP1OP) and 2-tertbutyl-4-chloro-5-(4-(2-(2-(2-(18)F-fluoroethoxy)ethoxy)ethoxy))benzyloxy-2H-pyridazin-3-one ((18)F-FP3OP) were synthesized, characterized, and evaluated as potential myocardial perfusion imaging (MPI) agents with PET. METHODS: The tosylate labeling precursors of 2-tert-butyl-4-chloro-5-(4-(2-tosyloxy-ethoxy))-benzyloxy-2H-pyridazin-3-one (OTs-P1OP), 2-tert-butyl-4-chloro-5-(4-(2-(2-(2-tosyloxy-ethoxy)ethoxy)ethoxy))-benzyloxy-2H-pyridazin-3-one (OTs-P3OP), and the corresponding nonradioactive compounds ((19)F-FP1OP and (19)F-FP3OP) were synthesized and characterized by infrared, (1)H nuclear magnetic resonance, (13)C nuclear magnetic resonance, and mass spectrometry analysis. (18)F-FP1OP and (18)F-FP3OP were obtained by 1-step nucleophilic substitution of tosyl with (18)F and evaluated as MPI agents in vitro (physicochemical properties, stability), ex vivo (autoradiography), and in vivo (toxicity and biodistribution in normal mice; cardiac PET in healthy Chinese mini swine and in acute myocardial infarction and chronic myocardial ischemia models). RESULTS: The total radiosynthesis time of both tracers, including final high-pressure liquid chromatography purification, was about 70-90 min. Typical decay-corrected radiochemical yields were about 50%, and the radiochemical purities were more than 98% after purification. (18)F-FP1OP had lower hydrophilicity and higher water stability than that of (18)F-FP3OP. In biodistribution studies, both (18)F-FP1OP and (18)F-FP3OP had high heart uptake (31.13 ± 6.24 and 31.10 ± 3.72 percentage injected dose per gram at 2 min after injection, respectively) and high heart-to-liver, heart-to-lung, and heart-to-blood ratios at all time points after injection. Further autoradiography evaluation of (18)F-FP1OP showed that the heart uptake could be blocked effectively by rotenone or nonradioactive (19)F-FP1OP. Clear cardiac PET images of (18)F-FP1OP were obtained in healthy Chinese mini swine at 2, 15, 30, 60, and 120 min after injection, and the uptake of perfusion deficit areas was much lower than in normal tissue in both acute myocardial infarction and chronic myocardial ischemia models. CONCLUSION: The (18)F-labeled pyridaben analogs reported in this study have high heart uptake and low background uptake in both the mouse model and the Chinese mini swine model. The tracer with the shorter radiolabeling side chain ((18)F-FP1OP) has better stability, faster clearance from the major organs, and a higher heart-to-liver ratio than the other tracer ((18)F-FP3OP). On the basis of the promising biologic properties, this mitochondrial complex I-targeted tracer ((18)F-FP1OP) is worthy to be developed as an MPI agent and to be compared with the other PET MPI agents in the future.

PMID: 22302832 [PubMed - as supplied by publisher]

A new sequence for single-shot diffusion-weighted NMR spectroscopy by the trace of the diffusion tensor.

Magn Reson Med. 2012 Feb 2;

Authors: Valette J, Giraudeau C, Marchadour C, Djemai B, Geffroy F, Ghaly MA, Le Bihan D, Hantraye P, Lebon V, Lethimonnier F

Abstract
Diffusion-weighted spectroscopy is a unique tool for exploring the intracellular microenvironment in vivo. In living systems, diffusion may be anisotropic, when biological membranes exhibit particular orientation patterns. In this work, a volume selective diffusion-weighted sequence is proposed, allowing single-shot measurement of the trace of the diffusion tensor, which does not depend on tissue anisotropy. With this sequence, the minimal echo time is only three times the diffusion time. In addition, cross-terms between diffusion gradients and other gradients are cancelled out. An adiabatic version, similar to localization by adiabatic selective refocusing sequence, is then derived, providing partial immunity against cross-terms. Proof of concept is performed ex vivo on chicken skeletal muscle by varying tissue orientation and intra-voxel shim. In vivo performance of the sequence is finally illustrated in a U87 glioblastoma mouse model, allowing the measurement of the trace apparent diffusion coefficient for six metabolites, including J-modulated metabolites. Although measurement performed along three separate orthogonal directions would bring similar accuracy on trace apparent diffusion coefficient under ideal conditions, the method described here should be useful for probing intimate properties of the cells with minimal experimental bias. Magn Reson Med, 2012. © 2012 Wiley Periodicals, Inc.

