The myeloperoxidase (MPO) system of activated phagocytes is central to normal host defense mechanisms, and dysregulated MPO contributes to the pathogenesis of inflammatory disease states ranging from atherosclerosis to cancer. Here we show that upon systemic administration, the small molecule luminol enables noninvasive bioluminescence imaging (BLI) of MPO activity in vivo. Luminol-BLI allowed quantitative longitudinal monitoring of MPO activity in animal models of acute dermatitis, mixed allergic contact hypersensitivity, focal arthritis and spontaneous large granular lymphocytic tumors. Bioluminescence colocalized with histological sites of inflammation and was totally abolished in gene-deleted Mpo −/− mice, despite massive tissue infiltration of neutrophils and activated eosinophils, indicating that eosinophil peroxidase did not contribute to luminol-BLI in vivo. Thus, luminol-BLI provides a noninvasive, specific and highly sensitive optical readout of phagocyte-mediated MPO activity in vivo and may enable new diagnostic applications in a wide range of acute and chronic inflammatory conditions. The heme-containing enzyme MPO is a key component of the cytotoxic armamentarium of phagocytic white blood cells 1,2 . MPO is by far the most abundant protein product in azurophilic granules of neutrophils (5%), constitutes approximately 1% of monocyte protein and is found in the lysosomes of other polymorphonuclear leukocytes and macrophages. The phagosomal oxidative burst is initiated by a stimulus-dependent assembly of the phagocytic NADPH oxidase (Phox), a multimeric protein complex located on the phagosomal membrane. Phox then reduces molecular oxygen to produce superoxide anion (O 2•− ), which further dismutates to yield the relatively unreactive hydrogen peroxide (H 2 O 2 ) 1 . Upon phagocytic activation, large quantities of active MPO are secreted into phagosomes, catalyzing the production of highly bactericidal hypochlorous acid (HOCl) with H 2 O 2 and chloride ions (Cl − ) as substrates (Fig. 1a) 1 .
Antivascular photodynamic therapy (PDT) of tumors with palladium-bacteriopheophorbide (TOOKAD) relies on in situ photosensitization of the circulating drug by local generation of cytotoxic reactive oxygen species, which leads to rapid vascular occlusion, stasis, necrosis and tumor eradication. Intravascular production of reactive oxygen species is associated with photoconsumption of O(2) and consequent evolution of paramagnetic deoxyhemoglobin. In this study we evaluate the use of blood oxygenation level-dependent (BOLD) contrast magnetic resonance imaging (MRI) for real-time monitoring of PDT efficacy. Using a solid tumor model, we show that TOOKAD-PDT generates appreciable attenuation (25-40%) of the magnetic resonance signal, solely at the illuminated tumor site. This phenomenon is independent of, though augmented by, ensuing changes in blood flow. These results were validated by immunohistochemistry and intravital microscopy. The concept of photosensitized BOLD-contrast MRI may have intraoperative applications in interactive guidance and monitoring of antivascular cancer therapy, PDT treatment of macular degeneration, interventional cardiology and possibly other biomedical disciplines.
The transcription factor NF-kappaB is a key regulator of cellular activation, proliferation and apoptosis. Defects in the NF-kappaB pathway contribute to a broad array of malignant, neurodegenerative and chronic inflammatory diseases. IKK-dependent IkappaB alpha degradation by the 26S proteasome is a critical NF-kappaB regulatory control point, which is emerging as an important target for drug development. To directly monitor regulation of IKK activation in intact organisms, we engineered an IkappaB alpha-firefly luciferase (IkappaB alpha-FLuc) fusion reporter. In cultured cells and living animals, the reporter provided a continuous, noninvasive readout of the kinetics of ligand-induced IKK activation and the pharmacodynamics of selective inhibitors of both IKK and the 26S proteasome. This IkappaB alpha-FLuc reporter now permits continuous readout of IKK activation in vivo, facilitates development and validation of target-specific therapeutics, and complements conventional NF-kappaB transcriptional reporters for more complete temporal and regional investigations of the NF-kappaB signaling pathway in health and disease.
Genetically encoded imaging reporters introduced into cells and transgenic animals enable noninvasive, longitudinal studies of dynamic biological processes in vivo. The most common reporters include firefly luciferase (bioluminescence imaging), green fluorescence protein (fluorescence imaging), herpes simplex virus-1 thymidine kinase (positron emission tomography), and variants with enhanced spectral and kinetic properties. When cloned into promoter/enhancer sequences or engineered into fusion proteins, imaging reporters allow transcriptional regulation, signal transduction, protein-protein interactions, oncogenic transformation, cell trafficking, and targeted drug action to be spatiotemporally resolved in vivo. Spying on cancer with genetically encoded imaging reporters provides insight into cancer-specific molecular machinery within the context of the whole animal.
