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Ching-Hsuan Tung

Publications and source records attributed to Ching-Hsuan Tung.

At least 37 records · Page 2Linked to original sources
PubMed2005

In vivo imaging of S-TRAIL-mediated tumor regression and apoptosis.

Therapeutic proteins with specific effector functions play an increasingly important role in drug therapy. For example, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) predominantly kills cancer cells, while sparing normal cells. Here, we report the use of a secreted version of TRAIL as a therapeutic protein that induces apoptosis and kills surrounding cells in vivo, thus resulting in the dramatic reduction of glioma burden in mouse tumor models. Using a caspase-3-activatable aminoluciferin, we were able to show the induction of apoptosis specifically in S-TRAIL vector-infected gliomas. We also show that S-TRAIL-mediated apoptosis and resulting changes in tumor burden can be imaged in the same animal by dual-substrate bioluminescence imaging. The use of S-TRAIL as a therapeutic protein and the ability to image noninvasively both apoptosis and any other cellular events in real time have important clinical implications.

Animals↗
PubMed2005

Near-infrared fluorescent imaging of cerebral thrombi and blood-brain barrier disruption in a mouse model of cerebral venous sinus thrombosis.

An intravital microscopy imaging method was developed to visualize active cerebral thrombus and blood-brain barrier (BBB) disruption using Near Infrared Fluorescent (NIRF) probes. A circular craniotomy was made in CD-1 mice. Thrombi were formed by applying 10%-FeCl3 to the entire exposed superior sagittal sinus (SSS, 5 mm), or to the posterior 2.5 mm of the SSS for 5 mins. Control animals were pretreated with heparin (50 U/kg) before thrombus induction. Three hours after thrombus formation, a FXIIIa-targeted NIRF imaging probe (A15) was intravenously injected, and the SSS was imaged by intravital microscopy. This was followed by injection of indocyanine green (ICG) to assess BBB permeability. The A15 optical probe bound to thrombus, and the fluorescent signal emitted by the bound agent corresponded well with histologically confirmed thrombus. A15 initially remained intravascular, followed by excretion and subsequent decrease in all tissues except for thrombus, where it was retained. The subsequent ICG was also intravascular immediately after injection, but then began to leak into the cerebral parenchyma at 3 to 5 mins. The sites of leakage were adjacent to thrombosed areas. Heparin pretreatment prevented thrombus formation and reduced ICG leakage significantly. This demonstrates the feasibility of simultaneous in vivo monitoring of thrombus and BBB permeability in an animal model of cerebral venous thrombosis.

Animals↗
PubMed2004

Molecular imaging of factor XIIIa activity in thrombosis using a novel, near-infrared fluorescent contrast agent that covalently links to thrombi.

BACKGROUND: Activated factor XIII (FXIIIa) mediates fibrinolytic resistance and is a hallmark of newly formed thrombi. In vivo imaging of FXIIIa activity could further elucidate the role of this molecule in thrombosis and other biological processes and aid in the clinical detection of acute thrombi. METHODS AND RESULTS: An FXIIIa-sensitive near-infrared fluorescence imaging agent (A15) was engineered by conjugating a near-infrared fluorochrome to a peptide ligand derived from the amino terminus of alpha2-antiplasmin. To evaluate the molecular specificity of A15 for FXIIIa, a control agent (C15) was also synthesized by modifying a single key glutamine residue in A15. Fluorescence imaging experiments with A15 demonstrated stronger thrombosis enhancement in human plasma clots in vitro (P<0.001 versus C15 clots and other controls). A15 was found to be highly specific for the active site of FXIIIa and was covalently bound to fibrin. In vivo murine experiments with A15 demonstrated significant signal enhancement in acute intravascular thrombi (P<0.05 versus C15 group). Minimal A15 enhancement was seen in older aged thrombi (>24 hours), consistent with an expected decline of FXIIIa activity over time. Imaging results were confirmed on correlative histopathology and fluorescence microscopy. CONCLUSIONS: A15 is a novel optical imaging agent that is specifically crosslinked to fibrin by FXIIIa, permitting detection of FXIIIa activity in experimental thrombi in vivo. This agent should permit assessment of FXIIIa activity in a broad range of biological processes and could aid in the clinical diagnosis of acute thrombi.

