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Biomedical subjects

Ralph Weissleder

Publications and source records attributed to Ralph Weissleder.

At least 127 records · Page 7Linked to original sources
PubMed2004

In vivo imaging of beta-galactosidase activity using far red fluorescent switch.

beta-Galactosidase (beta-gal) has been widely used as a transgene reporter enzyme, and several substrates are available for its in vitro detection. The ability to image beta-gal expression in living animals would further extend the use of this reporter. Here we show that DDAOG, a conjugate of beta-galactoside and 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) (DDAO), is not only a chromogenic beta-gal substrate but that the cleavage product has far-red fluorescence properties detectable by imaging. Importantly, the cleavage substrate shows a 50-nm red shift, enabling its specific detection in a background of intact probe, a highly desirable feature for in vivo imaging. Specifically, we show that beta-gal-expressing 9L gliomas are readily detectable by red fluorescence imaging in comparison with the native 9L gliomas. We furthermore show that herpes simplex virus amplicon-mediated LacZ gene transfer into tumors can be transiently and thus serially visualized over time. The results indicated that in vivo real-time detection of beta-gal activity is possible by fluorescence imaging technology.

Animals↗
PubMed2004

Imaging of stem cell recruitment to ischemic infarcts in a murine model.

BACKGROUND AND PURPOSE: Neural progenitor cells (NPC) have been reported to aid in the functional recovery from stroke and hold promise as a novel treatment for a variety of neurological diseases. There is a need for imaging tools to study the in vivo migratory behavior of these cells. METHODS: C17.2 NPC, stably transfected with firefly luciferase, were serially imaged through intact skull and skin by bioluminescence imaging over 2 to 3 weeks in nu/nu mice with closed-vessel middle cerebral artery infarcts, followed by contralateral intraparenchymal or intraventricular injections of NPC. RESULTS: NPC migrated to the site of infarct from the contraleral parenchyma, crossing the midline at 7 days. In control animals without infarcts, NPC remained at the site of administration. Intraventricular cell administration resulted in a wide distribution of cells, including the site of infarct. Within the infarct area, NPC colabeled with the neuronal marker NeuN and with astroglial marker glial fibrillary acidic protein. The time course and magnitude of NPC recruitment were longitudinally compared between the treatment groups. CONCLUSIONS: NPC recruitment to infarct can be assessed noninvasively by serial in vivo imaging. Images correlate well with histological cell distributions. NPC are recruited to infarcts with both parenchymal and cerebrospinal fluid administration, but higher initial photon counts suggest that cerebrospinal fluid administration is more efficient at early infarct seeding.

Animals↗
PubMed2004

Novel nanosensors for rapid analysis of telomerase activity.

Elevated telomerase levels are found in many malignancies, offering an attractive target for therapeutic intervention and diagnostic or prognostic purposes. Here we describe the use of a novel nanosensor developed for rapid screens of telomerase activity in biological samples. The technique utilizes magnetic nanoparticles that, on annealing with telomerase synthesized TTAGGG repeats, switch their magnet state, a phenomenon readily detectable by magnetic readers. We tested the efficacy of different telomerase inhibitors in crude human and murine samples and show that phosphorylation of telomerase regulates its activity. High-throughput adaptation of the technique by magnetic resonance imaging allowed processing of hundreds of samples within tens of minutes at ultrahigh sensitivities. Together, these studies establish and validate a novel and powerful tool for rapidly sensing telomerase activity and provide the rationale for developing analogous magnetic nanoparticles for in vivo sensing.

Biosensing Techniques↗
PubMed2004

In vivo imaging of HIV protease activity in amplicon vector-transduced gliomas.

