Images in clinical medicine. Leprosy.
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Adult↗
Biomedical subjects
C H Tung
Publications and source records attributed to C H Tung.
[Progress in optical imaging].
Different optical imaging technologies have significantly progressed over the last years. Besides advances in imaging techniques and image reconstruction, new "smart" optical contrast agents have been developed which can be used to detect molecular targets (such as endogenous enzymes) in vivo. The combination of novel imaging technologies coupled with smart agents bears great diagnostic potential both clinically and experimentally. This overview outlines the basic principles of optical imaging and summarizes the current state of the art.
Animals↗
In vivo molecular target assessment of matrix metalloproteinase inhibition.
A number of different matrix metalloproteinase (MMP) inhibitors have been developed as cytostatic and anti-angiogenic agents and are currently in clinical testing. One major hurdle in assessing the efficacy of such drugs has been the inability to sense or image anti-proteinase activity directly and non-invasively in vivo. We show here that novel, biocompatible near-infrared fluorogenic MMP substrates can be used as activatable reporter probes to sense MMP activity in intact tumors in nude mice. Moreover, we show for the first time that the effect of MMP inhibition can be directly imaged using this approach within hours after initiation of treatment using the potent MMP inhibitor, prinomastat (AG3340). The developed probes, together with novel near-infrared fluorescence imaging technology will enable the detailed analysis of a number of proteinases critical for advancing the therapeutic use of clinical proteinase inhibitors.
Animals↗
DNA binding chelates for nonviral gene delivery imaging.
Noninvasive in vivo monitoring of gene delivery would provide a critically important information regarding the spatial distribution, local concentration, kinetics of removal and/or biodegradation of the expression vector. We developed a novel approach to noninvasive gene delivery imaging using heterobifunctional peptide-based chelates (PBC) bearing double-stranded DNA-binding groups and a technetium-binding amino acid motif. One of such chelates: Gly-Cys(Acm)-Gly-Cys(Acm)-Gly-Lys(4)-Lys-(N-epsilon-[4-(psoralen-8-yloxy)]butyrate)-NH(2) has been characterized and labeled with reduced (99m)Tc pertechnetate (oxotechnetate). The psoralen moiety (a DNA binding group of PBC) allowed linking to double-stranded DNA upon short-term irradiation with the near UV range light (>320 nm). Approximately 30-40% of added (99m)Tc-labeled PBC was nonextractable and co-eluted with a model pCMV-GFP vector during the gel-permeation chromatography. Nuclear imaging of "naked" DNA and DNA complexes with lipid-based transfection reagents ("lipoplexes") has been performed after systemic or local administration of (99m)Tc-PBC-labeled DNA in mice. Imaging results were corroborated with the biodistribution using (99m)Tc-PBC and (32)P-labeled DNA and lipoplexes. A markedly different biodistribution of (99m)Tc PBC-labeled DNA and lipoplexes was observed with the latter being rapidly trapped in the liver, spleen and lung. (99m)Tc PBC-DNA was used as an imaging tracer during in vivo transfection of B16 melanoma by local injection of "naked" (99m)Tc PBC-DNA and corresponding lipoplexes. As demonstrated by nuclear imaging, (99m)Tc PBC-DNA lipoplexes showed a slower elimination from the site of injection than (99m)Tc PBC-DNA alone. This result correlated with a higher expression of marker mRNA and green fluorescent protein as determined using RT-PCR and immunohistochemistry, respectively.
Animals↗
Optical imaging of matrix metalloproteinase-2 activity in tumors: feasibility study in a mouse model.