PMID: 22302673 [PubMed - as supplied by publisher]

Complementary treatment of siTERT for improving the antitumor effect of TERT-specific I-131 therapy.

Cancer Gene Ther. 2012 Feb 3;

Authors: Kim S, Youn H, Song MG, Kang JH, Chung HK, Lee DS, Chung JK

Abstract
Sodium iodide symporter (NIS)-based radionuclide therapy provides an effective means of treating malignant tumors. However, it is sometimes inadequate because of limited effects on radio-resistant tumors, and thus, combination therapies with other therapeutic options have been requested to enhance its efficacy. Human telomerase reverse transcriptase (hTERT) has been reported to be involved in the progression of most cancers and also been viewed as a good candidate for targeting tumor. Application of TERT-specific radionuclide therapies using NIS gene transfer have been reported to treat TERT-positive tumors, but this approach only demonstrated tumor regression rather than eradication. As inhibiting TERT expression by introducing the hTERT-specific shRNA (siTERT) has been suggested as a therapeutic option, we investigated the complementary role of siTERT treatment after the TERT-specific I-131 therapy and its possibility as a novel anticancer therapeutic strategy. Retroviruses containing TERT promoter/NIS for TERT specific Radionuclide therapy and siTERT for TERT targeting antisense therapy were produced. Hep3B cells expressing TERT specific NIS (Hep3B-TERT/NIS) were xenografted into nude mouse and visualized with micro-SPECT/CT for monitoring NIS activity. The levels of hTERT mRNA, protein and its activity were confirmed by RT-PCR, Western blotting and Telomerase repeat amplification protocol assay. Cell proliferation was monitored by MTT assay and induced apoptosis was confirmed by Annexin-V-PI staining. Therapeutic effects of I-131 and/or siTERT were evaluated by clonogenic assay and mouse tumor model. Reduction of hTERT mRNA, protein and TERT activity by siTERT were observed in Hep3B-TERT/NIS cells. The viabilities of the infected cells were significantly decreased to 50% versus siScramble treated controls. The early apoptotic cell population was increased by siTERT. The survival rates of cells treated with siTERT or I-131 alone were 72.4±7.6% and 56.2±5.2%, respectively. However, the survival rate of cells treated with I-131 and siTERT were decreased to 22.1±2.8%. From mouse xenograft model, we also found that the siTERT gene therapy showed synergism to the radioiodine therapy for reducing tumor growth in vivo. Our Results suggested that complementary siTERT gene therapy offers a novel strategy of cancer therapy to improve the therapeutic efficacy of TERT-specific I-131.Cancer Gene Therapy advance online publication, 3 February 2012; doi:10.1038/cgt.2011.88.

PMID: 22301953 [PubMed - as supplied by publisher]

In vivo detection of oxidation-specific epitopes in atherosclerotic lesions using biocompatible manganese molecular magnetic imaging probes.

J Am Coll Cardiol. 2012 Feb 7;59(6):616-26

Authors: Briley-Saebo KC, Nguyen TH, Saeboe AM, Cho YS, Ryu SK, Volkava E, Dickson S, Leibundgut G, Weisner P, Green S, Casanada F, Miller YI, Shaw W, Witztum JL, Fayad ZA, Tsimikas S

