A novel ELISA test for laboratory diagnosis of Blastocystis spp. in human stool specimens.

Detection of Blastocystis is routinely performed by microscopy, culture, and formyl-ether (ethyl acetate) concentration technique (FECT). Yet, these methods require special skilled personnel, are time consuming, and often involve processing that may cause misdiagnosis. The aim of this work is to demonstrate the usefulness of a newly introduced ELISA test for the detection of Blastocystis antigens in stool samples (CoproELISA(TM) Blastocystis, Savyon Diagnostics) as a proper alternative to currently used methods, especially microscopy.

Interrogation of inhibitor of nuclear factor κB α/nuclear factor κB (IκBα/NF-κB) negative feedback loop dynamics: from single cells to live animals in vivo.

Full understanding of the biological significance of negative feedback processes requires interrogation at multiple scales as follows: in single cells, cell populations, and live animals in vivo. The transcriptionally coupled IκBα/NF-κB negative feedback loop, a pivotal regulatory node of innate immunity and inflammation, represents a model system for multiscalar reporters. Using a κB(5)→IκBα-FLuc bioluminescent reporter, we rigorously evaluated the dynamics of ΙκBα degradation and subsequent NF-κB transcriptional activity in response to diverse modes of TNFα stimulation.

Flavokawain B, the hepatotoxic constituent from kava root, induces GSH-sensitive oxidative stress through modulation of IKK/NF-kappaB and MAPK signaling pathways.

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.

T-cell activation promotes tumorigenesis in inflammation-associated cancer.

Chronic inflammation has long been associated with a wide range of malignancies, is now widely accepted as a risk factor for development of cancer, and has been implicated as a promoter of a variety of cancers including hematopoietic malignancies. We have described a mouse model uniquely suited to examine the link between inflammation and lymphoma in which the Tax oncogene, expressed in activated T and NK cells, perpetuates chronic inflammation that begins as microscopic intraepithelial lesions and develops into inflammatory nodules, subcutaneous tumors, and large granular lymphocytic leukemia. The use of bioluminescent imaging in these mice has expanded our ability to interrogate aspects of inflammation and tumorigenesis non-invasively.

Bioluminescence imaging of myeloperoxidase activity in vivo.

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.

Imaging spontaneous tumorigenesis: inflammation precedes development of peripheral NK tumors.

Early events in tumor development are spontaneous, microscopic, and affected by the microenvironment. We developed a mouse model of spontaneous lymphoma in which malignant transformation is coupled with light emission that can be detected noninvasively using bioluminescent imaging. The human T-cell leukemia virus (HTLV) type 1 transcriptional transactivator Tax is an oncogene sufficient to produce lymphoma in transgenic animal models.

Identification of a ligand-induced transient refractory period in nuclear factor-kappaB signaling.

In response to a variety of extracellular ligands, nuclear factor-kappaB (NF-kappaB) signaling regulates inflammation, cell proliferation, and apoptosis. It is likely that cells are not continuously exposed to stimulating ligands in vivo but rather experience transient pulses. To study the temporal regulation of NF-kappaB and its major regulator, inhibitor of NF-kappaBalpha (IkappaBalpha), in real time, we utilized a novel transcriptionally coupled IkappaBalpha-firefly luciferase fusion reporter and characterized the dynamics and responsiveness of IkappaBalpha processing upon a short 30-s pulse of tumor necrosis factor alpha (TNFalpha) or a continuous challenge of TNFalpha following a 30-s preconditioning pulse.

Veni, vidi, vici: in vivo molecular imaging of immune response.

"I came, I saw, I conquered," Julius Caesar proclaimed, highlighting the importance of direct visualization as a winning strategy. Continuing the "From the Field" series (see Editorial [2007] 26, 131), Gross et al. summarize how modern molecular imaging techniques can successfully dissect the complexities of immune response in vivo.

Continuous delivery of D-luciferin by implanted micro-osmotic pumps enables true real-time bioluminescence imaging of luciferase activity in vivo.

Bioluminescence imaging (BLI) of luciferase reporters in small animal models offers an attractive approach to monitor regulation of gene expression, signal transduction, and protein-protein interactions, as well as following tumor progression, cell engraftment, infectious pathogens, and target-specific drug action. Conventional BLI can be repeated within the same animal after bolus reinjections of a bioluminescent substrate. However, intervals between image acquisitions are governed by substrate pharmacokinetics and excretion, therefore restricting temporal resolution of reinjection protocols to the order of hours, limiting analyses of processes in vivo with short time constants.

Molecular imaging strategies for drug discovery and development.

Recent advances in non-invasive molecular imaging provide exciting opportunities for discovery, validation and development of novel therapeutics. As the arsenal of detection devices and strategies, injectable probes, genetically encoded reporters and animal models rapidly expands, molecular imaging is becoming indispensable for drug discovery and development. Not only do such strategies reduce the time, cost and workload associated with conventional destructive end-point assays, but they also enable spatial and temporal monitoring of in vivo gene expression, signaling pathways, biochemical reactions and targets as they relate to the pharmacokinetics and pharmacodynamics of novel drugs.

Spectral unmixing of multicolored bioluminescence emitted from heterogeneous biological sources.

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.

Monitoring proteasome activity in cellulo and in living animals by bioluminescent imaging: technical considerations for design and use of genetically encoded reporters.

The ubiquitin-proteasome pathway is the central mediator of regulated proteolysis, instrumental for switching on and off a variety of signaling cascades. Deregulation of proteasomal activity or improper substrate recognition and processing by the ubiquitin-proteasome machinery may lead to cancer, stroke, chronic inflammation, and neurodegenerative diseases. Quantifying total and substrate-specific proteasome activity in intact cells and living animals would enable analysis in vivo of proteasomal regulation and facilitate the screening and validation of potential modulators of the proteasome or its substrates.

Real-time imaging of ligand-induced IKK activation in intact cells and in living mice.

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.

Spying on cancer: molecular imaging in vivo with genetically encoded reporters.

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.

Monitoring photodynamic therapy of solid tumors online by BOLD-contrast MRI.

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.

Local photodynamic therapy (PDT) of rat C6 glioma xenografts with Pd-bacteriopheophorbide leads to decreased metastases and increase of animal cure compared with surgery.

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.