In vivo imaging of endogenously expressed mammalian proteases has been useful for the detection of cancer and preneoplastic lesions, for staging of inflammatory and autoimmune diseases, and for testing the efficacy of novel protease inhibitors. Here we report on the synthesis of a novel imaging probe that is specific for HIV-1 protease (PR). The probe was designed to be biocompatible, i.v. injectable, and detectable by fluorescence imaging. Human Gli36 glioblastoma cells infected with an human simplex virus amplicon vector expressing HIV-1PR showed specific fluorescence activation, an effect that could be inhibited by the HIV-1PR inhibitor, indinavir. The transfer of the HIV-1PR marker gene could be detected in vivo after intratumoral delivery of the human simplex virus-amplicon vector. These results are the first proof of principle that viral proteases can directly be imaged in vivo. These findings may be directly applicable in using viral protease expression as a transgene marker in tumor therapy and may have implications in testing the efficacy of HIV-1PR inhibitors in vivo.

Animals↗
PubMed2004

In vivo imaging of protease activity in arthritis: a novel approach for monitoring treatment response.

OBJECTIVE: Sensitive noninvasive strategies for monitoring treatment response in rheumatoid arthritis (RA) would be valuable for facilitating appropriate therapy and dosing, evaluating clinical outcome, and developing more effective drugs. Because different proteases are highly up-regulated in RA and contribute significantly to joint destruction, in the present study we investigated whether such enzymes are suitable in vivo imaging biomarkers for early evaluation of treatment response in a murine model of RA. METHODS: Using a protease-activated near-infrared fluorescence (NIRF) imaging "smart" probe, we examined the presence and distribution of fluorescence in arthritic joints of mice with collagen-induced arthritis by both noninvasive fluorescence imaging and histology. Proteases that target the Lys-Lys cleavage site, including cathepsin B, activate probe fluorescence. Treatment monitoring data were obtained following methotrexate (MTX) therapy. RESULTS: Twenty-four hours after intravenous injection of the protease sensor, affected toes and paws of arthritic mice showed significantly higher fluorescence intensity than did toes and paws of healthy mice. Fluorescence from the protease probe and cathepsin B antibody histologic staining were localized in the vast majority of cells in the inflamed synovium. In arthritic animals treated with MTX (35 mg of MTX/kg 48 hours prior to probe injection), a significantly lower fluorescent signal (inflamed paws 50%, inflamed toes 70%) was observed as compared with untreated arthritic animals. CONCLUSION: Protease-activated NIRF probes are sensitive means of imaging the presence of target enzymes in arthritic joints and can be used for early monitoring of treatment response to antirheumatic drugs such as MTX.

Animals↗
PubMed2004

Intracellular cargo delivery using tat peptide and derivatives.

Efficient delivery of therapeutic and diagnostic agents across the plasma membrane is crucial in developing novel therapies. Membrane permeating peptides, in particular HIV-1 tat peptide and its derivatives, have enabled the intracellular delivery of cargos of various sizes and physicochemical properties. This review summarizes the current knowledge of tat-derived cell permeating peptides in the transduction of exogenous molecules/complexes.

Animals↗
PubMed2004

Human myeloperoxidase: a potential target for molecular MR imaging in atherosclerosis.

Plaque rupture in atherosclerotic disease is the major cause of morbidity and correlates well with myeloperoxidase (MPO) secretion by activated neutrophils and macrophages in humans. We hypothesized that paramagnetic electron donor compounds that rapidly oxidize and polymerize in the presence of MPO could be designed to enable imaging of local MPO activity levels in arterial segments at risk. Several potential substrates for MPO were synthesized and tested. One lead compound consisting of a covalent conjugate of GdDOTA and serotonin (3-(2-aminoethyl)-5-hydroxyindole) was efficiently polymerized in the presence of human neutrophil MPO resulting in a 70-100% increase in proton relaxivity. As a result, we were able to demonstrate MPO activity in enzyme solutions and in a model tissue-like system. These studies suggest that activatable paramagnetic MR imaging agents can be used to directly image MPO activity.

Arteriosclerosis↗
PubMed2004

Design, synthesis, and characterization of urokinase plasminogen-activator-sensitive near-infrared reporter.