Abstract
OBJECTIVES: This study sought to evaluate the in vivo magnetic resonance imaging (MRI) efficacy of manganese [Mn(II)] molecular imaging probes targeted to oxidation-specific epitopes (OSE).
BACKGROUND: OSE are critical in the initiation, progression, and destabilization of atherosclerotic plaques. Gadolinium [Gd(III)]-based MRI agents can be associated with systemic toxicity. Mn is an endogenous, biocompatible, paramagnetic metal ion that has poor MR efficacy when chelated, but strong efficacy when released within cells.
METHODS: Multimodal Mn micelles were generated to contain rhodamine for confocal microscopy and conjugated with either the murine monoclonal IgG antibody MDA2 targeted to malondialdehyde (MDA)-lysine epitopes or the human single-chain Fv antibody fragment IK17 targeted to MDA-like epitopes ("targeted micelles"). Micelle formulations were characterized in vitro and in vivo, and their MR efficacy (9.4-T) evaluated in apolipoprotein-deficient (apoE(-/-)) and low-density lipoprotein receptor negative (LDLR(-/-)) mice (0.05 mmol Mn/kg dose) (total of 120 mice for all experiments). In vivo competitive inhibition studies were performed to evaluate target specificity. Untargeted, MDA2-Gd, and IK17-Gd micelles (0.075 mmol Gd/kg) were included as controls.
RESULTS: In vitro studies demonstrated that targeted Mn micelles accumulate in macrophages when pre-exposed to MDA-LDL with ∼10× increase in longitudinal relativity. Following intravenous injection, strong MR signal enhancement was observed 48 to 72 h after administration of targeted Mn micelles, with colocalization within intraplaque macrophages. Co-injection of free MDA2 with the MDA2-Mn micelles resulted in full suppression of MR signal in the arterial wall, confirming target specificity. Similar MR efficacy was noted in apoE(-/-) and LDLR(-/-) mice with aortic atherosclerosis. No significant differences in MR efficacy were noted between targeted Mn and Gd micelles.
CONCLUSIONS: This study demonstrates that biocompatible multimodal Mn-based molecular imaging probes detect OSE within atherosclerotic plaques and may facilitate clinical translation of noninvasive imaging of human atherosclerosis.

PMID: 22300697 [PubMed - in process]

Evaluation of selective tumor detection by clinical magnetic resonance imaging using antibody-conjugated superparamagnetic iron oxide.

J Control Release. 2012 Jan 26;

Authors: Koyama T, Shimura M, Minemoto Y, Nohara S, Shibata S, Iida Y, Iwashita S, Hasegawa M, Kurabayashi T, Hamada H, Kono K, Honda E, Aoki I, Ishizaka Y

Abstract
Active targeting by monoclonal antibodies (mAbs) combined with nanosize superparamagnetic iron oxide (SPIO) is a promising technology for magnetic resonance imaging (MRI) diagnosis. However, the clinical applicability of this technology has not been investigated using appropriate controls. It is important to evaluate the targeting technology using widely used clinical 1.5-Tesla MRI in addition to the high-Tesla experimental MRI. In this study, we measured mAb-conjugated dextran-coated SPIO nanoparticles (CMDM) in vivo using clinical 1.5-Tesla MRI. MRI of tumor-bearing mice was performed using a simple comparison between positive and negative tumors derived from the same genetic background in each mouse. The system provided significant tumor-targeting specificity of the target tumor. To the best of our knowledge, this is the first report on the specific detection of target tumors by mAb-conjugated SPIO using clinical 1.5-Tesla MRI. Our observations provide clues for reliable active targeting using mAb-conjugated SPIO in clinical applications.

PMID: 22300621 [PubMed - as supplied by publisher]

Transgene expression and local tissue distribution of naked and polymer-condensed plasmid DNA after intradermal administration in mice.

J Control Release. 2012 Jan 24;

Authors: Palumbo RN, Zhong X, Panus D, Han W, Ji W, Wang C

Abstract
DNA vaccination using cationic polymers as carriers has the potential to be a very powerful method of immunotherapy, but typical immune responses generated have been less than robust. To better understand the details of DNA vaccine delivery in vivo, we prepared polymer/DNA complexes using three structurally distinct cationic polymers and fluorescently labeled plasmid DNA and injected them intradermally into mice. We analyzed transgene expression (luciferase) and the local tissue distribution of the labeled plasmid at the injection site at various time points (from hours to days). Comparable numbers of luciferase expressing cells were observed in the skin of mice receiving naked plasmid or polyplexes one day after transfection. At day 4, however, the polyplexes appeared to result in more transfected skin cells than naked plasmid. Live animal imaging revealed that naked plasmid dispersed quickly in the skin of mice after injection and had a wider distribution than any of the three types of polyplexes. However, naked plasmid level dropped to below detection limit after 24h, whereas polyplexes persisted for up to 2weeks. The PEGylated polyplexes had a significantly wider distribution in the tissue than the nonPEGylated polyplexes. PEGylated polyplexes also distributed more broadly among dermal fibroblasts and allowed greater interaction with antigen-presenting cells (APCs) (dendritic cells and macrophages) starting at around 24h post-injection. By day 4, co-localization of polyplexes with APCs was observed at the injection site regardless of polymer structure, whereas small amounts of polyplexes were found in the draining lymph nodes. These in vivo findings demonstrate the superior stability of PEGylated polyplexes in physiological milieu and provide important insight on how cationic polymers could be optimized for DNA vaccine delivery.