PURPOSE: To develop an optical imaging method to determine the expression level of tumoral matrix metalloproteinase-2 (MMP-2) in vivo. MATERIALS AND METHODS: An optical contrast agent was developed that was highly activatable by means of MMP-2-induced conversion. Signal characteristics of the probe were quantified ex vivo with a recombinant enzyme. Animal tumor models were established with MMP-2-positive (human fibrosarcoma cell line, n = 4) and MMP-2-negative (well-differentiated mammary adenocarcinoma, n = 4) tumor cell lines. Both tumors were implanted into nude mice and were optically imaged after intravenous administration of the MMP-2-sensitive probe. RESULTS: The MMP-2-sensitive probe was activated by MMP-2 in vitro, producing up to an 850% increase in near-infrared fluorescent signal intensity. This activation could be blocked by MMP-2 inhibitors. MMP-2-positive tumors were easily identified as high-signal-intensity regions as early as 1 hour after intravenous injection of the MMP-2 probe, while contralateral MMP-2-negative tumors showed little to no signal intensity. A nonspecific control probe showed little to no activation in MMP-2-positive tumors. CONCLUSION: It is feasible to image MMP-2 enzyme activity in vivo by using near-infrared optical imaging technology and "smart" matrix metalloproteinase-sensitive probes.
Animals↗
In vivo imaging of proteolytic enzyme activity using a novel molecular reporter.
The single biggest challenge facing in vivo imaging techniques is to develop biocompatible molecular beacons that are capable of specifically and accurately measuring in vivo targets at the protein, RNA, or DNA level. Our efforts have focused on developing activatable imaging probes to measure specific enzyme activities in vivo. Using cathepsin D as a model target protease, we synthesized a long-circulating, synthetic graft copolymer bearing near-infrared (NIR) fluorochromes positioned on cleavable substrate sequences. In its native state, the reporter probe was essentially nonfluorescent at 700 nm due to energy resonance transfer among the bound fluorochromes (quenching) but became brightly fluorescent when the latter were released by cathepsin D. NIR fluorescence signal activation was linear over at least 4 orders of magnitude and specific when compared with scrambled nonsense substrates. Using matched rodent tumor models implanted into nude mice expressing or lacking the targeted protease, it could be shown that the former generated sufficient NIR signal to be directly detectable and that the signal was significantly different compared with negative control tumors. The developed probes should find widespread applications for real-time in vivo imaging of a variety of clinically relevant proteases, for example, to detect endogenous protease activity in disease, to monitor the efficacy of protease inhibitors, or to image transgene expression.
Animals↗
Synthetic glycopeptide-based delivery systems for systemic gene targeting to hepatocytes.
PURPOSE: To design, synthesize, and test synthetic glycopeptide-based delivery systems for gene targeting to hepatocytes by systemic administration. METHODS: All peptides were synthesized by the solid phase method developed using Fmoc chemistry on a peptide synthesizer. The binding of galactosylated peptides to HepG2 cells and accessibility of the galactose residues on particle surface was demonstrated by a competition assay using 125I-labeled asialoorosomucoid and RCA lectin agglutination assay, respectively. DNA plasmid encoding chloramphenicol acetyl transferase (CAT) gene was complexed with a tri-galactosylated peptide (GM245.3) or tri-galactosylated lipopeptide (GM246.3) in the presence of an endosomolytic peptide (GM225.1) or endosomolytic lipopeptide (GM227.3) to obtain DNA particles of 100-150 nm in size. The plasmid/peptide complexes were added to HepG2 cell cultures or intravenously administered by tail vein injection into normal mice or rats. Plasmid uptake and expression was quantified by qPCR and ELISA, respectively. RESULTS: Multiple antennary glycopeptides that have the ability to condense and deliver DNA plasmid to hepatocytes were synthesized and complexed with DNA plasmid to obtain colloidally stable DNA/peptide complexes. Addition of DNA/GM245.3/GM225.1 peptide complexes (1:3:1 (-/+/-)) to HepG2 cell cultures yielded CAT expression in transfected cells. The transfection efficiency was significantly reduced in the absence of galactose ligand or removal of endosomolytic peptide. Intravenous administration of DNA/GM245.3 peptide complexes (1:0.5 (-/+)) into the tail vein of normal rats yielded DNA uptake in the liver. Substitution of GM245.3 by galactosylated lipopeptide GM246.3 resulted in more stable DNA particles, and a 10-fold enhancement in liver plasmid uptake. CAT expression was detectable in liver following intravenous administration of DNA/GM246.3 complexes. Addition of endosomolytic lipopeptide GM227.3 into the complexes (DNA/ GM246.3/GM227.3 (1:0.5:1 (-/+/-))) yielded a 5-fold increase in CAT expression. Liver expression was 8-fold and 40-fold higher than lung and spleen, respectively, and localized in the hepatocytes only. The transfection efficiency in liver was enhanced by increasing DNA dose and injection volume. The plasmid uptake and expression in liver using DNA/GM246.3/GM227.3 complexes was 100-200-fold higher than DNA formulated in glucose. Tissue examination and serum biochemistry did not show any adverse effect of the DNA/GM246.3/ GM227.3 (1:0.5:1 (-/+/-)) complexes after intravenous delivery. CONCLUSIONS: Gene targeting to hepatocytes was achieved by systemic administration of a well-tolerated synthetic glycopeptide-based delivery system. The transfection efficiency of this glycopeptide delivery system was dependent on peptide structure, endosomolytic activity, colloidal particle stability, and injection volume.
