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Other Publications (90)

Articles by Uwe Himmelreich in JoVE

 JoVE Medicine

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

1Molecular Virology and Gene Therapy, KU Leuven, 2Department of Woman and Child, KU Leuven, 3Neurobiology and Gene Therapy, KU Leuven, 4Division of Nuclear Medicine, KU Leuven, 5Biomedical NMR Unit/ MoSAIC, KU Leuven


JoVE 4219

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

 JoVE Medicine

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

1Department of Molecular Microbiology, Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, VIB, KU Leuven, 2Biomedical MRI Unit/ MoSAIC, Department of Imaging & Pathology, KU Leuven


JoVE 52239

We present an experimental procedure of Candida albicans biofilm development in a mouse subcutaneous model. Fungal biofilms were quantified by determining the number of colony forming units and by a non-invasive bioluminescence imaging, where the amount of light that is produced corresponds with the number of viable cells.

Other articles by Uwe Himmelreich on PubMed

Cerebral Nocardiosis Characterized by Magnetic Resonance Spectroscopy in Vivo

We describe a patient with multiple cerebral lesions that initially were believed to be tumor metastases but were later found to be caused by Nocardia asteroides, after magnetic resonance spectroscopy (MRS) findings suggested that the lesions were infective in origin. This case report demonstrates the ability of MRS, a noninvasive imaging modality, to aid in the diagnosis of brain infection.

Heart Design: Free ADP Scales with Absolute Mitochondrial and Myofibrillar Volumes from Mouse to Human

Our aim was to estimate a number of bioenergetic parameters in the beating mouse, rat and guinea pig heart in situ and compare the values to those in hearts of mammals over a 2000-fold range in body mass. For the mouse, rat and guinea pig heart, we report a phosphorylation ratio of 1005+/-50 (n=16), 460+/-32 (n=10) and 330+/-22 (n=5) mM(-1) and a free cytosolic [ADP] concentration of 13, 18 and 22 microM, respectively. When each parameter was plotted against body mass, they scaled closely to the quarter power (-0.28, r=0.99 and -0.23, r=0.97). A similar regression slope was found when the inverse of free [ADP] was plotted against absolute mitochondrial (slope=-0.26, r=0.99) and myofibrillar volumes (slope=-0.24, r=0.99). The similar slopes indicate that the ratio of absolute mitochondria and myofibrillar volumes in the healthy mammalian heart is a constant, and independent of body size. In conclusion, our study supports the hypothesis that the mammalian heart has a number of highly conserved thermodynamic and kinetic parameters that obey quarter-power laws linking the phosphorylation ratio, ATP turnover rates, free [ADP] and absolute mitochondrial volumes to body size. The results are discussed in terms of possible mechanisms and potential deviations from these laws in some disease states.

Isolation and Characterization of Capsule Structure Mutant Strains of Cryptococcus Neoformans

The capsule of Cryptococcus neoformans is the most obvious virulence factor of this pathogenic yeast. The main capsule constituents are glucuronoxylomannans (GXM). Although several studies have focused on GXM composition and structure, very little is known about their genetics. To elucidate the relationship between the capsule structure and the pathophysiology of the cryptococcosis, genetic screening for mutant strains producing a structurally modified capsule was set up. Using monoclonal antibodies specific for different capsule sugar epitopes, we isolated strains with different mutated capsule structures (Cas mutants). According to their reactivities with various monoclonal antibodies, the mutants were classified into six groups (Cas1 to Cas6). One Cas2 mutant was used to clone the corresponding gene by complementation. This gene (USX1) encodes the previously identified UDP-xylose synthase. We demonstrated that it is necessary for both capsule xylosylation and C. neoformans virulence.

Pathology of Hepatocellular Carcinoma and Its Precursors Using Proton Magnetic Resonance Spectroscopy and a Statistical Classification Strategy

Apply the statistical classification strategy (SCS) to magnetic resonance spectroscopy (MRS) data from liver biopsies and test its potential to discriminate between normal liver, cirrhotic nodules and nodules of hepatocellular carcinoma with a high degree of accuracy.

Metabolites Released by Cryptococcus Neoformans Var. Neoformans and Var. Gattii Differentially Affect Human Neutrophil Function

Differences in the ability of Cryptococcus neoformans var. neoformans (CNVN) and var. gattii (CNVG) to establish localized lesions in the lungs of healthy humans remain unexplained. In this study, CNVG infection in a rat model was characterized by early neutrophil invasion into lung tissue, but phagocytosis of cryptococci was not observed. The chemical composition of non-enzymic components secreted by one strain of each variety (heat-inactivated supernatants from CNVN and CNVG, termed vns and vgs, respectively) were compared, using magnetic resonance spectroscopy. Effects on human neutrophil viability and functions at both pH 5.5 and 7.0 were investigated, as the pH of cryptococcomas was found to be 5.4-5.6 in vivo. The supernatants were similar in composition, although metabolites in vns were generally present in higher concentrations. In addition, vgs contained two novel metabolites-acetoin and dihydroxyacetone. Polyphosphate was observed in cells from both varieties and may be a source of extracellular inorganic phosphate. Superoxide production in the presence of phorbol ester was enhanced by treatment with vns and decreased by vgs. At pH 5.5, vns caused high levels of necrosis in neutrophils, as well as increased adhesion/migration through A549 lung epithelial cell monolayers. Individual supernatant components such as polyols, acetoin, dihydroxyacetone, and gamma-aminobutyric acid exhibited both pro- and anti-inflammatory properties. Overall, we found that vgs was potentially less pro-inflammatory than vns. Inhibition of neutrophil function by products of CNVG may promote survival of extracellular organisms, and local multiplication to form cryptococcomas.

Pathology of Barrett's Esophagus by Proton Magnetic Resonance Spectroscopy and a Statistical Classification Strategy

Barrett's esophagus is thought to be a precursor of adenocarcinoma. The incidence of adenocarcinoma of the lower esophagus in the Western world is rising and accounts for more than 40% of esophageal carcinomas in males. It is not possible to identify which Barrett's patients are at high risk of developing malignancy. Here we applied a statistical classification strategy to the analysis of magnetic resonance spectroscopy and histopathological data from esophageal biopsies to ascertain whether this risk could be identified in Barrett's patients.

Identification of Metabolites of Importance in the Pathogenesis of Pulmonary Cryptococcoma Using Nuclear Magnetic Resonance Spectroscopy

Primary lung infection with Cryptococcus neoformans is characterised by circumscribed lesions (cryptococcomas). To identify cryptococcal and/or host products of importance in pathogenesis, we applied proton nuclear magnetic resonance (NMR) spectroscopy, which identifies mobile compounds present in complex mixtures, to experimental pulmonary cryptococcomas from rats. Magnetic resonance experiments were performed on cryptococcomas (n = 10) and healthy lungs (n = 8). Signal assignment to key metabolites was confirmed by homo-nuclear and hetero-nuclear NMR correlation spectroscopy. Cryptococcal metabolites, dominating spectra from cryptococcomas included the stress protectants, trehalose and mannitol, acetate, and in some animals, ethanol. Glycerophosphorylcholine was also abundant in cryptococcomas, consistent with hydrolysis of phospholipids in vivo by the cryptococcal enzyme, phospholipase B (PLB). PLB has been identified by molecular studies as a cryptococcal virulence determinant. We propose that PLB secreted by cryptococci promotes tissue invasion by hydrolysing host phospholipids, such as dipalmitoyl phosphatidyl choline, which is abundant in pulmonary surfactant, and lung cell membrane phospholipids. Our results confirm the utility of NMR spectroscopy in studies of microbial pathogenesis.

Detergent-resistant Membrane Fractions Contribute to the Total 1H NMR-visible Lipid Signal in Cells

Leukocytes and other cells show an enhanced intensity of mobile lipid in their 1H NMR spectra under a variety of conditions. Such conditions include stimulation, which has recently been shown to involve detergent-resistant, plasma membrane domains (DRMs) often called lipid rafts. As there is much speculation surrounding the origin of cellular NMR-visible lipid, we analysed subcellular fractions, including DRMs, by NMR spectroscopy. We demonstrated that DRMs isolated by density gradient centrifugation from lymphoid (CEM-T4, stimulated Jurkat cells), and monocytoid (THP-1) cells produced NMR-visible, lipid signals. Large scale subfractionation of THP-1 cells determined that while cytoplasmic lipid droplets constituted much of the total NMR-visible lipid, the contribution of DRMs was significant. Qualitative and quantitative lipid analyses revealed that DRMs and lipid droplets differed in their lipid composition. DRMs were enriched in cholesterol and ganglioside GM1, and contained relatively unsaturated fatty acids compared with the lipid droplets. Both lipid droplets and DRMs contained neutral lipids (triacylgycerols, cholesterol ester, fatty acids in THP-1 cells) that could, in addition to phospholipids, contribute to the NMR-visible lipid. The lipid droplets also exhibited different protein profiles and contained 500-fold less protein than DRMs, confirming that DRMs and droplets were fractionated as separate entities. The NMR-visible lipid in DRMs is therefore unlikely to be a contaminant from lipid droplets. We propose a micropartitioning of the NMR-visible mobile lipid of whole cells between intracellular lipid droplets, where most of this lipid resides, and detergent-resistant plasma membrane domains.

Pathologic Characterization of Human Prostate Tissue with Proton MR Spectroscopy

To assess the accuracy of magnetic resonance (MR) spectroscopy in documenting the chemical features of human prostate tissue and to ascertain if there are chemical criteria of diagnostic importance.

Rapid Identification of Candida Species by Using Nuclear Magnetic Resonance Spectroscopy and a Statistical Classification Strategy

Nuclear magnetic resonance (NMR) spectra were acquired from suspensions of clinically important yeast species of the genus Candida to characterize the relationship between metabolite profiles and species identification. Major metabolites were identified by using two-dimensional correlation NMR spectroscopy. One-dimensional proton NMR spectra were analyzed by using a staged statistical classification strategy. Analysis of NMR spectra from 442 isolates of Candida albicans, C. glabrata, C. krusei, C. parapsilosis, and C. tropicalis resulted in rapid, accurate identification when compared with conventional and DNA-based identification. Spectral regions used for the classification of the five yeast species revealed species-specific differences in relative amounts of lipids, trehalose, polyols, and other metabolites. Isolates of C. parapsilosis and C. glabrata with unusual PCR fingerprinting patterns also generated atypical NMR spectra, suggesting the possibility of intraspecies discontinuity. We conclude that NMR spectroscopy combined with a statistical classification strategy is a rapid, nondestructive, and potentially valuable method for identification and chemotaxonomic characterization that may be broadly applicable to fungi and other microorganisms.

Determination of Grade and Receptor Status from the Primary Breast Lesion by Magnetic Resonance Spectroscopy

Magnetic resonance spectra (MRS) from fine needle aspiration biopsies (FNAB) from primary breast lesions were analysed using a pattern recognition method, Statistical Classification Strategy, to assess tumor grade and oestrogen receptor (ER) and progesterone receptor (PgR) status. Grade 1 and 2 breast cancers were separated from grade 3 cancers with a sensitivity and specificity of 96% and 95%, respectively. The ER status was predicted with a sensitivity of 91% and a specificity of 90%, and the PgR status with a sensitivity of 91% and a specificity of 86%. These classifiers provide rapid and reliable, computerized information and may offer an objective method for determining these prognostic indicators simultaneously with the diagnosis of primary pathology and lymph node involvement.