PMID: 22300619 [PubMed - as supplied by publisher]

In vivo functional and transcriptional profiling of bone marrow stem cells after transplantation into ischemic myocardium.

Arterioscler Thromb Vasc Biol. 2012 Jan;32(1):92-102

Authors: Sheikh AY, Huber BC, Narsinh KH, Spin JM, van der Bogt K, de Almeida PE, Ransohoff KJ, Kraft DL, Fajardo G, Ardigo D, Ransohoff J, Bernstein D, Fischbein MP, Robbins RC, Wu JC

Abstract
OBJECTIVE: Clinical trials of bone marrow-derived stem cell therapy for the heart have yielded variable results. The basic mechanism(s) that underlies their potential efficacy remains unknown. In the present study, we evaluated the survival kinetics, transcriptional response, and functional outcome of intramyocardial bone marrow mononuclear cell (BMMC) transplantation for cardiac repair in a murine myocardial infarction model.
METHODS AND RESULTS: We used bioluminescence imaging and high-throughput transcriptional profiling to evaluate the in vivo survival kinetics and gene expression changes of transplanted BMMCs after their engraftment into ischemic myocardium. Our results demonstrate short-lived survival of cells following transplant, with less than 1% of cells surviving by 6 weeks posttransplantation. Moreover, transcriptomic analysis of BMMCs revealed nonspecific upregulation of various cell regulatory genes, with a marked downregulation of cell differentiation and maturation pathways. BMMC therapy caused limited improvement of heart function as assessed by echocardiography, invasive hemodynamics, and positron emission tomography. Histological evaluation of cell fate further confirmed findings of the in vivo cell tracking and transcriptomic analysis.
CONCLUSIONS: Collectively, these data suggest that BMMC therapy, in its present iteration, may be less efficacious than once thought. Additional refinement of existing cell delivery protocols should be considered to induce better therapeutic efficacy.

PMID: 22034515 [PubMed - indexed for MEDLINE]

Self-aggregated pegylated poly (trimethylene carbonate) nanoparticles decorated with c(RGDyK) peptide for targeted paclitaxel delivery to integrin-rich tumors.

Biomaterials. 2011 Dec;32(35):9457-69

Authors: Jiang X, Sha X, Xin H, Chen L, Gao X, Wang X, Law K, Gu J, Chen Y, Jiang Y, Ren X, Ren Q, Fang X

Abstract
Cyclic RGD peptide-decorated polymeric micellar-like nanoparticles (MNP) based on PEGylated poly (trimethylene carbonate) (PEG-PTMC) were prepared for active targeting to integrin-rich cancer cells. An amphiphilic diblock copolymer, α-carboxyl poly (ethylene glycol)-poly (trimethylene carbonate) (HOOC-PEG-PTMC), was synthesized by ring-opening polymerization. The c(RGDyK) ligand, a cyclic RGD peptide that can bind to the integrin proteins predominantly expressed on the surface of tumor cells with high affinity and specificity, was conjugated to the NHS-Activated PEG terminus of the copolymer. The c(RGDyK)-functionalized PEG-PTMC micellar nanoparticles encapsulating PTX (c(RGDyK)-MNP/PTX) was fabricated by the emulsion/solvent evaporation technique and characterized in terms of morphology, size and zeta potential. Cellular uptake of c(RGDyK)-MNP/PTX was found to be higher than that of MNP/PTX due to the integrin protein-mediated endocytosis effect. In vitro cytotoxicity, cell apoptosis and cell cycle arrest studies also revealed that c(RGDyK)-MNP/PTX was more potent than those of MNP/PTX and Taxol. Pharmacokinetic study in rats demonstrated that the polymeric micellar nanoparticles significantly enhanced the bioavailability of PTX than Taxol. In vivo multispectral fluorescent imaging indicated that c(RGDyK)-MNP/PTX had high specificity and efficiency in tumor active targeting. Therefore, the results demonstrated that c(RGDyK)-decorated PEG-PTMC MNP developed in this study could be a potential vehicle for delivering hydrophobic chemotherapeutic agents to integrin-rich tumors.