The urokinase-type plasminogen activator (uPA) plays a critical role in malignancies, and its overexpression has been linked to poor clinical prognosis in breast cancer. The ability to noninvasively and serially map uPA expression as a biomarker would thus have significant potential in improving novel cancer therapies. Here, we describe the development of a selective uPA activatable near-infrared (NIR) fluorescent imaging probe. The probe consists of multiple peptide motifs, GGSGRSANAKC-NH2, terminally capped with different NIR fluorochromes (Cy5.5 or Cy7) and a pegylated poly-L-lysine graft copolymer. Upon addition of recombinant human uPA to the probe, significant fluorescence amplification was observed, up to 680% with the optimized preparation. No activation with negative control compounds and uPA inhibitors could be measured. These data indicate that the optimized preparation should be useful for imaging uPA in cancer.

Biomarkers, Tumor↗
PubMed2004

Catheter-based in vivo imaging of enzyme activity and gene expression: feasibility study in mice.

PURPOSE: To construct and evaluate an interventional catheter-based imaging system for intravital monitoring of molecularly sensitive near-infrared fluorescent probes and optical marker genes. MATERIALS AND METHODS: An imaging device that was based on a miniaturized fiberoptic sensor (MIFS) was built in which images created with a 2.7-F fiberoptic catheter were relayed through a dichroic mirror, through a bandpass filter, and on two independent cameras. This system permitted simultaneous recording of white-light and fluorescent images. Spatial resolution, spectral transmissions, and sensitivity were determined in vitro. In vivo testing was performed in nude mice bearing intraperitoneal tumors that express green fluorescent protein and in a mouse model of ovarian carcinoma with enzyme-activatable near-infrared probes sensitive to tumoral protease activity. Signal intensity on images of tumors and that on images of normal tissue were measured and compared with t test. RESULTS: The catheter, which was advanced through an 18-gauge sheath, showed resolution of 7 line pairs per millimeter and detection limit for fluorochrome Cy5.5 of 1-10 pmol. Detection of endogenous green fluorescent protein gene expression was feasible in tumor nodules smaller than 1 mm in diameter (mean tumor signal intensity, 153.26 +/- 26.45 [SD], compared with that of adjacent nontumoral tissue of 36.73 +/- 11.69; P <.008). Similarly, activation of the near-infrared probe by tumoral proteases could be detected in peritoneal tumor seeds of ovarian cancer model with mean tumor signal intensity of 246.33 +/- 7.77 compared with that of adjacent nontumoral tissue of 41.56 +/- 18.64 (P <.001). Mean contrast-to-noise ratio in the near-infrared channel exceeded white-light contrast-to-noise ratio by a factor of 6.7 (P <.02). CONCLUSION: With this system, in vivo MIFS imaging of gene expression, enzyme activity, and potentially other molecular events is feasible, through direct interventional access to several organs and body cavities and potentially through transvascular approaches.

Animals↗
PubMed2004

Imaging reactive oxygen species in arthritis.

Reactive oxygen species (ROS) have been shown to play a role in the pathogenesis of arthritides. Luminol was used as the primary reporter of ROS and photons resulting from the chemiluminescence reaction were detected using a super-cooled CCD photon counting system. Luminol was injected intravenously into groups of animals with different models of arthritis. Imaging signal correlated well with the severity of arthritis in focal and pan-arthritis as determined by histological measurement of ROS by formazan. Measurements were highly reproducible, sensitive, and repeatable. In vivo chemiluminescence imaging is expected to become a useful modality to elucidate the role of ROS in the pathogenesis of arthritides and in determining therapeutic efficacy of protective therapies.

Animals↗
PubMed2004

Singular-value analysis and optimization of experimental parameters in fluorescence molecular tomography.