Cas3p Belongs to a Seven-member Family of Capsule Structure Designer Proteins

The polysaccharide capsule is the main virulence factor of the basidiomycetous yeast Cryptococcus neoformans. Four genes (CAP10, CAP59, CAP60, and CAP64) essential for capsule formation have been previously identified, although their roles in the biosynthetic pathway remain unclear. A genetic and bioinformatics approach allowed the identification of six CAP64-homologous genes, named CAS3, CAS31, CAS32, CAS33, CAS34, and CAS35, in the C. neoformans genome. This gene family is apparently specific in a subclass of the basidiomycete fungi. Single as well as double deletions of these genes in all possible combinations demonstrated that none of the CAP64-homologous genes were essential for capsule formation, although the cas35Delta strains displayed a hypocapsular phenotype. The chemical structure of the glucuronomannan (GXM) produced by the CAS family deletants revealed that these genes determined the position and the linkage of the xylose and/or O-acetyl residues on the mannose backbone. Hence, these genes are all involved in assembly of the GXM structure in C. neoformans.

Identification of Staphylococcus Aureus Brain Abscesses: Rat and Human Studies with 1H MR Spectroscopy

To determine the feasibility of a statistical classification strategy (SCS) and the identity of metabolites of bacterial and host origins that potentially contributed to the most discriminatory regions of magnetic resonance (MR) spectra from Staphylococcus aureus abscesses of biopsy material from controls, gliomas, and staphylococcal abscesses.

Diagnosis of Cerebral Cryptococcoma Using a Computerized Analysis of 1H NMR Spectra in an Animal Model

Viable cryptococci load in biopsy material from an animal model of cerebral cryptococcoma were correlated with 1H NMR spectra and metabolite profiles. A statistical classification strategy was applied to distinguish among high-resolution 1H NMR spectra acquired from cryptococcomas, glioblastomas, and normal brain tissue. The overall classification accuracy was 100% when a genetic-algorithm-based optimal region selection preceded the development of linear discriminant analysis-based classifiers. The method remained robust despite differences in the microbial load of the cryptococcoma group when harvested at different time points. These results indicate the feasibility of the method for diagnosis without isolation of the pathogenic microorganism and its potential for in vivo diagnosis based on computerized analysis of magnetic resonance spectra.

Improved Stem Cell MR Detectability in Animal Models by Modification of the Inhalation Gas

In vivo monitoring of cells labeled with paramagnetic iron oxide particles by magnetic resonance imaging (MRI) is complicated by intrinsic contrast of blood vessels. Distinction between T2* effects caused by blood vessels from those caused by labeled cells was so far only possible after carefully following the location of hypointense regions through subsequent slices of T2*-weighted 3-D MRI datasets, a procedure that is time consuming and not always reliable in the case of smaller blood vessels. Here, we demonstrate that the modification of the inhalation gas mixture from the routinely used composition 35% O2 and 65% N2O to a mixture containing 95% O2 and 5% CO2 results in a contrast suppression of most small blood vessels and reduces the intrinsic T2* effect of large vessels dramatically in an animal model. This change in protocol of physiological conditions was well tolerated by all studied animals, even over prolonged experimental times. The changed inhalation gas mixture thus provides a more reliable identification method for small clusters of iron oxide labeled cells in vivo.

A Rapid Screening Test to Distinguish Between Candida Albicans and Candida Dubliniensis Using NMR Spectroscopy

Nuclear magnetic resonance (NMR) spectroscopy combined with a statistical classification strategy (SCS) successfully distinguished between Candida albicans and Candida dubliniensis. 96% of the isolates from an independent test set were identified correctly. This proves that this rapid approach is a valuable method for the identification and chemotaxonomic characterisation of closely related taxa. Most discriminatory regions were correlated with metabolite profiles, indicating biochemical differences between the two species.

Magnetic Resonance Spectroscopy Detects Biochemical Changes in the Brain Associated with Chronic Low Back Pain: a Preliminary Report

Magnetic resonance (MR) spectroscopy is a noninvasive technique that can be used to detect and measure the concentration of metabolites and neurotransmitters in the brain and other organs. We used in vivo (1)H MR spectroscopy in subjects with low back pain compared with control subjects to detect alterations in biochemistry in three brain regions associated with pain processing. A pattern recognition approach was used to determine whether it was possible to discriminate accurately subjects with low back pain from control subjects based on MR spectroscopy. MR spectra were obtained from the prefrontal cortex, anterior cingulate cortex, and thalamus of 32 subjects with low back pain and 33 control subjects without pain. Spectra were analyzed and compared between groups using a pattern recognition method (Statistical Classification Strategy). Using this approach, it was possible to discriminate between subjects with low back pain and control subjects with accuracies of 100%, 99%, and 97% using spectra obtained from the anterior cingulate cortex, thalamus, and prefrontal cortex, respectively. These results demonstrate that MR spectroscopy, in combination with an appropriate pattern recognition approach, is able to detect brain biochemical changes associated with chronic pain with a high degree of accuracy.

Application of Proton Nuclear Magnetic Resonance Spectroscopy to the Study of Cryptococcus and Cryptococcosis

Proton nuclear magnetic resonance spectroscopy is a nondestructive technique that identifies chemicals in solution and in living cells. It has been used in cryptococcal research to identify the primary structure of capsular glucuronoxylomannans, link cellular apoptosis susceptibility (CAS) genes to positioning of residues on the mannose backbone of glucuronoxylomannan, and verify that the cryptococcal virulence determinant, phospholipase B, is elaborated in vivo. Promising clinical applications include speciation (Cryptococcus neoformans and Cryptococcus gattii), with preliminary evidence that varieties neoformans and grubii can also be distinguished, non-invasive diagnosis of cerebral cryptococcomas, and, in cases of meningitis, monitoring therapeutic response by analysis of cerebrospinal fluid.

Antifungal Effects on Metabolite Profiles of Medically Important Yeast Species Measured by Nuclear Magnetic Resonance Spectroscopy

Drug-induced inhibition of fungal growth is used in the diagnostic laboratory to predict therapeutic efficacy but is relatively slow, and determination of endpoints can be problematic. Nuclear magnetic resonance (NMR) spectroscopy identifies the metabolic complement of microorganisms while monitoring utilization of constituents of the incubation medium. This technique may provide a rapid and objective indicator of antifungal effects. We evaluated the effects of caspofungin, amphotericin B (AMB), and voriconazole on metabolic profiles of yeast species cultured in RPMI-2% glucose-morpholinepropanesulfonic acid buffer in microtiter plates in a proof-of-principle study. 1H NMR spectra were obtained using Bruker NMR spectrometers at 1H frequencies of 600 and 360 MHz. Metabolites were identified by two-dimensional correlation NMR spectra, and relative peak integrals were calculated from one-dimensional 1H NMR spectra. MICs were determined by a modification of the Clinical and Laboratory Standards Institute broth microdilution method M27-A. Utilization of glucose and branched-chain and aromatic amino acid substrates was accompanied by fungal production of acetate, acetaldehyde, ethanol, formate, fumarate, glycerol, lactate, pyruvate, and succinate. Clear-cut metabolic endpoints indicating a greater than 50% reduction in substrate utilization and fungal metabolite production which correlated with MICs were noted at 16 and 24 h for all three drugs. At 8 h, reductions of greater than 50% for selected metabolites were noted for caspofungin and AMB. Direct NMR-based observation of metabolic alterations in yeast cultures reveals changes in key metabolic pathways and should be evaluated formally as a rapid technique for determining susceptibility to antifungal drugs.

Characterization and Regulation of the Trehalose Synthesis Pathway and Its Importance in the Pathogenicity of Cryptococcus Neoformans

The disaccharide trehalose has been found to play diverse roles, from energy source to stress protectant, and this sugar is found in organisms as diverse as bacteria, fungi, plants, and invertebrates but not in mammals. Recent studies in the pathobiology of Cryptococcus neoformans identified the presence of a functioning trehalose pathway during infection and suggested its importance for C. neoformans survival in the host. Therefore, in C. neoformans we created null mutants of the trehalose-6-phosphate (T6P) synthase (TPS1), trehalose-6-phophate phosphatase (TPS2), and neutral trehalase (NTH1) genes. We found that both TPS1 and TPS2 are required for high-temperature (37 degrees C) growth and glycolysis but that the block at TPS2 results in the apparent toxic accumulation of T6P, which makes this enzyme a fungicidal target. Sorbitol suppresses the growth defect in the tps1 and tps2 mutants at 37 degrees C, which supports the hypothesis that these sugars (trehalose and sorbitol) act primarily as stress protectants for proteins and membranes during exposure to high temperatures in C. neoformans. The essential nature of this pathway for disease was confirmed when a tps1 mutant strain was found to be avirulent in both rabbits and mice. Furthermore, in the system of the invertebrate C. elegans, in which high in vivo temperature is no longer an environmental factor, attenuation in virulence was still noted with the tps1 mutant, and this supports the hypothesis that the trehalose pathway in C. neoformans is involved in more host survival mechanisms than simply high-temperature stresses and glycolysis. These studies in C. neoformans and previous studies in other pathogenic fungi support the view of the trehalose pathway as a selective fungicidal target for use in antifungal development.

Bystander Killing of Malignant Glioma by Bone Marrow-derived Tumor-infiltrating Progenitor Cells Expressing a Suicide Gene

Adult stem cells are promising cellular vehicles for therapy of malignant gliomas as they have the ability to migrate into these tumors and even track infiltrating tumor cells. However, their clinical use is limited by a low passaging capacity that impedes large-scale production. In the present study, a bone marrow-derived, highly proliferative subpopulation of mesenchymal stem cells (MSCs)-here termed bone marrow-derived tumor-infiltrating cells (BM-TICs)-was genetically modified for the treatment of malignant glioma. Upon injection into the tumor or the vicinity of the tumor, BM-TICs infiltrated solid parts as well as the border of rat 9L glioma. After intra-tumoral injection, BM-TICs expressing the thymidine kinase of herpes simplex virus (HSV-tk) and enhanced green fluorescent protein (BM-TIC-tk-GFP) were detected by non-invasive positron emission tomography (PET) using the tracer 9-[4-[(18)F]fluoro-3-hydroxymethyl)butyl]guanine ([(18)F]FHBG). A therapeutic effect was demonstrated in vitro and in vivo by BM-TICs expressing HSV-tk through bystander-mediated glioma cell killing. Therapeutic efficacy was monitored by PET as well as by magnetic resonance imaging (MRI) and strongly correlated with histological analysis. In conclusion, BM-TICs expressing a suicide gene were highly effective in the treatment of malignant glioma in a rat model and therefore hold great potential for the therapy of malignant brain tumors in humans.

Switching on the Lights for Gene Therapy

Strategies for non-invasive and quantitative imaging of gene expression in vivo have been developed over the past decade. Non-invasive assessment of the dynamics of gene regulation is of interest for the detection of endogenous disease-specific biological alterations (e.g., signal transduction) and for monitoring the induction and regulation of therapeutic genes (e.g., gene therapy). To demonstrate that non-invasive imaging of regulated expression of any type of gene after in vivo transduction by versatile vectors is feasible, we generated regulatable herpes simplex virus type 1 (HSV-1) amplicon vectors carrying hormone (mifepristone) or antibiotic (tetracycline) regulated promoters driving the proportional co-expression of two marker genes. Regulated gene expression was monitored by fluorescence microscopy in culture and by positron emission tomography (PET) or bioluminescence (BLI) in vivo. The induction levels evaluated in glioma models varied depending on the dose of inductor. With fluorescence microscopy and BLI being the tools for assessing gene expression in culture and animal models, and with PET being the technology for possible application in humans, the generated vectors may serve to non-invasively monitor the dynamics of any gene of interest which is proportionally co-expressed with the respective imaging marker gene in research applications aiming towards translation into clinical application.

Cell Tracking Using Magnetic Resonance Imaging

Cell tracking by in vivo magnetic resonance imaging (MRI) requires strategies of labelling the cells with MRI contrast agents. The principal routes to achieve efficient cell labelling for neurological applications are discussed with methodological advantages and caveats. Beyond temporo-spatial localization of labelled cells, the investigation of functional cell status is of great interest to allow studies of functional cell dynamics. The two major approaches to reach this goal, use of responsive contrast agents and generation of transgenic cell lines, are discussed.