PMID: 21911250 [PubMed - indexed for MEDLINE]

A facile synthesis, in vitro and in vivo MR studies of d-glucuronic acid-coated ultrasmall Ln₂O₃ (Ln = Eu, Gd, Dy, Ho, and Er) nanoparticles as a new potential MRI contrast agent.

ACS Appl Mater Interfaces. 2011 Sep;3(9):3325-34

Authors: Kattel K, Park JY, Xu W, Kim HG, Lee EJ, Bony BA, Heo WC, Lee JJ, Jin S, Baeck JS, Chang Y, Kim TJ, Bae JE, Chae KS, Lee GH

Abstract
A facile one-pot synthesis of d-glucuronic acid-coated ultrasmall Ln(2)O(3) (Ln = Eu, Gd, Dy, Ho, and Er) nanoparticles is presented. Their water proton relaxivities were studied to address their possibility as a new potential MRI contrast agent. We focused on the d-glucuronic acid-coated ultrasmall Dy(2)O(3) nanoparticle because it showed the highest r(2) relaxivity among studied nanoparticles. Its performance as a T(2) MRI contrast agent was for the first time proved in vivo through its 3 T T(2) MR images of a mouse, showing that it can be further exploited for the rational design of a new T(2) MRI contrast agent at high MR fields.

PMID: 21853997 [PubMed - indexed for MEDLINE]

Biodistribution and human dosimetry of enantiomer-1 of the synthetic leucine analog anti-1-amino-2-fluorocyclopentyl-1-carboxylic acid.

Nucl Med Biol. 2011 Oct;38(7):1035-41

Authors: Nye JA, Jarkas N, Schuster DM, Savir-Baruch B, Voll RJ, Camp VM, Goodman MM

Abstract
INTRODUCTION: The enantiomerically enriched (ee=90%, enantiomer 1) synthetic amino acid (R,S)-anti-1-amino-2-fluorocyclopentyl-1-carboxylic acid (anti-2-[(18)F]FACPC-1) accumulates in malignant cells by elevated transport through the sodium-independent system-L (leucine preferring) amino acid transporter. The purpose of this study was to evaluate in vivo uptake and single-dose toxicity of anti-2-[(18)F]FACPC-1 in animals as well as the individual organ and whole-body dose in humans.
METHODS: A DU145 xenograft rodent model was used to measure anti-2-[(18)F]FACPC-1 uptake at 15, 30 and 60 min post-injection. Animals were sacrificed and organs harvested to measure the percent injected activity per organ and to calculate residence time. Anti-2-[(18)F]FACPC-1 toxicity was assessed using a single microdose (37-74 MBq/kg) in nonhuman primates. Their vital signs were monitored for 2 h post-injection for drug-related effects. Human biodistribution studies were collected by sequential whole-body PET/CT scans on six healthy volunteers (three male and three female) for 120 min following a single 247±61 MBq bolus injection of anti-2-[(18)F]FACPC-1. Estimates of radiation dose from anti-2-[(18)F]FACPC-1 to the human body were calculated using recommendations of the MIRD committee and MIRDOSE 3.0 software.
RESULTS: High anti-2-[(18)F]FACPC-1 residence time was observed in the pancreas of the rodent model compared to the human data. No abnormal treatment-related observations were made in the nonhuman primate toxicity studies. Human venous blood showed no metabolites of anti-2-[(18)F]FACPC-1 in the first 60 min post-injection. All volunteers showed initially high uptake in the kidneys followed by a rapid washout phase. The estimated effective dose equivalent was 0.0196 mSv/MBq.
CONCLUSION: Anti-2-[(18)F]FACPC-1 showed low background uptake in the brain, thoracic and abdominal cavities of humans, suggesting a possible use for detecting malignant tissues in these regions.

PMID: 21741251 [PubMed - indexed for MEDLINE]

Monitoring the glioma tropism of bone marrow-derived progenitor cells by 2-photon laser scanning microscopy and positron emission tomography.

