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Biomedical subjects

Lee Josephson

Publications and source records attributed to Lee Josephson.

At least 37 records · Page 2Linked to original sources
PubMed2004

Optimal modification of annexin V with fluorescent dyes.

The many uses of chemically modified annexin Vs necessitate an understanding of the optimal degree of modification and modification sites of the protein. When reacted with the N-hydroxysuccinimide ester of Cy5.5, annexin V with one modification per mole of protein retained its affinity for phosphatidylserine of apoptotic cells, whereas modification with two dyes per mole of protein caused a complete loss of activity. A tryptic digest LC/MS method was used to identify the modification sites as either of two closely spaced lysine residues, in position 286 or 290. The crystal structure indicated the location of these lysines was distal to the phosphatidylserine binding sites on annexin V. These results can be used to develop active or inactive fluorescent control annexin V proteins and to suggest strategies for attaining higher levels of modification with retention of bioactivity.

Annexin A5↗
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

Molecular imaging of cell-based therapies.

This article focuses on molecular imaging of novel cell-based therapies, particularly stem cell therapies and the adoptive transfer of immunocytes. The animal models,potential clinical applications, and likely future prospects of these therapies are discussed in the context of imaging.

Adoptive Transfer↗
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

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↗
PubMed2003

Near-infrared fluorescent imaging of tumor apoptosis.

Noninvasive imaging using radioactive annexin V is an emerging strategy for the assessment of cell death in vivo (F. G. Blankenberg, and H. W. Strauss. Apoptosis, 6: 117-123, 2001.). Therefore, we investigated whether annexin V labeled with the fluorophore Cy5.5 (Cy) could serve as a probe for imaging of tumor apoptosis using near infrared fluorescence (NIRF). We prepared active Cy-annexin (an equimolar dye:protein ratio) that bound to apoptotic Jurkat T cells and an inactive Cy-annexin probe (>2 dyes/mol protein) that did not. Active Cy annexin was used to image a 9L gliosarcoma, constitutively expressing green fluorescent protein marker, and the CR8 variant of Lewis lung carcinoma, stably transfected to express DsRed2. The expression of transfected fluorescent protein provided an indication of tumor margins and a means of defining tumor-associated NIRF signal intensity with both tumor models. Tumors were imaged with and without cyclophosphamide treatment. In both tumor models active Cy-annexin V tumor NIRF signal increased two to three times after the treatment. Tumor NIRF signal developed by 75 min after active Cy-annexin injection and remained for a 20-h observation period. Inactive annexin V was used as a control in the CR8 carcinoma experiments and resulted in a low nonspecific signal. With the 9L gliomosacrcoma model, active Cy-annexin V bound to both tumor cells (Cy-annexin V staining only) and endothelial cells (costained with Cy-annexin V and antibody to the endothelial marker CD31). Our results demonstrate that active Cy-annexin can be used as a NIRF probe to image apoptosis from outside an intact living animal and may provide nonradioactive method of measuring the antiproliferative effects of cancer chemotherapeutic regimens.

Animals↗
PubMed2003

Pan and sentinel lymph node visualization using a near-infrared fluorescent probe.

A number of different types of agents have been employed to aid in the visualization of lymph nodes, particularly the sentinel lymph node, and to decrease the tissue destruction associated with the diagnosis of nodal metastases. The current study was performed to see if a novel macromolecular near-infrared fluorescent (NIRF) probe could be used to visualize lymph nodes after intravenous administration (pan-node visualization) or subcutaneous administration (sentinel node visualization), and serve as method for guiding dissection with interventional radiologic and surgical procedures. Cy5.5-PGC, the near-infrared dye Cy5.5 coupled to a protected graft copolymer (PGC), was injected (i.v. or s.c.) into nude mice. Twenty-four hours later white light and NIRF images were obtained on (i) the live animal, (ii) a partially dissected animal, and (iii) tissue specimens. With Cy5.5-PGC administered intravenously, axillary nodes were visualized from outside a living mouse. With partial dissection, iliac and aortic nodes were visible as concentrated foci of high-intensity NIRF signals. With subcutaneous injection in the front extremity, axillary and brachial nodes draining the injection site were easily visualized. NIRF imaging provides a nonradioactive method of visualizing lymph nodes through layers of tissue that can be employed with intravenous or subcutaneous injection.

Animals↗
PubMed2002

Magnetic relaxation switches capable of sensing molecular interactions.