Normal Brain Cells Contribute to the Bystander Effect in Suicide Gene Therapy of Malignant Glioma

Lentiviral vectors pseudotyped with glycoproteins of the lymphocytic choriomeningitis virus (LCMV-GP) are promising candidates for gene therapy of malignant glioma, as they specifically and efficiently transduce glioma cells in vitro and in vivo. Here, we evaluated the therapeutic efficacy of LCMV-GP and vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped vectors.

Molecular and Cellular MR Imaging: Potentials and Challenges for Neurological Applications

This review presents the state of the art of molecular MRI and its application to experimental neurology and neuroscience. We do not repeat a broad, comprehensive overview over the rapidly growing literature in the field of "molecular MRI," which is achieved by several recent reviews. Instead, we focus here on the potential of this imaging technique and its challenges to achieve useful new information in various fields of application with the aim of visualizing cellular processes in the brain, in both the physiological and pathophysiological context. Particular attention will be given to the visualization of cells grafted into the brain. For this goal, the recent most exciting studies are selected as the best examples to elucidate the method's fast expanding potentials. Attention is also given to the aspects of producing synergies by combining molecular MRI with other molecular imaging modalities, thus generating the most complex pictures of cellular and molecular events in the brain under in vivo conditions.

Stem Cell Labeling for Magnetic Resonance Imaging

In vivo applications of cells for the monitoring of their cell dynamics increasingly use non-invasive magnetic resonance imaging. This imaging modality allows in particular to follow the migrational activity of stem cells intended for cell therapy strategies. All these approaches require the prior labeling of the cells under investigation for excellent contrast against the host tissue background in the imaging modality. The present review discusses the various routes of cell labeling and describes the potential to observe both cell localization and their cell-specific function in vivo. Possibilities for labeling strategies, pros and cons of various contrast agents are pointed out while potential ambiguities or problems of labeling strategies are emphasized.

Multimodal Imaging of Neural Progenitor Cell Fate in Rodents

For clinical application of stem cell-based therapies, noninvasive detection of applied stem cells is of high importance. We report on the feasibility of detecting implanted neural progenitor cells (NPCs) noninvasively and follow their fate and functional status by sequential multimodal molecular imaging and reporter gene technology. We investigated C17.2 cells stably expressing herpes simplex virus type 1-thymidine kinase (HSV-1-tk) and green fluorescent protein (gfp) (C17.2-tkIRESgfp = C17.2-TIG) or HSV-1-tk, gfp, and firefly luciferase (luc) (C17.2-lucIREStkgfp = C17.2-LITG) and determined the detection sensitivity of positron emission tomography (PET) and bioluminescence imaging (BLI) for these cells in culture and in vivo in subcutaneous and intracranial glioma models. In addition, PET and BLI were used to further investigate and follow the fate of implanted C17.2-LITG cells in an intracranial glioma model. We show that both imaging modalities are sensitive in detecting reporter gene expressing NPCs; however, PET, by the use of 9-[4-[(18)F]fluoro-3-hydroxymethyl)butyl]guanine ([(18)F]FHBG), detects NPCs only at sites of disrupted blood-brain barrier. Furthermore, both imaging modalities can be used to detect stem cell fate and migration and indicate excessive proliferation and aberrant migration. In conclusion, multimodal imaging can be used for longitudinal noninvasive monitoring of grafted NPCs in rodents.

Cell Labeling and Tracking for Experimental Models Using Magnetic Resonance Imaging

Magnetic Resonance Imaging (MRI), as one of the most powerful methods in clinical diagnosis, has emerged as an additional method in the field of molecular and cellular imaging. Compared to established molecular imaging methods, MRI provides in vivo images with high resolution. In particularly in the field of cell-based therapy, non-invasively acquired information on temporal changes of cell location linked to high-resolution anatomical information is of great interest. Relatively new approaches like responsive contrast agents or MR imaging reporter gene expression are MRI applications beyond temporal and spatial information on labeled cells towards investigations on functional changes of cells in vivo. MRI-based cell monitoring and tracking studies require prior labeling of the cells under investigation for excellent contrast against the background of host tissue. Here, an overview is provided on contrast generation strategies for MRI of cells. This includes MR contrast agents, various approaches of cell labeling and MRI as well as MR spectroscopic methods used for cell tracking in vivo. Advantages and disadvantages of the particular labeling approaches and methods are discussed. In addition to description of the methods, the emphasis is on the potential but also challenges and shortcomings of this imaging technique for applications that aim to visualize cellular processes in vivo.

Rapid Etiological Classification of Meningitis by NMR Spectroscopy Based on Metabolite Profiles and Host Response

Bacterial meningitis is an acute disease with high mortality that is reduced by early treatment. Identification of the causative microorganism by culture is sensitive but slow. Large volumes of cerebrospinal fluid (CSF) are required to maximise sensitivity and establish a provisional diagnosis. We have utilised nuclear magnetic resonance (NMR) spectroscopy to rapidly characterise the biochemical profile of CSF from normal rats and animals with pneumococcal or cryptococcal meningitis. Use of a miniaturised capillary NMR system overcame limitations caused by small CSF volumes and low metabolite concentrations. The analysis of the complex NMR spectroscopic data by a supervised statistical classification strategy included major, minor and unidentified metabolites. Reproducible spectral profiles were generated within less than three minutes, and revealed differences in the relative amounts of glucose, lactate, citrate, amino acid residues, acetate and polyols in the three groups. Contributions from microbial metabolism and inflammatory cells were evident. The computerised statistical classification strategy is based on both major metabolites and minor, partially unidentified metabolites. This data analysis proved highly specific for diagnosis (100% specificity in the final validation set), provided those with visible blood contamination were excluded from analysis; 6-8% of samples were classified as indeterminate. This proof of principle study suggests that a rapid etiologic diagnosis of meningitis is possible without prior culture. The method can be fully automated and avoids delays due to processing and selective identification of specific pathogens that are inherent in DNA-based techniques.

A Comparison of Random Forest and Its Gini Importance with Standard Chemometric Methods for the Feature Selection and Classification of Spectral Data

Regularized regression methods such as principal component or partial least squares regression perform well in learning tasks on high dimensional spectral data, but cannot explicitly eliminate irrelevant features. The random forest classifier with its associated Gini feature importance, on the other hand, allows for an explicit feature elimination, but may not be optimally adapted to spectral data due to the topology of its constituent classification trees which are based on orthogonal splits in feature space.

The Trehalose Synthesis Pathway is an Integral Part of the Virulence Composite for Cryptococcus Gattii

The trehalose pathway is essential for stress tolerance and virulence in fungi. We investigated the importance of this pathway for virulence of the pathogenic yeast Cryptococcus gattii using the highly virulent Vancouver Island, Canada, outbreak strain R265. Three genes putatively involved in trehalose biosynthesis, TPS1 (trehalose-6-phosphate [T6P] synthase) and TPS2 (T6P phosphatase), and degradation, NTH1 (neutral trehalose), were deleted in this strain, creating the R265tps1 Delta, R265tps2 Delta, and R265nth1 Delta mutants. As in Cryptococcus neoformans, cellular trehalose was reduced in the R265tps1 Delta and R265tps2 Delta mutants, which could not grow and died, respectively, at 37 degrees C on yeast extract-peptone-dextrose agar, suggesting that T6P accumulation in R265tps2 Delta is directly toxic. Characterizations of the cryptococcal hexokinases and trehalose mutants support their linkage to the control of glycolysis in this species. However, unlike C. neoformans, the C. gattii R265tps1 Delta mutant demonstrated, in addition, defects in melanin and capsule production, supporting an influence of T6P on these virulence pathways. Attenuated virulence of the R265tps1 Delta mutant was not due solely to its 37 degrees C growth defect, as shown in worm studies and confirmed by suppressor mutants. Furthermore, an intact trehalose pathway controls protein secretion, mating, and cell wall integrity in C. gattii. Thus, the trehalose synthesis pathway plays a central role in the virulence composites of C. gattii through multiple mechanisms. Deletion of NTH1 had no effect on virulence, but inactivation of the synthesis genes, TPS1 and TPS2, has profound effects on survival of C. gattii in the invertebrate and mammalian hosts. These results highlight the central importance of this pathway in the virulence composites of both pathogenic cryptococcal species.

Effects of MRI Contrast Agents on the Stem Cell Phenotype

The ultimate therapy for ischemic stroke is restoration of blood supply in the ischemic region and regeneration of lost neural cells. This might be achieved by transplanting cells that differentiate into vascular or neuronal cell types, or secrete trophic factors that enhance self-renewal, recruitment, long-term survival, and functional integration of endogenous stem/progenitor cells. Experimental stroke models have been developed to determine potential beneficial effect of stem/progenitor cell-based therapies. To follow the fate of grafted cells in vivo, a number of noninvasive imaging approaches have been developed. Magnetic resonance imaging (MRI) is a high-resolution, clinically relevant method allowing in vivo monitoring of cells labeled with contrast agents. In this study, labeling efficiency of three different stem cell populations [mouse embryonic stem cells (mESC), rat multipotent adult progenitor cells (rMAPC), and mouse mesenchymal stem cells (mMSC)] with three different (ultra)small superparamagnetic iron oxide [(U)SPIO] particles (Resovist, Endorem, Sinerem) was compared. Labeling efficiency with Resovist and Endorem differed significantly between the different stem cells. Labeling with (U)SPIOs in the range that allows detection of cells by in vivo MRI did not affect differentiation of stem cells when labeled with concentrations of particles needed for MRI-based visualization. Finally, we demonstrated that labeled rMAPC could be detected in vivo and that labeling did not interfere with their migration. We conclude that successful use of (U)SPIOs for MRI-based visualization will require assessment of the optimal (U)SPIO for each individual (stem) cell population to ensure the most sensitive detection without associated toxicity.

Pathogenesis of Pulmonary Cryptococcus Gattii Infection: a Rat Model

A model of pulmonary cryptococcosis in immunocompetent rats was developed to better understand the virulence of Cryptococcus gattii. Six isolates were studied, representing four molecular genotypes (VGI-MATα, VGIIa-MATα, VGIIa-MAT a, VGIIb-MATα), obtained from Australia, Vancouver (Canada) and Colombia. These originated from human patients, a cat and the environment and were administered intratracheally (i.t.) or transthoracically into Fischer 344 or Wistar-Furth rats in doses varying from 10(4) to 10(7) colony-forming units (CFU) in 0.1 ml of saline. With the exception of animals given the VGIIa-MAT a isolate, rats consistently became ill or died of progressive cryptococcal pneumonia following i.t. doses exceeding 10(7) CFU. Affected lungs increased in weight up to tenfold and contained numerous circumscribed, gelatinous lesions. These became larger and more extensive, progressing from limited hilar and/or tracheal lesions, to virtually confluent gelatinous masses. Disease was localized to the lungs for at least 3-4 weeks, with dissemination to the brain occurring in some animals after day 29. The dose-response relationship was steep for two VGI isolates studied (human WM179, environmental WM276); doses up to 10(6) CFU i.t. did not produce lesions, while 10(7) or more yeast cells produced progressive pneumonia. Intratracheal inoculation of rats with C. gattii provides an excellent model of human pulmonary cryptococcosis in healthy hosts, mimicking natural infections. Disease produced by C. gattii in rats is distinct from that caused by C. neoformans in that infections are progressive and ultimately fatal.