Neuro Oncol. 2012 Feb 1;

Authors: Hasenbach K, Wiehr S, Herrmann C, Mannheim J, Cay F, von Kürthy G, Bolmont T, Grathwohl SA, Weller M, Lengerke C, Pichler BJ, Tabatabai G

Abstract
Intracerebral experimental gliomas attract intravenously injected murine or human bone marrow-derived hematopoietic progenitor and stem cells (HPC) in vitro, ex vivo, and in vivo, indicating that these progenitor cells might be suitable vehicles for a cell-based delivery of therapeutic molecules to malignant gliomas. With regard to therapeutic application, it is important to investigate cell fates in vivo (i.e., the time-dependent intratumoral and systemic distribution after intravenously injection). Conventional histological analysis has limitations in this regard because longitudinal monitoring is precluded. Here, we used 2-photon laser scanning microscopy (2PLSM), positron emission tomography (PET), and MRI to study the fate of intravenously injected HPC carrying fluorescence, bioluminescence, and PET reporter genes in glioma-bearing mice. Our 2PLSM-based monitoring studies revealed that HPC homing to intracerebral experimental gliomas occurred already within the first 6 h and was most efficient within the first 24 h after intravenous injection. The highest PET signals were detected in intracerebral gliomas, whereas the tracer uptake in other organs, notably spleen, lung, liver, and muscle, remained at background levels. The results have important implications for designing schedules for therapeutic cell-based anti-glioma approaches. Moreover, the PET reporter-based imaging technique will allow noninvasive monitoring of cell fate in future cell-based therapeutic antiglioma approaches.

PMID: 22298526 [PubMed - as supplied by publisher]

Super-resolution methods in MRI: Can they improve the trade-off between resolution, signal-to-noise ratio, and acquisition time?

Magn Reson Med. 2012 Feb 1;

Authors: Plenge E, Poot DH, Bernsen M, Kotek G, Houston G, Wielopolski P, van der Weerd L, Niessen WJ, Meijering E

Abstract
Improving the resolution in magnetic resonance imaging comes at the cost of either lower signal-to-noise ratio, longer acquisition time or both. This study investigates whether so-called super-resolution reconstruction methods can increase the resolution in the slice selection direction and, as such, are a viable alternative to direct high-resolution acquisition in terms of the signal-to-noise ratio and acquisition time trade-offs. The performance of six super-resolution reconstruction methods and direct high-resolution acquisitions was compared with respect to these trade-offs. The methods are based on iterative back-projection, algebraic reconstruction, and regularized least squares. The algorithms were applied to low-resolution data sets within which the images were rotated relative to each other. Quantitative experiments involved a computational phantom and a physical phantom containing structures of known dimensions. To visually validate the quantitative evaluations, qualitative experiments were performed, in which images of three different subjects (a phantom, an ex vivo rat knee, and a postmortem mouse) were acquired with different magnetic resonance imaging scanners. The results show that super-resolution reconstruction can indeed improve the resolution, signal-to-noise ratio and acquisition time trade-offs compared with direct high-resolution acquisition. Magn Reson Med, 2012. © 2012 Wiley Periodicals, Inc.

PMID: 22298247 [PubMed - as supplied by publisher]

Intravital microscopy of the spleen: quantitative analysis of parasite mobility and blood flow.

J Vis Exp. 2012;(59)

Authors: Ferrer M, Martin-Jaular L, Calvo M, Del Portillo HA

Abstract
The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions (1,2). Thus, in vivo imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes (3), the complete development of the parasite in the skin (4), and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream (5). Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria (6). Thus, intravital imaging has radically changed our view on key events in Plasmodium development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints. Using the murine model of malaria Plasmodium yoelii in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii 17X as opposed to infections with the P.yoelii 17XL lethal parasite line (7). To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen. Ethics statement All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.

PMID: 22298018 [PubMed - in process]

Integrated multimodal endomicroscopy platform for simultaneous en face optical coherence and two-photon fluorescence imaging.