Highly sensitive, efficient, and high-throughput biosensors are required for genomic and proteomic data acquisition in complex biological samples and potentially for in vivo applications. To facilitate these studies, we have developed biocompatible magnetic nanosensors that act as magnetic relaxation switches (MRS) to detect molecular interactions in the reversible self-assembly of disperse magnetic particles into stable nanoassemblies. Using four different types of molecular interactions (DNA-DNA, protein-protein, protein-small molecule, and enzyme reactions) as model systems, we show that the MRS technology can be used to detect these interactions with high efficiency and sensitivity using magnetic relaxation measurements including magnetic resonance imaging (MRI). Furthermore, the magnetic changes are detectable in turbid media and in whole-cell lysates without protein purification. The developed magnetic nanosensors can be used in a variety of biological applications such as in homogeneous assays, as reagents in miniaturized microfluidic systems, as affinity ligands for rapid and high-throughput magnetic readouts of arrays, as probes for magnetic force microscopy, and potentially for in vivo imaging.

Base Sequence↗
PubMed2002

DNA-based magnetic nanoparticle assembly acts as a magnetic relaxation nanoswitch allowing screening of DNA-cleaving agents.

Monodisperse magnetic nanoparticles conjugated with complementary oligonucleotide sequences self-assemble into stable magnetic nanoassemblies resulting in a decrease of the spin-spin relaxation times (T2) of neighboring water protons. When these nanoassemblies are treated with a DNA cleaving agent, the nanoparticles become dispersed, switching the T2 of the solution back to original values. These qualities render the developed nanoparticles and their nanoassemblies as magnetic relaxation switches capable of screening for DNA-cleaving compounds by magnetic resonance methods such as MRI and NMR.

Biosensing Techniques↗
PubMed2002

Detection of lymph node metastases by contrast-enhanced MRI in an experimental model.

Lymph node size, the accumulation of a nodal lymphotrophic contrast agent (LCDIO), and MRI were compared as methods for detecting nodal metastases in an experimental murine model. Lymph node metastases (B16-F1 melanoma expressing green fluorescent protein (GFP) and C57BL/6 mice) were generated to obtain a wide spectrum of nodes, including normal nodes and nodes bearing micrometastases, small metastases, or large metastases. Nodal uptake of LCDIO was measured using (111)Indium-labeled LCDIO and was found to be lower in micrometastastic nodes (4.20 +/- 1.4%ID/gm) than in normal nodes (8.60 +/- 0.22% ID dose/gram, P < 0.005). Nodal tumor burden was quantified from the amount of GFP present in nodes measured using the Western blot method, and was found to correlate with the decrease of LCDIO uptake. By MRI, nodes bearing small and large metastases contained regions of high signal intensity (SI) that corresponded to the visual pattern of tumor in nodes. Micrometastatic nodes were distinguishable from normal nodes based on a diffuse pattern of inhomogeneous SI. The signal-to-background ratio (SBR) of normal nodes (0.0112 +/- 0.0061) was different from micrometastatic nodes (0.179 +/- 0.080, P < 0.00046) and nodes bearing small metastases (0.723 +/- 0.269, P < 0.00013), with high degrees of significance.

Animals↗
PubMedDate not supplied

The transferrin receptor: a potential molecular imaging marker for human cancer.

Noninvasive imaging of differences between the molecular properties of cancer and normal tissue has the potential to enhance the detection of tumors. Because overexpression of endogenous transferrin receptor (TfR) has been qualitatively described for various cancers and is presumably due to malignant transformation of cells, TfR may represent a suitable target for application of molecular imaging technologies to increase detection of smaller tumors. In the work reported here, investigation into the biology of this receptor using electron microscopy has demonstrated that iron oxide particles targeted to TfR are internalized and accumulate in lysosomal vesicles within cells. Biochemical analysis of the interaction of imaging probes with cells overexpressing the TfR demonstrated that the extent of accumulation, and therefore probe efficacy, is dependent on the nature of the chemical cross-link between transferrin and the iron oxide particle. These data were utilized to design and synthesize an improved imaging probe. Experiments demonstrate that the novel magnetic resonance imaging (MRI) probe is sensitive enough to detect small differences in endogenous TfR expression in human cancer cell lines. Quantitative measurement of TfR overexpression in a panel of 27 human breast cancer patients demonstrated that 74% of patient cancer tissues overexpressed the TfR and that the sensitivity of the new imaging agent was suitable to detect TfR overexpression in greater than 40% of these cases. Based on a biochemical and cell biological approach, these studies have resulted in the synthesis and development of an improved MRI probe with the best in vitro and in vivo imaging properties reported to date.