Efficient Gene Transfer into the Mouse Lung by Fetal Intratracheal Injection of RAAV2/6.2

Fetal gene therapy is one of the possible new therapeutic strategies for congenital or perinatal diseases with high mortality or morbidity. We developed a novel delivery strategy to inject directly into the fetal mouse trachea. Intratracheal (i.t.) injection at embryonic day 18 (E18) was more efficient in targeting the fetal lung than conventional intra-amniotic (i.a.) delivery. Viral vectors derived from adeno-associated virus serotype 6.2, with tropism for the airway epithelium and not earlier tested in the fetal mouse lung, were injected into the fetal trachea. Bioluminescence (BL) imaging (BLI) was combined with magnetic resonance (MR) imaging (MRI) for noninvasive and accurate localization of transgene expression in vivo. Histological analysis for β-galactosidase (β-gal) revealed 17.5% of epithelial cells transduced in the conducting airways and 1.5% in the alveolar cells. Stable gene expression was observed up to 1 month after injection. This study demonstrates that direct injection of rAAV2/6.2 in the fetal mouse trachea is superior to i.a. delivery for transducing the lung. Second, as stable gene transfer was detected up to 1 postnatal month, this approach may be useful to evaluate fetal gene therapy for pulmonary diseases such as cystic fibrosis, requiring both substantial numbers of transduced cells as well as prolonged gene expression to obtain a stable phenotypic effect.

Age-associated Adaptations in Murine Adipose Tissues

Ageing is associated with an increase in visceral obesity in men and women. Although wild-type mice with a C57Bl/6 genetic background are extensively used in studies on obesity and metabolism, little information is available on age-associated changes in their adipose tissues. We have evaluated development and composition of subcutaneous (SC) and gonadal (GON) adipose tissue in male C57Bl/6 mice at the ages of 10 weeks, 12 months or 24 months, while kept on normal chow. Total body weight as well as SC and GON fat mass significantly increased between 10 weeks and 12 months, but markedly decreased again up to 24 months of age. Adipocyte size in both fat depots and blood vessel size in GON fat followed this trend. Plasma leptin levels correlated positively with body weight and SC or GON fat mass. Both 12 and 24 months old mice displayed better insulin sensitivity as compared to 10 weeks old counterparts, reflected by significantly decreased plasma levels of insulin and/or glucose. Thus, ageing of C57Bl/6 male mice is associated with a biphasic pattern (increase up to 12 months followed by a decrease up to 24 months) of body weight, SC and GON fat mass, adipocyte and blood vessel size.

Intracellular Nanoparticle Coating Stability Determines Nanoparticle Diagnostics Efficacy and Cell Functionality

Iron oxide nanoparticles (NPs) are frequently employed in biomedical research as magnetic resonance (MR) contrast agents where high intracellular levels are required to clearly depict signal alterations. To date, the toxicity and applicability of these particles have not been completely unraveled. Here, we show that endosomal localization of different iron oxide particles results in their degradation and in reduced MR contrast, the rate of which is governed mainly by the stability of the coating. The release of ferric iron generates reactive species, which greatly affect cell functionality. Lipid-coated NPs display the highest stability and furthermore exhibit intracellular clustering, which significantly enhances their MR properties and intracellular persistence. These findings are of considerable importance because, depending on the nature of the coating, particles can be rapidly degraded, thus completely annihilating their MR contrast to levels not detectable when compared to controls and greatly impeding cell functionality, thereby hindering their application in functional in vivo studies.

Characterization of the Inflammatory Response in a Photothrombotic Stroke Model by MRI: Implications for Stem Cell Transplantation

The aim of this study was to evaluate the specificity of magnetic resonance imaging (MRI) contrast in a photothrombotic (PT) stroke model with and without engraftment of superparamagnetic iron oxide (SPIO)-labeled stem cells.

Cytotoxic Effects of Iron Oxide Nanoparticles and Implications for Safety in Cell Labelling

The in vitro labelling of cultured cells with iron oxide nanoparticles (NPs) is a frequent practice in biomedical research. To date, the potential cytotoxicity of these particles remains an issue of debate. In the present study, 4 different NP types (dextran-coated Endorem, carboxydextran-coated Resovist, lipid-coated magnetoliposomes (MLs) and citrate-coated very small iron oxide particles (VSOP)) are tested on a variety of cell types, being C17.2 neural progenitor cells, PC12 rat pheochromocytoma cells and human blood outgrowth endothelial cells. Using different NP concentrations, the effect of the NPs on cell morphology, cytoskeleton, proliferation, reactive oxygen species, functionality, viability and cellular homeostasis is investigated. Through a systematic study, the safe concentrations for every particle type are determined, showing that MLs can lead up to 67.37 ± 5.98 pg Fe/cell whereas VSOP are the most toxic particles and only reach 18.65 ± 2.07 pg Fe/cell. Using these concentrations, it is shown that for MRI up to 500 cells/μl labelled with VSOP are required to efficiently visualize in an agar phantom in contrast to only 50 cells/μl for MLs and 200 cells/μl for Endorem and Resovist. These results highlight the importance of in-depth cytotoxic evaluation of cell labelling studies as at non-toxic concentrations, some particles appear to be less suitable for the MR visualization of labelled cells.

Impact of Clock Gene Bmal1 Deficiency on Nutritionally Induced Obesity in Mice

To evaluate the hypothesis that the clock gene Bmal1 (brain and muscle arnt like protein-1) plays a role in the development of obesity, 5-week-old male Bmal1-deficient (Bmal1(-/-)) mice and wild-type littermates (Bmal1(+/+)) were kept on a high-fat diet (HFD) for 15 weeks. Despite an initial accelerated weight gain of Bmal1(-/-) mice, body weight and subcutaneous (SC) and gonadal (GON) adipose tissue mass were comparable to Bmal1(+/+) mice at the end of the diet period. Noninvasive magnetic resonance imaging scanning revealed a modest increase in fat content in Bmal1(-/-) mice after 10 weeks of HFD, whereas at the start and the end of the HFD feeding no differences were observed between both genotypes. After 15 weeks of HFD, adipocyte and blood vessel size and density were similar for Bmal1(+/+) and Bmal1(-/-) mice. However, the weight of major organs was significantly reduced in Bmal1(-/-) mice, confirming the premature ageing phenotype. Thus, we hypothesize that an initial accelerated increase in body weight and fat mass of Bmal1(-/-) mice on HFD may have been offset by the effect of premature ageing on organ weight, resulting in comparable weights after 15 weeks of HFD.

MR Perfusion and Diffusion Imaging in the Follow-up of Recurrent Glioblastoma Treated with Dendritic Cell Immunotherapy: a Pilot Study

This study aims to determine the potential value of MR-PWI and MR-DWI to differentiate immune therapy-induced inflammatory response from recurrent glioblastoma tumour growth. Both can present as contrast-enhancing lesions on conventional magnetic resonance imaging (MRI).

The Labeling of Cationic Iron Oxide Nanoparticle-resistant Hepatocellular Carcinoma Cells Using Targeted Magnetoliposomes

The in vitro labeling of cultured cells with nanomaterials is a frequent practice but the efficiency, specificity and cytotoxicity of labeling specific cell types using targeted nanoparticles has only rarely been investigated. In the present work, functionalized anionic lipid-coated iron oxide cores (magnetoliposomes (MLs)) bearing galactose moieties were used for the specific labeling of asialoglycoprotein receptor 1 (ASGPR-1)-expressing HepG2 cells. The optimal number of galactose moieties per particle (± 26) was determined and uptake efficiency was compared with galactose-lacking anionic and cationic MLs. Using a blocking assay with free galactose, electron microscopy and co-cultures of HepG2 and non-ASGPR-1 expressing C17.2 cells, the specificity of the particles for the ASGPR-1 receptor was demonstrated. The intracellular localization of the galactose-bearing MLs was further verified by confocal microscopy. The non-toxic ML concentration was determined to be 400 μg Fe/ml. Finally, the use of these MLs for visualization of labelled cells by magnetic resonance imaging (MRI) was demonstrated. The data show a high uptake and specificity of the galactose-bearing MLs, whereas the cationic MLs remain primarily surface-associated. Thus, targeted MLs offer a successful alternative for cell labeling when cationic particles fail to be efficiently internalized.

HRG Inhibits Tumor Growth and Metastasis by Inducing Macrophage Polarization and Vessel Normalization Through Downregulation of PlGF

Polarization of tumor-associated macrophages (TAMs) to a proangiogenic/immune-suppressive (M2-like) phenotype and abnormal, hypoperfused vessels are hallmarks of malignancy, but their molecular basis and interrelationship remains enigmatic. We report that the host-produced histidine-rich glycoprotein (HRG) inhibits tumor growth and metastasis, while improving chemotherapy. By skewing TAM polarization away from the M2- to a tumor-inhibiting M1-like phenotype, HRG promotes antitumor immune responses and vessel normalization, effects known to decrease tumor growth and metastasis and to enhance chemotherapy. Skewing of TAM polarization by HRG relies substantially on downregulation of placental growth factor (PlGF). Besides unveiling an important role for TAM polarization in tumor vessel abnormalization, and its regulation by HRG/PlGF, these findings offer therapeutic opportunities for anticancer and antiangiogenic treatment.

MRI Assessment of Blood Outgrowth Endothelial Cell Homing Using Cationic Magnetoliposomes

The use of contrast material to stimulate magnetic resonance imaging (MRI) of migrating cells has become an important area of research. In the present study, cationic magnetoliposomes (MLs) were used to magnetically label human blood outgrowth endothelial cells (BOECs) and follow their homing by magnetic resonance imaging (MRI). The biodistribution and functional integration capacity of BOECs, which have shown extensive promise as gene delivery vehicles, have thus far only rarely been investigated. MLs were avidly internalized by BOECs giving clear MRI contrast in phantom studies and the magnetic labeling did not affect cell proliferation, viability, morphology or homeostasis and elicited only minor reactive oxygen species levels. Intravenous injection of labeled BOECs was compared with injection of free MLs and unlabeled BOECs, resulting in homing of BOECs toward the liver and spleen, which was confirmed by histology. The MLs used offer great potential for cellular tracking studies by MRI when low levels of widely distributed cells are present. In particular, the use of these MLs will allow to evaluate the efficacy of new methods to enhance BOEC homing and integration to optimize their use as efficient vehicles for gene therapy.

Site-specific 68Ga-labeled Annexin A5 As a PET Imaging Agent for Apoptosis

Two variants of Annexin A5 (Cys2-AnxA5 and Cys165-AnxA5) were labelled with Gallium-68 in order to evaluate their biological properties.

Magnetoliposomes As Magnetic Resonance Imaging Contrast Agents

Among the wide variety in iron oxide nanoparticles which are routinely used as magnetic resonance imaging (MRI) contrast agents, magnetoliposomes (MLs) take up a special place. In the present work, the two main types (large and small MLs) are defined and their specific features are commented. For both types of MLs, the flexibility of the lipid coating allows for efficient functionalization, enabling bimodal imaging (e.g., MRI and fluorescence) or the use of MLs as theranostics. These features are especially true for large MLs, where several magnetite cores are encapsulated within a single large liposome, which were found to be highly efficient theranostic agents. By carefully fine-tuning the number of magnetite cores and attaching Gd(3+) -complexes onto the liposomal surface, the large MLs can be efficiently optimized for dynamic MRI. A special type of MLs, biogenic MLs, can also be efficiently used in this regard, with potential applications in cancer treatment and imaging. Small MLs, where the lipid bilayer is immediately attached onto a solid magnetite core, give a very high r2 /r1 ratio. The flexibility of the lipid bilayer allows the incorporation of poly(ethylene glycol)-lipid conjugates to increase blood circulation times and be used as bone marrow contrast agents. Cationic lipids can also be incorporated, leading to high cell uptake and associated strong contrast generation in MRI of implanted cells. Unique for these small MLs is the high resistance the particles exhibit against intracellular degradation compared with dextran- or citrate-coated particles. Additionally, intracellular clustering of the iron oxide cores enhances negative contrast generation and enables longer tracking of labeled cells in time.