Animals↗
PubMed2004

Inducible release of TRAIL fusion proteins from a proapoptotic form for tumor therapy.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can selectively kill neoplastic cells and control of its activity could enhance tumor therapy. We have developed means to control the secretion of a novel recombinant (r) TRAIL fusion protein using a viral protease. This system uses the endoplasmic reticulum (ER) as a storage depot for rTRAIL, because TRAIL acts by binding to its cognate receptors on the cell surface. We have engineered two TRAIL variants: (a) a secretable form that enhances apoptosis via a bystander effect; and (b) an ER-targeted TRAIL that is retained in the ER until selectively released by the viral protease. Gene delivery can be monitored in vivo by systemic administration of a near infrared fluorescent (NIRF) probe activated by the protease. This study serves as a template for design of recombinant proteins to enhance and control apoptosis of tumor cells via specific viral proteases and for use of viral proteases as in vivo reporters for cancer therapy.

Animals↗
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

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

Fluorescent peptide probes for in vivo diagnostic imaging.

Recently, many novel peptide-based near-infrared (NIR) fluorescent molecular probes have been developed for in vivo biomedical imaging. To report specific information of biological targets, the probes were individually designed according to the unique property or functions of their targets. These peptide-based probes can be classified into targeting, crosslinking, and enzyme-activatable probes. Several of them have been tested in various in vitro and in vivo models, and the obtained imaging information has been applied to disease detection, medical diagnosis, and drug evaluations.

Diagnostic Imaging↗
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

Early diagnosis of osteoarthritis using cathepsin B sensitive near-infrared fluorescent probes.

OBJECTIVE: Osteoarthritis is currently diagnosed utilizing X-ray and MRI-techniques, both of which are based on the morphological changes of tissue. However, once changes are detected, the tissue has an irreversible defect. This study investigates early diagnosis of OA on a molecular basis using a recently developed cathepsin B sensitive near-infrared (NIR) fluorescent probe. METHOD: Twelve male nude mice were induced osteoarthritis by intra-articular injection of collagenase (1.0%, w/v) into the right knee joint. The left knee joint served as the negative control. The cathepsin B NIR probe is activated by arthritis-associated cathepsin B, thus resulting in the emission of an intensive NIR fluorescence signal which can be detected in vivo. NIR fluorescence signals were acquired on an optical imaging system using an excitation wavelength of 610-650 nm and an emission wavelength of 680-720 nm. RESULTS: Mild to moderate degenerative cartilage was observed 1 month after collagenase injection. NIR fluorescence imaging of mice showed approximately a 3-fold difference in signal intensity between osteoarthritic and normal joints 24 h after intravenous injection of the reporter probe. Immunohistochemical evaluation also revealed cathepsin B expression in the arthritic lesion of femorotibial joints, and not in the control contra-lateral knee joints. CONCLUSION: As the cathepsin B activatable NIR fluorescent imaging showed a significant difference between the osteoarthritic and normal joints, the cathepsin B activatable NIR fluorescent probe thus offers a potential new imaging technology for early OA diagnosis.

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↗
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

Arginine containing peptides as delivery vectors.

Recently several membrane translocalizational signals (MTS) have been identified and applied to various applications. These peptide signals, ranging between nine and 30 amino acid residues in length, have the capability of crossing plasma membrane, in addition to delivering other biological molecules into cells. To date, small molecules, peptides, proteins, oligonucleotides, plasmids and even nanometer-sized particles have been delivered. These MTS sequences vary from hydrophobic to purely hydrophilic, and, surprisingly, all of them are able to penetrate cellular membrane in an energy-independent pathway. Potentially, MTS could be used as delivery vectors for a number of therapeutic agents. In this review, we specifically focus on arginine-containing MTS, and their properties, characteristics, in vitro and in vivo applications are discussed in detail.