The advent of specific molecular markers and probes employing optical reporters has encouraged the application of in vivo diffuse tomographic imaging at greater spatial resolutions and hence data-set volumes. This study applied singular-value analysis (SVA) of the fluorescence tomographic problem to determine optimal source and detector distributions that result in data sets that are balanced between information content and size. Weight matrices describing the tomographic forward problem were constructed for a range of source and detector distributions and fields of view and were decomposed into their associated singular values. These singular-value spectra were then compared so that we could observe the effects of each parameter on imaging performance. The findings of the SVA were then confirmed by examining reconstructions of simulated and experimental data acquired with the same optode distributions as examined by SVA. It was seen that for a 20-mm target width, which is relevant to the small-animal imaging situation, the source and detector fields of view should be set at approximately 30 mm. Equal numbers of sources and detectors result in the best imaging performance in the parallel-plate geometry and should be employed when logistically feasible. These data provide guidelines for the design of small-animal diffuse optical tomographic imaging systems and demonstrate the utility of SVA as a simple and efficient means of optimizing experimental parameters in problems for which a forward model of the data collection process is available.

Animals↗
PubMed2003

A multimodal nanoparticle for preoperative magnetic resonance imaging and intraoperative optical brain tumor delineation.

The determination of brain tumor margins both during the presurgical planning phase and during surgical resection has long been a challenging task in the therapy of brain tumor patients. Using a model of gliosarcoma with stably green fluorescence protein-expressing 9L glioma cells, we explored a multimodal (near-infrared fluorescent and magnetic) nanoparticle as a preoperative magnetic resonance imaging contrast agent and intraoperative optical probe. Key features of nanoparticle metabolism, namely intracellular sequestration by microglia and the combined optical and magnetic properties of the probe, allowed delineation of brain tumors both by preoperative magnetic resonance imaging and by intraoperative optical imaging. This prototypical multimodal nanoparticle has unique properties that may allow radiologists and neurosurgeons to see the same probe in the same cells and may offer a new approach for obtaining tumor margins.

Animals↗
PubMed2003

In vivo high resolution three-dimensional imaging of antigen-specific cytotoxic T-lymphocyte trafficking to tumors.

Magnetic resonance imaging (MRI) allows noninvasive and three-dimensional visualization of whole organisms over time, and, therefore, would be ideally suited to monitor cell trafficking in vivo. Until now, systemically injected cells had been difficult to visualize by MRI because of relatively inefficient labeling methods. We developed a novel, biocompatible, and physiologically inert nanoparticle (highly derivatized cross-linked iron oxide nanoparticle; CLIO-HD) for highly efficient intracellular labeling of a variety of cell types that now allows in vivo MRI tracking of systemically injected cells at near single-cell resolution. CD8+ cytotoxic T lymphocytes labeled with CLIO-HD were detectable via MRI with a detection threshold of 2 cells/voxel in vitro and approximately 3 cells/voxel in vivo in live mice. Using B16-OVA melanoma and CLIO-HD-labeled OVA-specific CD8+ T cells, we have demonstrated for the first time high resolution imaging of T-cell recruitment to intact tumors in vivo. We have revealed the extensive three-dimensional spatial heterogeneity of T-cell recruitment to target tumors and demonstrated a temporal regulation of T-cell recruitment within the tumor. Significantly, our data indicate that serial administrations of CD8+ T cells appear to home to different intratumoral locations, and may, therefore, provide a more effective treatment regimen than a single bolus administration. Together, these results demonstrate that CLIO-HD is uniquely suited for quantitative repetitive MRI of adoptively transferred cells and that this approach may be particularly useful for evaluating novel cell-based therapies in vivo.