Polyelectrolyte Coating of Iron Oxide Nanoparticles for MRI-based Cell Tracking

Iron oxide-based magnetic nanoparticles (MNPs) offer unique properties for cell tracking by magnetic resonance imaging (MRI) in cellular immunotherapy. In this study, we investigated the uptake of chemically engineered NPs into antigen-presenting dendritic cells (DCs). DCs are expected to perceive MNPs as foreign antigens, thus exhibiting the capability to immunologically sense MNP surface chemistry. To systematically evaluate cellular uptake and T2/T2(⁎) MR imaging properties of MNPs, we synthesized polymer-based MNPs by employing layer-by-layer (LbL) technology. Thereby, we achieved modification of particle shell parameters, such as size, surface charge, and chemistry. We found that subcellular packaging of MNPs rather than MNP content in DCs influences MR imaging quality. Increased local intracellular electron density as inferred from transmission electron microscopy (TEM) strongly correlated with enhanced contrast in MRI. Thus, LbL-tailoring of MNP shells using polyelectrolytes that impact on uptake and subcellular localization can be used for modulating MR imaging properties.

(19)F-heptuloses As Tools for the Non-invasive Imaging of GLUT2-expressing Cells

Suitable analogs of d-mannoheptulose are currently considered as possible tools for the non-invasive imaging of pancreatic islet insulin-producing cells. Here, we examined whether (19)F-heptuloses could be used for non-invasive imaging of GLUT2-expressing cells. After 20 min incubation, the uptake of (19)F-heptuloses (25 mM) by rat hepatocytes, as assessed by (19)F NMR spectroscopy, ranged from 0.50 (1-deoxy-1-fluoro-d-mannoheptulose and 3-deoxy-3-fluoro-d-mannoheptulose) to 0.25 (1,3-dideoxy-1,3-difluoro-d-mannoheptulose) and 0.13 (1-deoxy-1-fluoro-d-glucoheptulose, 3-deoxy-3-fluoro-d-glucoheptulose and 1,3-dideoxy-1,3-difluoro-d-glucoheptulose) μmol per 3×10(6)cells. (19)F MRI experiments also allowed the detection of 1-deoxy-1-fluoro-d-mannoheptulose in rat hepatocytes. All three (19)F-mannoheptuloses cited above, as well as 7-deoxy-7-fluoro-d-mannoheptulose and 1-deoxy-1-fluoro-d-glucoheptulose inhibited insulin release evoked in rat isolated pancreatic islets by 10mM d-glucose to the same extent as that observed with an equivalent concentration (10mM) of d-mannoheptulose, while 3-deoxy-3-fluoro-d-glucoheptulose and 1,3-dideoxy-1,3-difluoro-d-glucoheptulose (also 10mM) were less potent than d-mannoheptulose in inhibiting insulin release. The 1-deoxy-1-fluoro-d-mannoheptulose and 3-deoxy-3-fluoro-d-mannoheptulose only marginally affected INS-1 cell viability. These findings are compatible with the view that selected (19)F-heptuloses may represent suitable tools for the non-invasive imaging of hepatocytes and insulin-producing cells by (19)F MRI.

A Review on Various Targeted Anticancer Therapies

Translational oncology aims to translate laboratory research into new anticancer therapies. Contrary to conventional surgery, chemotherapy, and radiotherapy, targeted anticancer therapy (TAT) refers to systemic administration of drugs with particular mechanisms that specifically act on well-defined targets or biologic pathways that, when activated or inactivated, may cause regression or destruction of the malignant process, meanwhile with minimized adverse effects on healthy tissues. In this article, we intend to first give a brief review on various known TAT approaches that are deemed promising for clinical applications in the current trend of personalized medicine, and then we will introduce our newly developed approach namely small molecular sequential dual targeting theragnostic strategy as a generalized class of TAT for the management of most solid malignancies, which, after optimization, is expected to help improve overall cancer treatability and curability.

Gliomas: Diffusion Kurtosis MR Imaging in Grading

To assess the diagnostic accuracy of diffusion kurtosis magnetic resonance imaging parameters in grading gliomas.

In Vivo Imaging of Inhibitory, GABAergic Neurons by MRI

The unambiguous detection of specific neuronal subtypes is up to now only possible with invasive techniques or optical imaging after genetic modification. High field magnetic resonance imaging (MRI) has the ability to visualize the brain structure and anatomy noninvasively, with high resolution--but missing the cell specific and functional information. Here we present a new tool for neuroimaging with MRI, enabling the selective detection of GABAergic neurons under in vivo conditions. The specific imaging contrast is achieved by a novel paramagnetic contrast agent, which responds to the activity of the enzyme glutamic acid decarboxylase--expressed solely by inhibitory neurons. The relaxivity of the complex is increased upon decarboxylation of two glutamic acid moieties, thus allowing increased water access to the inner and outer coordination spheres of the paramagnetic ion. The mechanism and specificity of activation were proven with tissue lysates and further applied to a differentiation protocol for murine embryonic stem cells. The relaxation enhancement was studied quantitatively and revealed decreased longitudinal relaxation times in the inhibitory neuron samples compared to the naïve stem cells in vitro and in vivo. Furthermore, this approach offers not only the discrimination of inhibitory, GABAergic neurons in the brain but also may expand the usefulness of MRI for functional imaging on a cellular level.

Quantitative Evaluation of MRI-based Tracking of Ferritin-labeled Endogenous Neural Stem Cell Progeny in Rodent Brain

Endogenous neural stem cells have the potential to facilitate therapy for various neurodegenerative brain disorders. To increase our understanding of neural stem and progenitor cell biology in healthy and diseased brain, methods to label and visualize stem cells and their progeny in vivo are indispensable. Iron oxide particle based cell-labeling approaches enable cell tracking by MRI with high resolution and good soft tissue contrast in the brain. However, in addition to important concerns about unspecific labeling and low labeling efficiency, the dilution effect upon cell division is a major drawback for longitudinal follow-up of highly proliferating neural progenitor cells with MRI. Stable viral vector-mediated marking of endogenous stem cells and their progeny with a reporter gene for MRI could overcome these limitations. We stably and efficiently labeled endogenous neural stem/progenitor cells in the subventricular zone in situ by injecting a lentiviral vector expressing ferritin, a reporter for MRI. We developed an image analysis pipeline to quantify MRI signal changes at the level of the olfactory bulb as a result of migration of ferritin-labeled neuroblasts along the rostral migratory stream. We were able to detect ferritin-labeled endogenous neural stem cell progeny into the olfactory bulb of individual animals with ex vivo MRI at 30 weeks post injection, but could not demonstrate reliable in vivo detection and longitudinal tracking of neuroblast migration to the OB in individual animals. Therefore, although LV-mediated labeling of endogenous neural stem and progenitor cells resulted in efficient and stable ferritin-labeling of stem cell progeny in the OB, even with quantitative image analysis, sensitivity remains a limitation for in vivo applications.

Magnetically Triggered Clustering of Biotinylated Iron Oxide Nanoparticles in the Presence of Streptavidinylated Enzymes

This work deals with the production and characterization of water-compatible, iron oxide based nanoparticles covered with functional poly(ethylene glycol) (PEG)-biotin surface groups (SPIO-PEG-biotin). Synthesis of the functionalized colloids occurred by incubating the oleate coated particles used as precursor magnetic fluid with anionic liposomes containing 14 mol% of a phospholipid-PEG-biotin conjugate. The latter was prepared by coupling dimyristoylphosphatidylethanolamine (DC(14:0)PE) to activated α-biotinylamido-ω -N-hydroxy-succinimidcarbonyl-PEG (NHS-PEG-biotin). Physical characterization of the oleate and PEG-biotin iron oxide nanocolloids revealed that they appear as colloidal stable clusters with a hydrodynamic diameter of 160 nm and zeta potentials of - 39 mV (oleate coated particles) and - 14 mV (PEG-biotin covered particles), respectively, as measured by light scattering techniques. Superconducting quantum interference device (SQUID) measurements revealed specific saturation magnetizations of 62-73 emu g(-1) Fe(3)O(4) and no hysteresis was observed at 300 K. MR relaxometry at 3 T revealed very high r(2) relaxivities and moderately high r(1) values. Thus, both nanocolloids can be classified as small, superparamagnetic, negative MR contrast agents. The capacity to functionalize the particles was illustrated by binding streptavidin alkaline phosphatase (SAP). It was found, however, that these complexes become highly aggregated after capturing them on the magnetic filter device during high-gradient magnetophoresis, thereby reducing the accessibility of the SAP.

Small-animal PET Imaging of the Type 1 and Type 2 Cannabinoid Receptors in a Photothrombotic Stroke Model

Recent ex vivo and pharmacological evidence suggests involvement of the endocannabinoid system in the pathophysiology of stroke, but conflicting roles for type 1 and 2 cannabinoid receptors (CB(1) and CB(2)) have been suggested. The purpose of this study was to evaluate CB(1) and CB(2) receptor binding over time in vivo in a rat photothrombotic stroke model using PET.

Quantification of Lung Fibrosis and Emphysema in Mice Using Automated Micro-computed Tomography

In vivo high-resolution micro-computed tomography allows for longitudinal image-based measurements in animal models of lung disease. The combination of repetitive high resolution imaging with fully automated quantitative image analysis in mouse models of lung fibrosis lung benefits preclinical research. This study aimed to develop and validate such an automated micro-computed tomography analysis algorithm for quantification of aerated lung volume in mice; an indicator of pulmonary fibrosis and emphysema severity.

In Situ Labeling and Imaging of Endogenous Neural Stem Cell Proliferation and Migration

Endogenous neural stem cells (eNSCs) reside in defined regions of the adult brain and have the potential to generate new brain cells, including neurons. Stimulation of adult neurogenesis presents an enormous potential for regenerative therapies in the central nervous system. However, the methods used to monitor the proliferation, migration, differentiation, and functional integration of eNSCs and their progeny are often invasive and limited in studying dynamic processes. To overcome this limitation, novel techniques and contrast mechanisms for in vivo imaging of neurogenesis have recently been developed and successfully applied. In vivo labeling of endogenous neuronal progenitor cells in situ with contrast agents or tracers enables longitudinal visualization of their proliferation and/or migration. Labeling of these cells with magnetic nanoparticles has proven to be very useful for tracking neuroblast migration with MRI. Alternatively, genetic labeling using reporter gene technology has been demonstrated for optical and MR imaging, leading to the development of powerful tools for in vivo optical imaging of neurogenesis. More recently, the iron storage protein ferritin has been used as an endogenously produced MRI contrast agent to monitor neuroblast migration. The use of specific promoters for neuronal progenitor cell imaging increases the specificity for visualizing neurogenesis. Further improvements of detection sensitivity and neurogenesis-specific contrast are nevertheless required for each of these imaging techniques to further improve the already high utility of this toolbox for preclinical neurogenesis research.

Mapping of Oxygen by Imaging Lipids Relaxation Enhancement: A Potential Sensitive Endogenous MRI Contrast to Map Variations in Tissue Oxygenation

PURPOSE: Because of its paramagnetic properties, oxygen may act as an endogenous magnetic resonance imaging contrast agent by changing proton relaxation rates. Changes in tissue oxygen concentrations have been shown to produce changes in relaxation rate R(1) of water. The aim of the study was to improve the sensitivity of oxygen enhanced R(1) imaging by exploiting the higher solubility of oxygen in lipids (as compared with water) to sensitively monitor changes in tissue oxygen levels by selectively measuring the R(1) of lipids. METHODS: The method, with the acronym "MOBILE" (mapping of oxygen by imaging lipids relaxation enhancement), was applied in different mouse models of hypoxic processes on a 11.7 T magnetic resonance imaging system. MOBILE was compared with R*(2) , R(1) of water, and with pO(2) measurements (using electron paramagnetic resonance oximetry). MOBILE was also applied in the brain of healthy human volunteers exposed to an oxygen breathing challenge on a 3 T magnetic resonance imaging system. RESULTS: MOBILE was shown to be able to monitor changes in oxygenation in tumor, peripheral, liver, and brain tissues. The clinical translation was demonstrated in human volunteers. CONCLUSION: MOBILE arises as a promising noninvasive and sensitive tool for diagnosis and therapeutic guidance in disorders involving hypoxia. Magn Reson Med, 2012. © 2012 Wiley Periodicals, Inc.