Amino Acid Sequence↗
Tat peptide-derivatized magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells.
The ability to track the distribution and differentiation of progenitor and stem cells by high-resolution in vivo imaging techniques would have significant clinical and research implications. We have developed a cell labeling approach using short HIV-Tat peptides to derivatize superparamagnetic nanoparticles. The particles are efficiently internalized into hematopoietic and neural progenitor cells in quantities up to 10-30 pg of superparamagnetic iron per cell. Iron incorporation did not affect cell viability, differentiation, or proliferation of CD34+ cells. Following intravenous injection into immunodeficient mice, 4% of magnetically CD34+ cells homed to bone marrow per gram of tissue, and single cells could be detected by magnetic resonance (MR) imaging in tissue samples. In addition, magnetically labeled cells that had homed to bone marrow could be recovered by magnetic separation columns. Localization and retrieval of cell populations in vivo enable detailed analysis of specific stem cell and organ interactions critical for advancing the therapeutic use of stem cells.
Animals↗
In vivo imaging of tumors with protease-activated near-infrared fluorescent probes.
We have developed a method to image tumor-associated lysosomal protease activity in a xenograft mouse model in vivo using autoquenched near-infrared fluorescence (NIRF) probes. NIRF probes were bound to a long circulating graft copolymer consisting of poly-L-lysine and methoxypolyethylene glycol succinate. Following intravenous injection, the NIRF probe carrier accumulated in solid tumors due to its long circulation time and leakage through tumor neovasculature. Intratumoral NIRF signal was generated by lysosomal proteases in tumor cells that cleave the macromolecule, thereby releasing previously quenched fluorochrome. In vivo imaging showed a 12-fold increase in NIRF signal, allowing the detection of tumors with submillimeter-sized diameters. This strategy can be used to detect such early stage tumors in vivo and to probe for specific enzyme activity.
Animals↗
Requirement for cell-to-cell contact in Epstein-Barr virus infection of nasopharyngeal carcinoma cells and keratinocytes.
Two nasopharyngeal carcinoma (NPC) cell lines and one keratinocyte cell line could be infected with Epstein-Barr virus (EBV) by cocultivation with virus-producing cells but not by cell-free virus. Using porous culture inserts to manipulate the cell-to-cell contact, we demonstrated that contact between EBV donor B cells and EBV recipient epithelial cells was required for the infection. Cell-to-cell contact not only provided a CR2-independent route of infection but also enhanced CR2-mediated infection in a synergistic manner. Activity of two EBV promoters (Cp and Wp) and expression of EBNA2 were detected in the infected population. A small proportion of the infected cells spontaneously entered an EBV lytic state, which could be induced prominently by chemical treatment. This study provides information on how EBV may infect epithelial cells in vivo, such as at the onset of NPC development.