Animals↗
PubMed2003

Real-time imaging of TRAIL-induced apoptosis of glioma tumors in vivo.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to induce apoptosis in neoplastic cells. While many previous studies have been performed in cell culture, the delivery and efficiency of TRAIL variants in vivo is less well established. Using dual substrate/reporter bioluminescence imaging (Fluc: firefly luciferase-luciferin and Rluc: Renilla luciferase-coelenterazine), we tested the efficacy of TRAIL using replication-deficient herpes simplex virus (HSV) type 1 amplicon vectors in gliomas. The cDNA for complete TRAIL and the extracellular domain of TRAIL (aa 114-281) were cloned into HSV amplicons and packaged into helper virus-free vectors. Both forms of TRAIL induced similar degrees of apoptosis in human glioma cells (Gli36) in culture within 24 h of infection with TRAIL amplicon vectors. Growth of tumors stably transfected with Fluc (Gli36fluc+) was readily monitored in vivo by bioluminescence imaging following luciferin administration. HSV amplicon vectors bearing the genes for TRAIL and Rluc injected directly into Gli36fluc(+)-expressing subcutaneous gliomas revealed peak Rluc activity 36 h after intratumoral injection as determined by coelenterazine injection followed by imaging. TRAIL-treated gliomas regressed in size over a period of 4 weeks as compared to the mock-injected gliomas. These results show the efficacy of vector delivered TRAIL in treating tumors in vivo and offer a unique way to monitor both gene delivery and efficacy of TRAIL-induced apoptosis in tumors in vivo in real time by dual enzyme substrate (Rluc/Fluc) imaging.

Animals↗
PubMed2003

Protease sensors for bioimaging.

Optical imaging of specific molecular targets and pathways in vivo has recently become possible through continued developments in imaging equipment, reconstruction algorithms, and more importantly the availability of imaging reporter molecules. These reporter molecules encompass photoproteins expressed in vivo and exogenously administered probes detectable by fluorescence and/or bioluminescence imaging. One particularly enticing aspect of optical imaging is the ability to design activatible probes with inherent amplification. This review summarizes our experience in developing novel near-infrared fluorescent (NIRF) imaging agents that report on protease activities. These agents are designed to be biocompatible, highly activatible, and able to produce bright NIRF following protease cleavage.

Animals↗
PubMed2003

In vivo tracking of neural progenitor cell migration to glioblastomas.

The ability to noninvasively track the migration, engraftment, and proliferation of neural progenitor cells (NPCs) has significant clinical and research implications. The purpose of our study was to explore the macroscopic migratory capabilities of NPCs toward brain tumors after implantation into nude mice. We stably transfected C17.2 NPCs with the firefly luciferase gene (F-luc) and implanted cells into (1) the contralateral brain parenchyma (2 x 10(6) cells), (2) the ventricles (2 x 10(6) cells), (3) the vasculature (1 x 10(5) cells), or (4) the intraperitoneal cavity (5 x 10(6) cells) of mice bearing intracranial gliomas (Gli36). Using serial bioluminescence imaging, migration of parenchymally injected cells was observed across the corpus callosum, first detected at 1 week, with maximal density at the tumor site 2-3 weeks after implantation. Similar patterns were also observed with intraventricular injections; however, tumors were populated earlier, presumably because of the shorter distance to travel. Intravenous injections resulted in more modest tumoral NPC populations, whereas virtually no cells could be identified in tumors after intraperitoneal injection. These results confirm the migratory capability of NPCs over considerable distances and their preferential accumulation in brain tumors on CNS rather than peripheral injection.

Animals↗
PubMed2003

Viral-induced self-assembly of magnetic nanoparticles allows the detection of viral particles in biological media.

Monodisperse magnetic nanoparticles conjugated with virus-surface-specific antibodies self-assemble in the presence of specific viral particles to create supramolecular structures with enhanced magnetic properties, as detected by magnetic resonance methods (NMR/MRI). The observed magnetic relaxation changes that occur upon viral-induced assembly allowed for highly sensitive and selective detection of a virus in complex biological media. The developed method was shown to specifically detect adenovirus-5 and herpes simplex virus-1 at concentrations of 5 viral particles/10 muL without the need of extensive sample preparation. The applications of this new method span from high-throughput NMR detection of viruses in biological samples to potential MR imaging of viral distribution in vivo.

Adenoviridae↗