A Single-dose Toxicity Study on Non-radioactive Iodinated Hypericin for a Targeted Anticancer Therapy in Mice

Hypericin (Hyp) and its radio-derivatives have been investigated in animal models with ischemic heart diseases and malignancies for diagnostic and therapeutic purposes. Before radioiodinated Hyp ((123)I-Hyp or (131)I-Hyp) can be considered as a clinically useful drug, vigorous evaluations on its chemotoxicity are necessary. In the present study, we examined the toxicity of a single dose of non-radioactive (127)I-Hyp in normal mice for 24 h and 14 d.

Exploring Theranostic Potentials of Radioiodinated Hypericin in Rodent Necrosis Models

The present animal experiments were conducted to evaluate radioiodinated Hypericin (Hyp) for its regional distribution as well as theranostic potentials.

Hierarchical Non-negative Matrix Factorization (hNMF): a Tissue Pattern Differentiation Method for Glioblastoma Multiforme Diagnosis Using MRSI

MRSI has shown potential in the diagnosis and prognosis of glioblastoma multiforme (GBM) brain tumors, but its use is limited by difficult data interpretation. When the analyzed MRSI data present more than two tissue patterns, conventional non-negative matrix factorization (NMF) implementation may lead to a non-robust estimation. The aim of this article is to introduce an effective approach for the differentiation of GBM tissue patterns using MRSI data. A hierarchical non-negative matrix factorization (hNMF) method that can blindly separate the most important spectral sources in short-TE ¹H MRSI data is proposed. This algorithm consists of several levels of NMF, where only two tissue patterns are computed at each level. The method is demonstrated on both simulated and in vivo short-TE ¹H MRSI data in patients with GBM. For the in vivo study, the accuracy of the recovered spectral sources was validated using expert knowledge. Results show that hNMF is able to accurately estimate the three tissue patterns present in the tumoral and peritumoral area of a GBM, i.e. normal, tumor and necrosis, thus providing additional useful information that can help in the diagnosis of GBM. Moreover, the hNMF results can be displayed as easily interpretable maps showing the contribution of each tissue pattern to each voxel.

Reproducibility of Rapid Short Echo Time CSI at 3 Tesla for Clinical Applications

To validate the reproducibility of a chemical shift imaging (CSI) acquisition protocol with parallel imaging, using automated repositioning software.

Unsupervised Nosologic Imaging for Glioma Diagnosis

In this letter a novel approach to create nosologic images of the brain using magnetic resonance spectroscopic imaging (MRSI) data in an unsupervised way is presented. Different tissue patterns are identified from the MRSI data using nonnegative matrix factorization and are then coded as different primary colors (i.e. red, green, and blue) in an RGB image, so that mixed tissue regions are automatically visualized as mixtures of primary colors. The approach is useful in assisting glioma diagnosis, where several tissue patterns such as normal, tumor, and necrotic tissue can be present in the same voxel/spectrum. Error-maps based on linear least squares estimation are computed for each nosologic image to provide additional reliability information, which may help clinicians in decision making. Tests on in vivo MRSI data show the potential of this new approach.

Does Rosiglitazone Affect Adiposity and Cardiac Function in Genetic Diabetic Mice?

Rosiglitazone ((RS)-5-[4-(2-[methyl(pyridin-2-yl)amino]ethoxy)benzyl]thiazolidine-2,4-dione, RGZ)-induced adverse drug effects in diabetic patients were not adequately predicted by current preclinical rodent models. Therefore, we have used the Akita mouse with genetic predisposition to diabetes to unravel the underlying molecular mechanisms. The effect of RGZ on adipose tissue and on cardiac function was evaluated in diabetic Akita mice kept on a high fat-high cholesterol diet (HF-HCD) for 4 months. When compared to wild-type (WT) mice with the same C57BL/6J genetic background, Akita mice gained significantly less weight (4.4±1.4 g versus 12±0.97 g for WT; P=0.002) and developed less fat (volume of 3.1±1.2 ml versus 16±2.1 ml for WT; P=0.004), associated with adipocyte hypotrophy. Upon treatment with RGZ (10mg/kg/day), Akita mice showed enhanced weight gain (11±0.70 g; P=0.004 versus untreated Akita mice) and fat volume (7.4±0.63 ml; P<0.05 versus untreated Akita mice), without effects on adipocyte or blood vessel size or on macrophage infiltration in adipose tissues. Akita mice kept on HF-HCD for 4 months with administration of RGZ (30 mg/kg/day) showed increased intraventricular septum thickness and cardiac output, without, however, an effect on fractional shortening or ejection fraction. In conclusion, RGZ promotes adiposity and early signs of hypertrophic cardiomyopathy in the diabetic Akita mouse. Thus, this genetically manipulated model may be suitable to test safety of anti-diabetic drugs.

Preclinical Evaluation of Carbon-11 and Fluorine-18 Sulfonamide Derivatives for in Vivo Radiolabeling of Erythrocytes

Diabetes Mellitus and the Metabolic Syndrome Do Not Abolish, but Might Reduce, the Cardioprotective Effect of Ischemic Postconditioning

Ischemic preconditioning fails to protect the diabetic heart against lethal reperfusion injury. Because the pathways of ischemic pre- and postconditioning partially overlap, we evaluated the cardioprotective effect of ischemic postconditioning in mouse models of type 2 diabetes (ObOb) and the metabolic syndrome (DKO).

Design and Evaluation of Theranostic Perfluorocarbon Particles for Simultaneous Antigen-loading and ¹⁹F-MRI Tracking of Dendritic Cells

Perfluorocarbon (PFC) particles are currently on the rise as cell labeling agents for ¹⁹F-MRI tracking of dendritic cell (DC)-based vaccines. In this work, we design theranostic PFC particles for single-step loading of DCs with both antigenic protein and with a liquid PFC for ¹⁹F-MRI detection of the antigen-loaded cells. Upon addition to DCs in vitro, the antigen-loaded PFC particles are efficiently internalized, resulting in intracellular presence of up to 40 pmol ¹⁹F atoms per cell. At the same time, the DCs become loaded with antigenic proteins, that can be efficiently processed, without important effects on cell viability or altering the DC's phenotype and the cell's capacity to respond to danger signals. In addition, antigen-loaded PFC particle containing DCs are capable of inducing extensive proliferation of antigen-specific CD8⁺ T cells in vitro. Importantly, the antigen-coated PFC particles allow in vitro ¹⁹F-MRI-based detection of the antigen-containing DCs with detection limits as low as 10³ cells μl⁻¹. The dual-modality characteristics of the designed particles could assure that only those DCs that have taken up the antigen, and hence are responsible for an immune response, are traceable via ¹⁹F-MRI. Taken together, these novel dual-modality particles represent an interesting strategy in the development of a traceable DC vaccine.

2-D Strain Assessment in the Mouse Through Spatial Compounding of Myocardial Velocity Data: in Vivo Feasibility

Ultrasound assessment of myocardial strain can provide valuable information on regional cardiac function. However, Doppler-based methods often used in practice for strain estimation suffer from angle dependency. In this study, a partial solution to that fundamental limitation is presented. We have previously reported using simulated data sets that spatial compounding of axial velocities obtained at three steering angles can theoretically outperform 2-D speckle tracking for 2-D strain estimation in the mouse heart. In this study, the feasibility of the method was analyzed in vivo using spatial compounding of Doppler velocities on six mice with myocardial infarction and five controls, and results were compared with those of tagged microscopic magnetic resonance imaging (μMRI). Circumferential estimates quantified by means of both ultrasound and μMRI could detect regional dysfunction. Between echocardiography and μMRI, a good regression coefficient was obtained for circumferential strain estimates (r = 0.69), whereas radial strain estimates correlated only moderately (r = 0.37). A second echocardiography was performed after μMRI to test the reproducibility of the compounding method. This yielded a higher correlation coefficient for the circumferential component than for the radial component (r = 0.74 circumferentially, r = 0.49 radially).

Unique Gene Expression and MR T2 Relaxometry Patterns Define Chronic Murine Dextran Sodium Sulphate Colitis As a Model for Connective Tissue Changes in Human Crohn's Disease

Chronically relapsing inflammation, tissue remodeling and fibrosis are hallmarks of inflammatory bowel diseases. The aim of this study was to investigate changes in connective tissue in a chronic murine model resulting from repeated cycles of dextran sodium sulphate (DSS) ingestion, to mimic the relapsing nature of the human disease.

Reporter Gene Approaches for Mapping Cell Fate Decisions by MRI: Promises and Pitfalls

The central dogma of molecular biology, namely the process by which information encoded in the DNA serves as the template for transcriptional activation of specific mRNA resulting in temporal and spatial control of the translation of specific proteins, stands at the basis of normal and pathological cellular processes. Serving as the primary mechanism linking genotype to phenotype, it is clearly of significant interest for in vivo imaging. While classically, imaging revolutionized the ability to phenotype the anatomical and physiological impact of induction of changes in gene expression, the preceding molecular events remained invisible. Reporter gene-based imaging techniques provide a window for in vivo visualization of such transcriptional activation events. In addition to the widespread use of fluorescent and bioluminescent reporter genes and development of a number of reporter genes for positron emission tomography (PET) imaging, there has been significant progress in the development of reporter genes for MRI. With the development of strategies for cellular based therapies, such imaging tools could become central components for personalized patient monitoring.

Variability in Contrast Agent Uptake by Different but Similar Stem Cell Types

The need to track and evaluate the fate of transplanted cells is an important issue in regenerative medicine. In order to accomplish this, pre-labelling cells with magnetic resonance imaging (MRI) contrast agents is a well-established method. Uptake of MRI contrast agents by non-phagocytic stem cells, and factors such as cell homeostasis or the adverse effects of contrast agents on cell biology have been extensively studied, but in the context of nanoparticle (NP)-specific parameters. Here, we have studied three different types of NPs (Endorem®, magnetoliposomes [MLs], and citrate coated C-200) to label relatively larger, mesenchymal stem cells (MSCs) and, much smaller yet faster proliferating, multipotent adult progenitor cells (MAPCs). Both cell types are similar, as they are isolated from bone marrow and have substantial regenerative potential, which make them interesting candidates for comparative experiments. Using NPs with different surface coatings and sizes, we found that differences in the proliferative and morphological characteristics of the cells used in the study are mainly responsible for the fate of endocytosed iron, intracellular iron concentration, and cytotoxic responses. The quantitative analysis, using high-resolution electron microscopy images, demonstrated a strong relationship between cell volume/surface, uptake, and cytotoxicity. Interestingly, uptake and toxicity trends are reversed if intracellular concentrations, and not amounts, are considered. This indicates that more attention should be paid to cellular parameters such as cell size and proliferation rate in comparative cell-labeling studies.