Photodynamic therapy (PDT), locally applied to solid C6 rat glioma tumors in the foot of CD1 nude mice, eradicated the primary tumor and also decreased the rate of groin and lung metastases. Pd-Bacteriopheophorbide (Pd-Bpheid), a novel photosensitizer synthesized in our laboratory, was used in our study. The primary lesion in the hind leg was treated by an i.v. injection of 5 mg/kg of Pd-Bpheid and immediate illumination (650 -800 nm, 360 J/cm 2 ). This protocol and the surgical amputation of the leg were compared for local and metastasis responses. Following PDT, hemorrhage, inflammation with tumor necrosis and flattening were observed and histologically verified in the photodynamically treated tumor. Whereas local tumor control rates were up to 64% following PDT, in surgically treated animals, local tumor control was absolute. The rates of metastases in the groin and the lungs were at least 12-fold lower in the photodynamically treated animals compared with untreated or surgerytreated groups. The overall cure rates after PDT or surgery were 36% and 6%, respectively, at 8 weeks. These findings suggest that local PDT with Pd-Bpheid, which acts primarily on the tumor vasculature, efficiently eradicates the solid C6 tumors. In addition, the local PDT of the primary lesion has beneficial therapeutic effects on remote C6 metastasis, which is not obtained with surgery. It is therefore suggested, that although surgery is highly efficient for the immediate removal of the primary tumor, it lacks such systemic, therapeutic effects on distant metastases. Pd-Bpheid-PDT may thus offer a potentially superior curative therapy for C6 glioma tumors in the limb by eradicating the target tumor and by reducing the rate of metastasis in the groin and lung, possibly due to innate immunity. © 2002 Wiley-Liss, Inc. Key words: photodynamic therapy; surgery; Pd-bacteriopheophorbide; C6 glioma tumor; metastasisPhotodynamic therapy (PDT) is based on the destruction of tumors by cytotoxic reactive oxygen species (ROS) produced upon local tumor illumination in patients administered with a photosensitizer. 1-3 Following health agency approval for photofrin-based PDT in many countries, this anti-cancer treatment modality entered clinical use for the local treatment of an increasing number of indications including skin, esophageal, lung, gastric, cervical and bladder cancers. 4 PDT is usually considered a local anti-tumor treatment modality. However, reports from several laboratories suggest that PDT also induces beneficial systemic effects. Following in vitro hematoporphyrin-based PDT, adhesiveness and metastatic potential decline in DHD-K12-cultured colon carcinoma cells. Moreover, intravenous or s.c. injection of these PDT-treated cells to rats resulted in a reduced number of lung metastases compared with untreated cell injection. 5,6 Although this observation may be due to local photodynamic damage, the potential beneficial effect may be viewed as systemic. Other in vivo studies showed that local PDT with various photosensitizers mediates ...
A wide variety of bioluminescent luciferase proteins are available for use in transcriptional or biochemical reporter assays. However, spectral overlap normally prevents them from being monitored simultaneously. To address this problem, a Java plug-in for ImageJ was written to deconvolute bioluminescent images composed of signals from multiple luciferases. The methodology was validated by testing the program with both simulated and real luciferase images. Bioluminescent images were acquired using a CCD camera equipped with optical filters, and the images were deconvoluted using the ImageJ plug-in. HeLa cells were transfected with either click beetle red luciferase (CBR), click beetle green luciferase (CBG99), or Renilla luciferase (Rluc), and mixed lysates were imaged in varying proportions in a 96-well plate to biochemically validate the methodology. After spectral deconvolution, the predicted, pure luciferase signals could be recovered with maximal cross-talk errors of +/-1.5%. In addition, live cells expressing CBR, CBG99, and Rluc were simultaneously imaged and deconvoluted in 96-well plates to demonstrate the feasibility of applying this methodology to high-throughput applications. Finally, multicolor transcriptional and posttranslational modification reporters were simultaneously imaged and shown to deconvolute normalized IkappaBeta kinase activity in longitudinal assays. Thus, our software provided a rapid, simple, and accurate method for simultaneously measuring multiple bioluminescent reporters in living cells.
Genetically encoded imaging reporters introduced into cells and transgenic animals enable noninvasive, longitudinal studies of dynamic biological processes in vivo. The most common reporters include firefly luciferase (bioluminescence imaging), green fluorescence protein (fluorescence imaging), herpes simplex virus-1 thymidine kinase (positron emission tomography), and variants with enhanced spectral and kinetic properties. When cloned into promoter/enhancer sequences or engineered into fusion proteins, imaging reporters allow transcriptional regulation, signal transduction, protein-protein interactions, oncogenic transformation, cell trafficking, and targeted drug action to be spatiotemporally resolved in vivo. Spying on cancer with genetically encoded imaging reporters provides insight into cancer-specific molecular machinery within the context of the whole animal.
Kava (Piper methysticum Foster, Piperaceae) organic solvent-extract has been used to treat mild to moderate anxiety, insomnia, and muscle fatigue in Western countries, leading to its emergence as one of the 10 best-selling herbal preparations. However, several reports of severe hepatotoxicity in kava consumers led the U.S. Food and Drug Administration and authorities in Europe to restrict sales of kava-containing products. Herein we demonstrate that flavokawain B (FKB), a chalcone from kava root, is a potent hepatocellular toxin, inducing cell death in HepG2 (LD(50)=15.3 ± 0.2 μM) and L-02 (LD(50)=32 μM) cells. Hepatocellular toxicity of FKB is mediated by induction of oxidative stress, depletion of reduced glutathione (GSH), inhibition of IKK activity leading to NF-κB transcriptional blockade, and constitutive TNF-α-independent activation of mitogen-activated protein kinase (MAPK) signaling pathways, namely, ERK, p38, and JNK. We further demonstrate by noninvasive bioluminescence imaging that oral consumption of FKB leads to inhibition of hepatic NF-κB transcriptional activity in vivo and severe liver damage. Surprisingly, replenishment with exogenous GSH normalizes both TNF-α-dependent NF-κB as well as MAPK signaling and rescues hepatocytes from FKB-induced death. Our data identify FKB as a potent GSH-sensitive hepatotoxin, levels of which should be specifically monitored and controlled in kava-containing herb products.
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