Cell Line↗
Near-infrared optical imaging of protease activity for tumor detection.
PURPOSE: To build and test an optical imaging system that is sensitive to near-infrared fluorescent molecular probes activated by specific enzymes in tumor tissues in mice. MATERIALS AND METHODS: The imaging system consisted of a source that delivered 610-650-nm excitation light within a lighttight chamber, a 700-nm longpass filter for selecting near-infrared fluorescence emission photons from tissues, and a charge-coupled device (CCD) for recording images. The molecular probe was a biocompatible autoquenched near-infrared fluorescent compound that was activated by tumor-associated proteases for cathepsins B and H. Imaging experiments were performed 0-72 hours after intravenous injection of the probe in nude mice that bore human breast carcinoma (BT-20). RESULTS: The imaging system had a maximal spatial resolution of 60 microns, with a field of view of 14 cm2. The detection threshold of the nonquenched near-infrared fluorescent dye was subpicomolar in the imaging phantom experiments. In tissue, 250 pmol of fluorochrome was easily detected during the 10-second image acquisition. After intravenous injection of the probe into the tumor-bearing animals, tumors as small as 1 mm became detectable because of tumor-associated enzymatic activation of the quenched compound. CONCLUSION: Tumor proteases can be used as molecular targets, allowing visualization of millimeter-sized tumors. The development of this technology, probe design, and optical imaging systems hold promise for molecular imaging, cancer detection, and evaluation of treatment.
Adenocarcinoma↗
Hybridization properties of oligodeoxynucleotide pairs bridged by polyarginine peptides.
The hybridization properties of a series of probes, based on two 9mer oligodeoxynucleotides (designated as I and II) having an appended oligoarginine chain (Rn) to produce peptide-oligonucleotide conjugates or peptide-bridged oligonucleotide pairs (e.g. Rn-I or II-Rn-I), were investigated. For the double-linked probes, we found that the peptide bridge induces the two 9mers to bind complementary single-stranded DNA or RNA targets with substantially enhanced thermal stability. The resulting hybrid with complementary DNA was found to assume a 1:1 complex in the B conformation as judged by UV mixing curves and CD spectroscopy. Complexes of single or double-linked probes with complementary RNA exhibited sensitivity to RNase H digestion. The influence of the identity and chirality of the repeating unit in the bridge, the length of the bridge, the gap size and the salt concentration on the hybridization properties of this new class of oligonucleotide probes was also studied. Our data reveal that these compounds exhibit properties that should prove useful in the development of antisense strategies.
Amino Acid Sequence↗
Application of convergent-beam collimation and simultaneous transmission emission tomography to cardiac single-photon emission computed tomography.
Single-photon emission computed tomography (SPECT) is the most commonly performed imaging technique for perfusion studies of the heart and brain. However, these organs are much smaller than the crystal surface of gamma cameras. SPECT sensitivity and resolution can be improved by using fan- and cone-beam collimators to magnify the image of these organs over a larger portion of the crystal face. Special orbits and reconstruction algorithms must be used with convergent-beam acquisitions to prevent image distortion. Differential attenuation of source activity in the chest is one of the most vexing problems in cardiac SPECT, especially with Thallium-201. Multi-headed cameras equipped with convergent-beam collimators allow a transmission image to be obtained at the same time as emission images. Applying a transmission map of the chest attenuation values to the emission images produces a truer picture of source distribution in the heart. This article reviews the technical problems associated with convergent-beam geometry and simultaneous transmission emission tomography SPECT imaging of the heart.
Algorithms↗
A simulation of emission and transmission noise propagation in cardiac SPECT imaging with nonuniform attenuation correction.