Towards Non-invasive Monitoring of Pathogen-host Interactions During Candida Albicans Biofilm Formation Using in Vivo Bioluminescence

Candida albicans is a major human fungal pathogen causing mucosal and deep tissue infections of which the majority is associated with biofilm formation on medical implants. Biofilms have a huge impact on public health, as fungal biofilms are highly resistant against most antimycotics. Animal models of biofilm formation are indispensable for improving our understanding of biofilm development inside the host, their antifungal resistance and their interaction with the host immune defence system. In currently used models, evaluation of biofilm development or the efficacy of antifungal treatment is limited to ex vivo analyses, requiring host sacrifice, which excludes longitudinal monitoring of dynamic processes during biofilm formation in the live host. In this study, we have demonstrated for the first time that non-invasive, dynamic imaging and quantification of in vitro and in vivo C. albicans biofilm formation including morphogenesis from the yeast to hyphae state is feasible by using growth-phase dependent bioluminescent C. albicans strains in a subcutaneous catheter model in rodents. We have shown the defect in biofilm formation of a bioluminescent bcr1 mutant strain. This approach has immediate applications for the screening and validation ofantimycotics under in vivo conditions, for studying host-biofilm interactions in different transgenic mouse models and for testing the virulence of luminescent C. albicans mutants, hereby contributing to a better understanding of the pathogenesis of biofilm-associated yeast infections.

Magnetic Layer-by-layer Coated Particles for Efficient MRI of Dendritic Cells and Mesenchymal Stem Cells

Cell detection by MRI requires high doses of contrast agent for generating image contrast. Therefore, there is a constant need to develop improved systems that further increase sensitivity, and which could be used in clinical settings. In this study, we devised layer-by-layer particles and tested their potential for cell labeling.

A Pictorial Essay on Fetal Rabbit Anatomy Using Micro-ultrasound and Magnetic Resonance Imaging

With this pictorial essay, we aimed to provide gestational age specific reference ranges of relevant fetal structures using micro-ultrasound, as well as its correlation with postmortem MRI and whole body sections.

Bioluminescence Imaging of Fungal Biofilm Development in Live Animals

Fungal biofilms formed on various types of medical implants represent a major problem for hospitalized patients. These biofilms and related infections are usually difficult to treat because of their resistance to the classical antifungal drugs. Animal models are indispensable for investigating host-pathogen interactions and for identifying new antifungal targets related to biofilm development. A limited number of animal models is available that can be used for testing novel antifungal drugs in vivo against C. albicans, one of the most common pathogens causing fungal biofilms. Fungal load in biofilms in these models is traditionally analyzed postmortem, requiring host sacrifice and enumeration of microorganisms from individual biofilms in order to evaluate the amount of colony forming units and the efficacy of antifungal treatment. Bioluminescence imaging (BLI) made compatible with small animal models for in vivo biofilm formation is a valuable noninvasive tool to follow-up biofilm development and its treatment longitudinally, reducing the number of animals needed for such studies. Due to the nondestructive and noninvasive nature of BLI, the imaging procedure can be repeated in the same animal, allowing follow-up of the biofilm growth in vivo without removing the implanted device or detaching the biofilm from its substrate. The method described here introduces BLI of C. albicans biofilm formation in vivo on subcutaneously implanted catheters in mice. One of the main challenges to overcome for BLI of fungi is the hampered intracellular substrate delivery through the fungal cell wall, which is managed by using extracellularly located Gaussia luciferase. Although detecting a quantifiable in vivo BLI signal from biofilms formed on the inside of implanted catheters is challenging, BLI proved to be a practical tool in the study of fungal biofilms. This method describing the use of BLI for in vivo follow-up of device-related fungal biofilm formation has the potential for efficient in vivo screening for interesting genes of the pathogen and the host involved in C. albicans biofilm formation as well as for testing novel antifungal therapies.

In Vivo Hepatocyte MR Imaging Using Lactose Functionalized Magnetoliposomes

The aim of this study was to assess a novel lactose functionalized magnetoliposomes (MLs) as an MR contrast agent to target hepatocytes as well as to evaluate the targeting ability of MLs for in vivo applications. In the present work, 17 nm sized iron oxide cores functionalized with anionic MLs bearing lactose moieties were used for targeting the asialoglycoprotein receptor (ASGP-r), which is highly expressed in hepatocytes. Non-functionalized anionic MLs were tested as negative controls. The size distribution of lactose and anionic MLs was determined by transmission electron microscopy (TEM) and dynamic light scattering (DLS). After intravenous administration of both MLs, contrast enhancement in the liver was observed by magnetic resonance imaging (MRI). Label retention was monitored non-invasively by MRI and validated with Prussian blue staining and TEM for up to eight days post MLs administration. Although the MRI signal intensity did not show significant differences between functionalized and non-functionalized particles, iron-specific Prussian blue staining and TEM analysis confirmed the uptake of lactose MLs mainly in hepatocytes. In contrast, non-functionalized anionic MLs were mainly taken up by Kupffer and sinusoidal cells. Target specificity was further confirmed by high-resolution MR imaging of phantoms containing isolated hepatocytes, Kupffer cell (KCs) and hepatic stellate cells (HSCs) fractions. Hypointense signal was observed for hepatocytes isolated from animals which received lactose MLs but not from animals which received anionic MLs. These data demonstrate that galactose-functionalized MLs can be used as a hepatocyte targeting MR contrast agent to potentially aid in the diagnosis of hepatic diseases if the non-specific uptake by KCs is taken into account.

Sensitive in Vivo Cell Detection Using Size-optimized Superparamagnetic Nanoparticles

Magnetic nanoparticle (MNP) enabled cell visualization with magnetic resonance imaging (MRI) is currently an intensively studied area of research. In the present study, we have synthesized polyethylene glycolated (PEG) MNPs and validated their suitability as MR cell labeling agents in in vitro and in vivo experiments. The labeling of therapeutic potent mesenchymal stem cells (MSCs) with small core and large core MNPs was evaluated. Both MNPs were, in combination with a transfection agent, stably internalized into the MSCs and didn't show an effect on cell metabolism. The labeled cells showed high contrast in MRI phantom studies. For quantification purposes, the MRI contrast generating properties of cells labeled with small core MNPs were compared with large core MNPs and with the commercial contrast agent Endorem. MSCs labeled with the large core MNPs showed the highest contrast generating properties in in vitro phantom studies and in in vivo intracranial stereotactic injection experiments, confirming the size-relaxivity relationship in biological systems. Finally, the distribution of MSCs pre-labeled with large core PEGylated MNPs was visualized non-invasively with MRI in a glioma model.

Integrating Diffusion Kurtosis Imaging, Dynamic Susceptibility-weighted Contrast-enhanced MRI, and Short Echo Time Chemical Shift Imaging for Grading Gliomas

We assessed the diagnostic accuracy of diffusion kurtosis imaging (DKI), dynamic susceptibility-weighted contrast-enhanced (DSC) MRI, and short echo time chemical shift imaging (CSI) for grading gliomas.

Synthetic Antiferromagnetic Nanoparticles As Potential Contrast Agents in MRI

We present the top-down synthesis of a novel type of MRI T2 contrast agent with great control over size and shape using a colloidal lithography technique. The resulting synthetic antiferromagnetic nanoparticles (SAF-NPs) yield improved relaxivities compared to superparamagnetic iron oxide alternatives (SPIONs). For T2 weighted imaging, the outer sphere relaxation theory has shown that the sensitivity of a T2 contrast agent is dependent on the particle size with an optimal size that exceeds the superparamagnetic limit of SPIONs. With the use of the interlayer exchange coupling effect, the SAF-NPs presented here do not suffer from this limit. Adjusting the outer sphere relaxation theory for spherical particles to SAF-NPs, we show both theoretically and experimentally that the SAF-NP size can be optimized to reach the r2 maximum. With measured r2 values up to 355 s(-1) mM(-1), our SAF-NPs show better performance than commercial alternatives and are competitive with the state-of-the-art. This performance is confirmed in an in vitro MRI study on SKOV3 cells.

A Role for LHC1 in Higher Order Structure and Complement Binding of the Cryptococcus Neoformans Capsule

Polysaccharide capsules are important virulence factors for many microbial pathogens including the opportunistic fungus Cryptococcus neoformans. In the present study, we demonstrate an unusual role for a secreted lactonohydrolase of C. neoformans, LHC1 in capsular higher order structure. Analysis of extracted capsular polysaccharide from wild-type and lhc1Δ strains by dynamic and static light scattering suggested a role for the LHC1 locus in altering the capsular polysaccharide, both reducing dimensions and altering its branching, density and solvation. These changes in the capsular structure resulted in LHC1-dependent alterations of antibody binding patterns, reductions in human and mouse complement binding and phagocytosis by the macrophage-like cell line J774, as well as increased virulence in mice. These findings identify a unique molecular mechanism for tertiary structural changes in a microbial capsule, facilitating immune evasion and virulence of a fungal pathogen.

Cytotoxicity of Cadmium-free Quantum Dots and Their Use in Cell Bioimaging

The use of quantum dots (QDots) as bright and photostable probes for long-term fluorescence imaging is gaining more interest. Thus far, (pre)clinical use of QDots remains limited, which is primarily caused by the potential toxicity of QDots. Most QDots consist of Cd2+ ions, which are known to cause high levels of toxicity. In order to overcome this problem, several strategies have been tested, such as the generation of cadmium-free QDots. In the present study, two types of cadmium-free QDots, composed of ZnSe/ZnS (QDotZnSe) and InP/ZnS (QDotInP), were studied with respect to their cytotoxicity and cellular uptake in a variety of cell types. A multiparametric cytotoxicity approach is used, where the QDots are studied with respect to cell viability, oxidative stress, cell morphology, stem cell differentiation, and neurite outgrowth. The data reveal slight differences in uptake levels for both types of QDots (maximal for QDotZnSe), but clear differences in cytotoxicity and cell functionality effects exist, with highest toxicity for QDotZnSe. Differences between cell types and between both types of QDots can be explained by the intrinsic sensitivity of certain cell types and chemical composition of the QDots. At concentrations at which no toxic effects can be observed, the functionality of the QDots for fluorescence cell visualization is evaluated, revealing that the higher brightness of QDotZnSe overcomes most of the toxicity issues compared to that of QDotInP. Comparing the results obtained with common Cd2+-containing QDots tested under identical conditions, the importance of particle functionality is demonstrated, revealing that cadmium-free QDots tested in this study are not significantly better than Cd2+-containing QDots for long-term cell imaging and that more work needs to be performed in optimizing the brightness and surface chemistry of cadmium-free QDots for them to replace currently used Cd2+-containing QDots.

Magnetic Resonance Imaging for Noninvasive Assessment of Lung Fibrosis Onset and Progression: Cross-validation and Comparison of Different Magnetic Resonance Imaging Protocols with Micro-computed Tomography and Histology in the Bleomycin-induced Mouse Model

Bleomycin instillation is frequently used to model lung fibrosis, although the onset and severity of pathology varies highly between mice. This makes non-invasive fibrosis detection and quantification essential to obtain a comprehensive analysis of the disease course and to validate novel therapies. Magnetic resonance imaging (MRI) of lung disease progression and therapy may provide such a sensitive in vivo readout of lung fibrosis, bypassing radiotoxicity concerns (when using micro-CT [μCT]) and elaborate invasive end point measurements (histology). We aimed to optimize and evaluate 3 different lung MRI contrast and acquisition methods to visualize disease onset and progression in the bleomycin-induced mouse model of lung fibrosis using a small-animal MRI scanner. For validation, we compared the MRI results with established μCT and histological measures of lung fibrosis.