Opt Lett. 2012 Feb 1;37(3):362-4

Authors: Xi J, Chen Y, Zhang Y, Murari K, Li MJ, Li X

Abstract
We report an all-fiber-optic scanning, multimodal endomicroscope capable of simultaneous optical coherence tomography (OCT) and two-photon fluorescence (TPF) imaging. Both imaging modalities share the same miniature fiber-optic scanning endomicroscope, which consists of a double-clad fiber with a core operating in single mode at both the OCT (1310 nm) and two-photon excitation (1550 nm) wavelengths, a piezoelectric two-dimensional fiber-optic beam scanner, and a miniature aspherical compound lens suitable for simultaneous acquisition of en face OCT and TPF images. A fiber-optic wavelength division multiplexer was employed in the integrated platform to combine the low coherence OCT light source and the femtosecond two-photon excitation laser into the same optical path. Preliminary imaging results of cell cultures and mouse tissue ex vivo demonstrate the feasibility of simultaneous real-time OCT and TPF imaging in a scanning endomicroscopy setting for the first time.

PMID: 22297353 [PubMed - in process]

Organ specific mapping of in vivo redox state in control and cigarette smoke-exposed mice using EPR/NMR co-imaging.

J Magn Reson. 2011 Nov 7;

Authors: Caia GL, Efimova OV, Velayutham M, El-Mahdy MA, Abdelghany TM, Kesselring E, Petryakov S, Sun Z, Samouilov A, Zweier JL

Abstract
In vivo mapping of alterations in redox status is important for understanding organ specific pathology and disease. While electron paramagnetic resonance imaging (EPRI) enables spatial mapping of free radicals, it does not provide anatomic visualization of the body. Proton MRI is well suited to provide anatomical visualization. We applied EPR/NMR co-imaging instrumentation to map and monitor the redox state of living mice under normal or oxidative stress conditions induced by secondhand cigarette smoke (SHS) exposure. A hybrid co-imaging instrument, EPRI (1.2GHz)/proton MRI (16.18MHz), suitable for whole-body co-imaging of mice was utilized with common magnet and gradients along with dual EPR/NMR resonators that enable co-imaging without sample movement. The metabolism of the nitroxide probe, 3-carbamoyl-proxyl (3-CP), was used to map the redox state of control and SHS-exposed mice. Co-imaging allowed precise 3D mapping of radical distribution and reduction in major organs such as the heart, lungs, liver, bladder and kidneys. Reductive metabolism was markedly decreased in SHS-exposed mice and EPR/NMR co-imaging allowed quantitative assessment of this throughout the body. Thus, in vivo EPR/NMR co-imaging enables in vivo organ specific mapping of free radical metabolism and redox stress and the alterations that occur in the pathogenesis of disease.

PMID: 22296801 [PubMed - as supplied by publisher]

Cancer cell-selective in vivo near infrared photoimmunotherapy targeting specific membrane molecules.

Nat Med. 2011 Dec;17(12):1685-91

Authors: Mitsunaga M, Ogawa M, Kosaka N, Rosenblum LT, Choyke PL, Kobayashi H

Abstract
Three major modes of cancer therapy (surgery, radiation and chemotherapy) are the mainstay of modern oncologic therapy. To minimize the side effects of these therapies, molecular-targeted cancer therapies, including armed antibody therapy, have been developed with limited success. In this study, we have developed a new type of molecular-targeted cancer therapy, photoimmunotherapy (PIT), that uses a target-specific photosensitizer based on a near-infrared (NIR) phthalocyanine dye, IR700, conjugated to monoclonal antibodies (mAbs) targeting epidermal growth factor receptors. Cell death was induced immediately after irradiating mAb-IR700-bound target cells with NIR light. We observed in vivo tumor shrinkage after irradiation with NIR light in target cells expressing the epidermal growth factor receptor. The mAb-IR700 conjugates were most effective when bound to the cell membrane and produced no phototoxicity when not bound, suggesting a different mechanism for PIT as compared to conventional photodynamic therapies. Target-selective PIT enables treatment of cancer based on mAb binding to the cell membrane.

PMID: 22057348 [PubMed - indexed for MEDLINE]

High mobility group box 1 promotes endothelial cell angiogenic behavior in vitro and improves muscle perfusion in vivo in response to ischemic injury.