Transmission computed tomography provides information needed for nonuniform attenuation correction of cardiac single photon emission computed tomography (SPECT). Nonuniform attenuation correction is accomplished using an iterative ML-EM algorithm and a projection-backprojection operation that incorporates attenuation factors measured from the reconstructed transmission map. The precision and accuracy of the attenuation corrected emission reconstruction is a function of emission and transmission statistics. This paper presents an error propagation analysis that uses a mathematical cardiac chest phantom to simulate various combinations of total emission counts C and transmission flux I0 under ideal imaging conditions (without geometric response distortion and without scatter). The spatial average, spatial variance, and accuracy measures for a 4 x 4 pixel region in the heart are tabulated after 30 iterations of the ML-EM algorithm. The confidence intervals for these measures were determined from 1000 realizations of reconstructions from projections randomly generated with the same transmission and emission statistics. It can be shown empirically from the simulation results that the spatial %rms uncertainty for the simulated cardiac region has a simple expression: %rms2 = K1/C+K2/I0(2)+B2 where K1 and K2 are least-square estimates based on the simulation results, and B is the measured spatial %rms uncertainty for the simulation at infinite statistics. For a transmission incident flux of 1500 events per projection bin of 0.712 cm and typical clinical emission events totaling 1 x 10(5), the spatial %rms uncertainty is approximately 14%. At clinical transmission and emission statistics, the statistical noise in the simulated attenuation-corrected reconstructions are dominated by the emission statistics.
Algorithms↗
Polyamine-linked oligonucleotides for DNA triple helix formation.
The concept of antigene therapy of disease is based on the ability of an oligonucleotide (the therapeutic agent) to bind to double-stranded genomic DNA (the target associated with the disease). Examples are herein given of the linkage of a series of polyamines to a 21-mer homopyrimidine oligonucleotide. These conjugated 21-mers can each form a triple helix with an appropriate double-stranded homopurine-homopyrimidine DNA according to Hoogsteen base-pairing rules. No triple helix was found when unmodified third strand was used at 10 mM sodium phosphate, pH 6.5, 100 mM sodium chloride solution. In contrast, the spermine-conjugated oligonucleotide had a melting temperature of 42 degrees C. According to the melting profile, the appended spermine moiety was found to affect the Tm only of the triple helix, but not of the subsequent melting of the underlying double helix. The Tm enhancing ability of the spermine-conjugate was found to be better than that of other polyamine-conjugates.
Base Sequence↗
Oligonucleotide-poly-L-ornithine conjugates: binding to complementary DNA and RNA.
On the basis of the reported enhanced antisense activity of polylysine-oligonucleotide conjugates, a synthetic 12-mer oligodeoxyribonucleotide has been coupled at its 5' terminus to a series of positively charged (delta-ornithine)n cysteine peptides. Binding between the nucleic acid-peptide conjugate and its complementary DNA target sequence was detected by the impact of complexation on the melting temperature (Tm). It was found that the Tm for the nucleic acid-peptide gradually increased with increasing net charge on the conjugated peptide. Site-directed cleavage with RNase H demonstrates that the peptide-modified oligomer also hybridizes with its RNA target sequence. Increased affinity for target mRNA with net charge was shown by a cell-free translation arrest assay.
Base Sequence↗
Preparation and physical properties of conjugates of oligodeoxynucleotides with poly(delta)ornithine peptides.
Oligodeoxynucleotides (12-mers) having either a 3' aminolinker or both 3' and 5' aminolinker groups were synthesized and then reacted with N-iodoacetoxysuccinimide. Separately, a series of peptides, consisting of cysteine carboxyamide and a varying number of residues of ornithine coupled through their side-chain amino groups, was prepared, leaving on the final Fmoc protecting group. Reaction of each Fmoc-peptide with an activated oligodeoxynucleotide yielded the desired conjugates, which were purified by an Fmoc-on/Fmoc-off two-step reversed-phase HPLC procedure. On hybridization with a complementary unmodified 12-mer oligodeoxynucleotide, it was found that there is a gradual increase in melting temperature with the number of ornithine residues, whereas the appended peptide did not perturb the B form of the duplex.
Base Sequence↗
Design, synthesis and assay of tetrapeptide-acridine mimics of ribonuclease.
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Acridines↗