Bioluminescence Imaging of Stroke-induced Endogenous Neural Stem Cell Response

Brain injury following stroke affects neurogenesis in the adult mammalian brain. However, a complete understanding of the origin and fate of the endogenous neural stem cells (eNSCs) in vivo is missing. Tools and technology that allow non-invasive imaging and tracking of eNSCs in living animals will help to overcome this hurdle. In this study, we aimed to monitor eNSCs in a photothrombotic (PT) stroke model using in vivo bioluminescence imaging (BLI). In a first strategy, inducible transgenic mice expressing firefly luciferase (Fluc) in the eNSCs were generated. In animals that received stroke, an increased BLI signal originating from the infarct region was observed. However, due to histological limitations, the identity and exact origin of cells contributing to the increased BLI signal could not be revealed. To overcome this limitation, we developed an alternative strategy employing stereotactic injection of conditional lentiviral vectors (Cre-Flex LVs) encoding Fluc and eGFP in the subventricular zone (SVZ) of Nestin-Cre transgenic mice, thereby specifically labeling the eNSCs. Upon induction of stroke, increased eNSC proliferation resulted in a significant increase in BLI signal between 2days and 2weeks after stroke, decreasing after 3months. Additionally, the BLI signal relocalized from the SVZ towards the infarct region during the 2weeks following stroke. Histological analysis at 90days post stroke showed that in the peri-infarct area, 36% of labeled eNSC progeny differentiated into astrocytes, while 21% differentiated into mature neurons. In conclusion, we developed and validated a novel imaging technique that unequivocally demonstrates that nestin(+) eNSCs originating from the SVZ respond to stroke injury by increased proliferation, migration towards the infarct region and differentiation into both astrocytes and neurons. In addition, this new approach allows non-invasive and specific monitoring of eNSCs over time, opening perspectives for preclinical evaluation of candidate stroke therapeutics.

The Performance of Gradient Alloy Quantum Dots in Cell Labeling

The interest in using quantum dots (QDots) as highly fluorescent and photostable nanoparticles in biomedicine is vastly increasing. One major hurdle that slows down the (pre)clinical translation of QDots is their potential toxicity. Several strategies have been employed to optimize common core-shell QDots, such as the use of gradient alloy (GA)-QDots. These particles no longer have a size-dependent emission wavelength, but the emission rather depends on the chemical composition of the gradient layer. Therefore, particles of identical sizes but with emission maxima spanning the entire visible spectrum can be generated. In the present study, two types of GA-QDots are studied with respect to their cytotoxicity and cellular uptake. A multiparametric cytotoxicity approach reveals concentration-dependent effects on cell viability, oxidative stress, cell morphology and cell functionality (stem cell differentiation and neurite outgrowth), where the particles are very robust against environmentally-induced breakdown. Non-toxic concentrations are defined and compared to common core-shell QDots analyzed under identical conditions. Additionally, this value is translated into a functional value by analyzing the potential of the particles for cell visualization. Interestingly, these particles result in clear endosomal localization, where different particles result in identical intracellular distributions. This is in contrast with CdTe QDots with the same surface coating, which resulted in clearly distinct intracellular distributions as a result of differences in nanoparticle diameter. The GA-QDots are therefore ideal platforms for cell labeling studies given their high brightness, low cytotoxicity and identical sizes, resulting in highly similar intracellular particle distributions which offer a lot of potential for optimizing drug delivery strategies.

In Vivo and Ex Vivo Assessment of the Blood Brain Barrier Integrity in Different Glioblastoma Animal Models

Blood brain barrier (BBB) disruption is used (pre)clinically as a measure for brain tumor malignancy and grading. During treatment it is one of the parameters followed rigorously to assess therapeutic efficacy. In animal models, both invasive and non-invasive methods are used to determine BBB disruption, among them Evans blue injection prior to sacrifice and T1-weighted magnetic resonance imaging (MRI) post contrast injection. In this study, we have assessed the BBB integrity with the methods mentioned above in two experimental high grade glioma models, namely the GL261 mouse glioblastoma model and the Hs683 human oligodendroglioma model. The GL261 model showed clear BBB integrity loss with both, contrast-enhanced (CE) MRI and Evans blue staining. In contrast, the Hs683 model only displayed BBB disruption with CE-MRI, which was not evident on Evans blue staining, indicating a limited BBB disruption. These results clearly indicate the importance of assessing the BBB integrity status using appropriate methods. Especially when using large therapeutic molecules that have difficulties crossing the BBB, care should be taken with the appropriate BBB disruption assessment studies.

Early Decrease of Type 1 Cannabinoid Receptor Binding and Phosphodiesterase 10A Activity In vivo in R6/2 Huntington Mice

Several lines of evidence imply early alterations in endocannabinoid and phosphodiesterase 10A (PDE10A) signaling in Huntington disease (HD). Using [(18)F]MK-9470 and [(18)F]JNJ42259152 small-animal positron emission tomography (PET), we investigated for the first time cerebral changes in type 1 cannabinoid (CB1) receptor binding and PDE10A levels in vivo in presymptomatic, early symptomatic, and late symptomatic HD (R6/2) mice, in relation to glucose metabolism ([(18)F]FDG PET), brain morphology (magnetic resonance imaging) and motor function. Ten R6/2 and 16 wild-type (WT) mice were investigated at 3 different time points between the age of 4 and 13 weeks. Parametric CB1 receptor and PDE10A images were anatomically standardized to Paxinos space and analyzed voxelwise. Volumetric microMRI imaging was performed to assess HD pathology. In R6/2 mice, CB1 receptor binding was decreased in comparison with WT in a cluster comprising the bilateral caudate-putamen, globus pallidus, and thalamic nucleus at week 5 (-8.1% ± 2.6%, p = 1.7 × 10(-5)). Longitudinal follow-up showed further progressive decline compared with controls in a cluster comprising the bilateral hippocampus, caudate-putamen, globus pallidus, superior colliculus, thalamic nucleus, and cerebellum (late vs. presymptomatic age: -13.7% ± 3.1% for R6/2 and +1.5% ± 4.0% for WT, p = 1.9 × 10(-5)). In R6/2 mice, PDE10A binding potential also decreased over time to reach significance at early and late symptomatic HD (late vs. presymptomatic age: -79.1% ± 1.9% for R6/2 and +2.1% ± 2.7% for WT, p = 1.5 × 10(-4)). The observed changes in CB1 receptor and PDE10A binding were correlated to anomalies exhibited by R6/2 animals in motor function, whereas no correlation was found with magnetic resonance imaging-based striatal volume. Our findings point to early regional dysfunctions in endocannabinoid and PDE10A signaling, involving the caudate-putamen and lateral globus pallidus, which may play a role in the progression of the disease in R6/2 animals. PET quantification of in vivo CB1 and/or PDE10A binding may thus be useful early biomarkers for HD. Our results also provide evidence of subtle motor deficits at earlier stages than previously described.

Controlling and Monitoring Stem Cell Safety in Vivo in an Experimental Rodent Model

Adult stem cells have been investigated increasingly over the past years for multiple applications. Although they have a more favorable safety profile compared to pluripotent stem cells, they are still capable of self-renewal and differentiate into several cell types. We investigated the behavior of Oct4-positive (Oct4(+)) and Oct4-negative (Oct4(-) ) murine or rat bone marrow (BM)-derived stem cells in the healthy brain of syngeneic mice and rats. Engraftment of mouse and rat Oct4-positive BM-derived hypoblast-like stem cells (m/rOct4(+) BM-HypoSCs) resulted in yolk-sac tumor formation in the healthy brain which was monitored longitudinally using magnetic resonance imaging (MRI) and bioluminescence imaging (BLI). Contrast enhanced MRI confirmed the disruption of the blood brain barrier. In contrast, m/r Oct4-negative BM-derived multipotent adult progenitor cells (m/rOct4(-) BM-MAPCs) did not result in mass formation after engraftment into the brain. mOct4(+) BM-HypoSCs and mOct4(-) BM-MAPCs were transduced to express enhanced green fluorescent protein, firefly luciferase (fLuc), and herpes simplex virus-thymidine kinase to follow up suicide gene expression as a potential "safety switch" for tumor-forming stem cells by multimodal imaging. Both cell lines were eradicated efficiently in vivo by ganciclovir administration indicating successful suicide gene expression in vivo, as assessed by MRI, BLI, and histology. The use of suicide genes to prevent tumor formation is in particular of interest for therapeutic approaches where stem cells are used as vehicles to deliver therapeutic genes.

High-content Imaging and Gene Expression Analysis to Study Cell-nanomaterial Interactions: the Effect of Surface Hydrophobicity

The effects of nanoparticle (NP)-related parameters on cellular interactions are currently uncertain as analysis is complicated by the combinatorial diversity arising from the array of size, shape and surface properties. Here, we present a validated multiparametric high-content imaging method, with the utility of this approach demonstrated by in-depth analysis of the role of hydrophobicity on the interaction of Au NPs with cultured cells. In this methodology, we evaluate cell viability, membrane damage, induction of reactive oxygen species, mitochondrial health, cell area, skewness and induction of autophagy. High-content cell cycle phase studies and in-depth gene expression studies then serve to elucidate the underlying mechanisms. The data reveal a clear influence of the degree of NP surface hydrophobicity with membrane damage and autophagy induction, which is stronger than the effect of surface charge, for charges ranging between -50 and +20 mV. All labeling experiments occur in the same format, and can be further supplemented with additional parameters providing a broadly accessible format for studying cell-NP interactions under highly reproducible conditions.

FMRP Regulates Multipolar to Bipolar Transition Affecting Neuronal Migration and Cortical Circuitry

Deficiencies in fragile X mental retardation protein (FMRP) are the most common cause of inherited intellectual disability, fragile X syndrome (FXS), with symptoms manifesting during infancy and early childhood. Using a mouse model for FXS, we found that Fmrp regulates the positioning of neurons in the cortical plate during embryonic development, affecting their multipolar-to-bipolar transition (MBT). We identified N-cadherin, which is crucial for MBT, as an Fmrp-regulated target in embryonic brain. Furthermore, spontaneous network activity and high-resolution brain imaging revealed defects in the establishment of neuronal networks at very early developmental stages, further confirmed by an unbalanced excitatory and inhibitory network. Finally, reintroduction of Fmrp or N-cadherin in the embryo normalized early postnatal neuron activity. Our findings highlight the critical role of Fmrp in the developing cerebral cortex and might explain some of the clinical features observed in patients with FXS, such as alterations in synaptic communication and neuronal network connectivity.

Longitudinal Assessment of Infarct Progression, Brain Metabolism and Behavior Following Anterior Cerebral Artery Occlusion in Rats

Stroke patients suffering from occlusion of the anterior cerebral artery (ACAo) develop cognitive and executive deficits. Experimental models to investigate such functional impairments and recovery are rare and not satisfyingly validated.

Longitudinal Follow-up and Characterization of a Robust Rat Model for Parkinson's Disease Based on Overexpression of Alpha-synuclein with Adeno-associated Viral Vectors

Testing of new therapeutic strategies for Parkinson's disease (PD) is currently hampered by the lack of relevant and reproducible animal models. Here, we developed a robust rat model for PD by injection of adeno-associated viral vectors (rAAV2/7) encoding α-synuclein into the substantia nigra, resulting in reproducible nigrostriatal pathology and behavioral deficits in a 4-week time period. Progressive dopaminergic dysfunction was corroborated by histopathologic and biochemical analysis, motor behavior testing and in vivo microdialysis. L-DOPA treatment was found to reverse the behavioral phenotype. Non-invasive positron emission tomography imaging and magnetic resonance spectroscopy allowed longitudinal monitoring of neurodegeneration. In addition, insoluble α-synuclein aggregates were formed in this model. This α-synuclein rat model shows improved face and predictive validity, and therefore offers the possibility to reliably test novel therapeutics. Furthermore, it will be of great value for further research into the molecular pathogenesis of PD and the importance of α-synuclein aggregation in the disease process.

Enhanced β-adrenergic Cardiac Reserve in Trpm4-/- Mice with Ischemic Heart Failure

Heart failure (HF) is a complex syndrome characterized by critically reduced cardiac contractility and function. We have shown previously that Transient Receptor Potential Melastatin 4 protein (TRPM4) functions as a Ca2+-activated non-selective cation channel and constitutes a novel regulator of ventricular contractility. In healthy Trpm4-deficient (Trpm4-/-) mice, we observed increased cardiac contractile function after β-adrenergic stimulation. In the current study, cardiac performance was examined in wild-type (WT) and Trpm4-/- mice with severe ischemic HF.

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