Animals↗
PubMedDate not supplied

Optical imaging of apoptosis as a biomarker of tumor response to chemotherapy.

A rapid and accurate assessment of the antitumor efficacy of new therapeutic drugs could speed up drug discovery and improve clinical decision making. Based on the hypothesis that most effective antitumor agents induce apoptosis, we developed a near-infrared fluorescent (NIRF) annexin V to be used for optical sensing of tumor environments. To demonstrate probe specificity, we developed both an active (i.e., apoptosis-recognizing) and an inactive form of annexin V with very similar properties (to account for nonspecific tumor accumulation), and tested the agents in nude mice each bearing a cyclophosphamide (CPA) chemosensitive (LLC) and a chemoresistant LLC (CR-LLC). After injection with active annexin V, the tumor-annexin V ratio (TAR; tumor NIRF/background NIRF) for untreated mice was 1.22+/-0.34 for LLC and 1.43+/-0.53 for CR-LLC (n=4). The LLC of CPA-treated mice had significant elevations of TAR (2.56+/-0.29, P=.001, n=4), but only a moderate increase was obtained for the CR-LLC (TAR=1.89+/-0.19, P=.183). The in vivo measurements correlated well with terminal deoxyribosyl transferase-mediated dUTP nick end labeling indexes. When inactive Cy-annexin V was used, with or without CPA treatment and in both CCL and CR-CCL tumors, tumor NIRF values ranged from 0.91 to 1.17 (i.e., tumor were equal to background). We conclude that active Cy-annexin V and surface reflectance fluorescence imaging provide a nonradioactive, semiquantitative method of determining chemosensitivity in LLC xenografts. The method maybe used to image pharmacologic responses in other animal models and, potentially, may permit the clinical imaging of apoptosis with noninvasive or minimally invasive instrumentation.

Animals↗
PubMedDate not supplied

High throughput magnetic resonance imaging for evaluating targeted nanoparticle probes.

The ability to image specific molecular targets in vivo would have significant impact in allowing earlier disease detection and in tailoring molecular therapies. One of the rate-limiting steps in the development of novel compounds as reporter probes has been the lack of cell-based, biologically relevant, high throughput screening methods. Here we describe the development and validation of magnetic resonance imaging (MRI) as a technique to rapidly screen compounds that are potential MR reporter agents for their interaction with specific cellular targets. We show that MR imaging can (1) evaluate thousands of samples simultaneously and rapidly, (2) provide exceedingly accurate measurements, and (3) provide receptor binding/internalization data as validated by radioactive assays. The technique allows the screening of libraries of peptide-nanoparticle conjugates against target cells and the identification of conjugates that may be subsequently used as reporter agents in vivo. The technology should greatly accelerate the development of target-specific or cell-specific MR contrast agents.

Cell Line↗
PubMedDate not supplied

Magnetic resonance imaging of inducible E-selectin expression in human endothelial cell culture.

Covalent conjugates of the cross-linked iron oxide nanoparticles (CLIO) and high-affinity (K(d)(app) = 8.5 nM) anti-human E-selectin (CD62E) F(ab')(2) fragments were prepared and tested in vitro to establish feasibility of endothelial proinflammatory marker magnetic resonance (MR) imaging. The conjugates were obtained by using thiol-disulfide exchange reaction between 3-(2-pyridyl)propionyl-CLIO and S-acetylthioacetate-modified F(ab')(2) fragments. The purified CLIO-F(ab')(2) conjugates (average hydrodynamic diameter 40.6 nm) were used in experiments with the live human endothelial umbilical vein cells (HUVEC). Cells treated with IL-1 beta expressed E-selectin and showed a 100-200 times higher binding of CLIO particles (83-104 ng iron/million cells) than control cells. The binding resulted in a high superparamagnetism of HUVEC with the transverse water proton relaxation time (T2) decrease to 30-40 ms in cell precipitates. Cells did not bind/internalize CLIO-F(ab')(2) conjugates prepared using a control fragment or nonconjugated iron oxide particles before or after treatment with IL-1 beta. MR imaging of cells showed a highly specific T2-weighted signal darkening associated with cells treated with IL-1 beta and incubated with anti-E selectin. Demonstration of MR imaging of E-selectin expression justifies further development of MR-targeted agents for monitoring tumor vascular endothelial proliferation, angiogenesis, and atherosclerosis.

Cells, Cultured↗