J Vasc Surg. 2012 Jan;55(1):180-91

Authors: Sachdev U, Cui X, Hong G, Namkoong S, Karlsson JM, Baty CJ, Tzeng E

Abstract
OBJECTIVES: The angiogenic drive in skeletal muscle ischemia remains poorly understood. Innate inflammatory pathways are activated during tissue injury and repair, suggesting that this highly conserved pathway may be involved in ischemia-induced angiogenesis. We hypothesize that one of the endogenous ligands for innate immune signaling, high mobility group box 1 (HMGB1), in combination with autophagic responses to hypoxia or nutrient deprivation, plays an important role in angiogenesis.
METHODS: Human dermal microvascular endothelial cells (ECs) were cultured in normoxia or hypoxia (1% oxygen). Immunocytochemical analysis of HMGB1 subcellular localization, evaluation of tube formation, and Western blot analysis of myotubule light-chain 3I (LC3I) conversion to LC3II, as a marker of autophagy, were conducted. 3-Methyladenine (3MA), chloroquine, or rapamycin were administered to inhibit or promote autophagy, respectively. In vivo, a murine hind limb ischemia model was performed. Muscle samples were collected at 4 hours to evaluate for nuclear HMGB1 and at 14 days to examine endothelial density. Perfusion recovery in the hind limbs was calculated by laser Doppler perfusion imaging (LDPI).
RESULTS: Hypoxic ECs exhibited reduced nuclear HMGB1 staining compared with normoxic cells (mean fluorescence intensity, 186.9 ± 17.1 vs 236.0 ± 1.6, P = .01) with a concomitant increase in cytosolic staining. HMGB1 treatment of ECs enhanced tube formation, an angiogenic phenotype of ECs. Neutralization of endogenous HMGB1 markedly impaired tube formation and inhibited LC3II formation. Inhibition of autophagy with 3MA or chloroquine abrogated tube formation, whereas its induction with rapamycin enhanced tubing and promoted HMGB1 translocation. In vivo, ischemic skeletal muscle showed reduced numbers of HMGB1-positive myocyte nuclei compared with nonischemic muscle (34.9% ± 1.9% vs 51.7% ± 2.0%, P < .001). Injection of HMGB1 into ischemic hind limbs increased perfusion recovery by 21% and increased EC density (49.2 ± 4.1 vs 34.2 ± 3.4 ECs/high-powered field, respectively; P = .02) at 14 days compared with control hind limbs.
CONCLUSIONS: Nuclear release of HMGB1 and autophagy occur in ECs in response to hypoxia or serum depletion. HMGB1 and autophagy are necessary and likely play an interdependent role in promoting the angiogenic behavior of ECs. In vivo, HMGB1 promotes perfusion recovery and increased EC density after ischemic injury. These findings suggest a possible mechanistic link between autophagy and HMGB1 in EC angiogenic behavior and support the importance of innate immune pathways in angiogenesis.

PMID: 21944908 [PubMed - indexed for MEDLINE]

Pharmacological characterization of a recombinant, fluorescent somatostatin receptor agonist.

Bioconjug Chem. 2011 Sep 21;22(9):1768-75

Authors: Sreenivasan VK, Stremovskiy OA, Kelf TA, Heblinski M, Goodchild AK, Connor M, Deyev SM, Zvyagin AV

Abstract
Somatostatin (SST) is a peptide neurotransmitter/hormone found in several mammalian tissue types. Apart from its natural importance, labeled SST/analogues are utilized in clinical applications such as targeting/diagnosis of neuroendocrine tumors. We report on the development and characterization of a novel, recombinant, fluorescent somatostatin analogue that has potential to elucidate somatostatin-activated cell signaling. SST was genetically fused with a monomeric-red fluorescent protein (mRFP) as the fluorescent label. The attachment of SST to mRFP had no detectable effect on its fluorescent properties. This analogue's potency to activate the endogenous and transfected somatostatin receptors was characterized using assays of membrane potential and Ca(2+) mobilization and immunocytochemistry. SST-mRFP was found to be an effective somatostatin receptor agonist, able to trigger the membrane hyperpolarization, mobilization of the intracellular Ca(2+) and receptor-ligand internalization in cells expressing somatostatin receptors. This complex represents a novel optical reporter due to its red emission spectral band suitable for in vivo imaging and tracking of the somatostatin receptor signaling pathways, affording higher resolution and sensitivity than those of the state-of-the-art radiolabeling bioassays.

PMID: 21823634 [PubMed - indexed for MEDLINE]