Arginine↗
PubMed2003

Synthesis and properties of sulfhydryl-reactive near-infrared cyanine fluorochromes for fluorescence imaging.

Near-infrared fluorochromes (NIRF) are useful compounds for diverse biotechnology applications and for in vivo biomedical imaging. Such NIRF must have high quantum yield, be biocompatible, and be conjugatable to a wide variety of proteins, peptides, and other affinity ligands. Here, we describe the synthesis of four new nonsymmetrical sulfhydryl-reactive cyanine NIRF with excellent optical and chemical properties. Each fluorochrome was designed to contain an iodoacetamido group that reacts specifically with sulfhydryl-containing molecules. The synthesized fluorochromes were used to label model peptides and sulfhydryl-containing biomolecules.

Carbocyanines↗
PubMed2002

In vivo imaging of thrombin activity in experimental thrombi with thrombin-sensitive near-infrared molecular probe.

OBJECTIVE: Thrombin, a serine protease, plays an important role in thrombosis as well as other cellular and developmental processes. In this study, we investigated the ability of a novel thrombin-activatable molecular probe to provide in vivo images of thrombin activity in experimental thrombi. METHODS AND RESULTS: The thrombin probe consists of a near-infrared (NIR) fluorochrome attached to a delivery vehicle via a thrombin-specific oligopeptide substrate. In human blood, endogenous thrombin activated the thrombin probe and increased the fluorescence signal by 18-fold (P=0.008). Hirudin, a specific thrombin inhibitor, suppressed probe activation by 82% (P=0.007). Imaging of in vivo thrombin activity was then investigated in acute experimental murine thrombosis models up to 12 hours. After systemic thrombin probe injection, focal NIR fluorescence signal enhancement was rapidly detected within acute and subacute thrombi. In contrast, no thrombosis signal enhancement was seen in similar experiments with a control NIR fluorochrome. CONCLUSIONS: Thrombin activity can be imaged in vivo by using a novel thrombin-activatable and thrombin-specific NIR molecular probe. The thrombin probe could enhance the understanding of the role of thrombin in thrombogenesis and other homeostatic and pathological conditions.

Adult↗
PubMed2002

Coxiella burnetii survival in THP-1 monocytes involves the impairment of phagosome maturation: IFN-gamma mediates its restoration and bacterial killing.

The subversion of microbicidal functions of macrophages by intracellular pathogens is critical for their survival and pathogenicity. The replication of Coxiella burnetii, the agent of Q fever, in acidic phagolysosomes of nonphagocytic cells has been considered as a paradigm of intracellular life of bacteria. We show in this study that C. burnetii survival in THP-1 monocytes was not related to phagosomal pH because bacterial vacuoles were acidic independently of C. burnetii virulence. In contrast, virulent C. burnetii escapes killing in resting THP-1 cells by preventing phagosome maturation. Indeed, C. burnetii vacuoles did not fuse with lysosomes because they were devoid of cathepsin D, and did not accumulate lysosomal trackers; the acquisition of markers of late endosomes and late endosomes-early lysosomes was conserved. In contrast, avirulent variants of C. burnetii were eliminated by monocytes and their vacuoles accumulated late endosomal and lysosomal markers. The fate of virulent C. burnetii in THP-1 monocytes depends on cell activation. Monocyte activation by IFN-gamma restored C. burnetii killing and phagosome maturation as assessed by colocalization of C. burnetii with active cathepsin D. In addition, when IFN-gamma was added before cell infection, it was able to stimulate C. burnetii killing but it also induced vacuolar alkalinization. These findings suggest that IFN-gamma mediates C. burnetii killing via two distinct mechanisms, phagosome maturation, and phagosome alkalinization. Thus, the tuning of vacuole biogenesis is likely a key part of C. burnetii survival and the pathophysiology of Q fever.

Blood Bactericidal Activity↗