The immune system responds to pathogens by a variety of pattern recognition molecules such as the Toll-like receptors (TLRs), which promote recognition of dangerous foreign pathogens. However, recent evidence indicates that normal intestinal microbiota might also positively influence immune responses, and protect against the development of inflammatory diseases. One of these elements may be short-chain fatty acids (SCFAs), which are produced by fermentation of dietary fibre by intestinal microbiota. A feature of human ulcerative colitis and other colitic diseases is a change in 'healthy' microbiota such as Bifidobacterium and Bacteriodes, and a concurrent reduction in SCFAs. Moreover, increased intake of fermentable dietary fibre, or SCFAs, seems to be clinically beneficial in the treatment of colitis. SCFAs bind the G-protein-coupled receptor 43 (GPR43, also known as FFAR2), and here we show that SCFA-GPR43 interactions profoundly affect inflammatory responses. Stimulation of GPR43 by SCFAs was necessary for the normal resolution of certain inflammatory responses, because GPR43-deficient (Gpr43(-/-)) mice showed exacerbated or unresolving inflammation in models of colitis, arthritis and asthma. This seemed to relate to increased production of inflammatory mediators by Gpr43(-/-) immune cells, and increased immune cell recruitment. Germ-free mice, which are devoid of bacteria and express little or no SCFAs, showed a similar dysregulation of certain inflammatory responses. GPR43 binding of SCFAs potentially provides a molecular link between diet, gastrointestinal bacterial metabolism, and immune and inflammatory responses.

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Regulation of inflammatory responses by gut microbiota and chemoattractant receptor GPR43

New technologies for imaging molecules, particularly optical technologies, are increasingly being used to understand the complexity, diversity and in vivo behaviour of cancers. 'Omic' approaches are providing comprehensive 'snapshots' of biological indicators, or biomarkers, of cancer, but imaging can take this information a step further, showing the activity of these markers in vivo and how their location changes over time. Advances in experimental and clinical imaging are likely to improve how cancer is understood at a systems level and, ultimately, should enable doctors not only to locate tumours but also to assess the activity of the biological processes within these tumours and to provide 'on the spot' treatment.

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Imaging in the era of molecular oncology

A review of NIR dyes in cancer targeting and imaging.

Medical imaging technologies have undergone explosive growth over the past few decades and now play a central role in clinical oncology. But the truly transformative power of imaging in the clinical management of cancer patients lies ahead. Today, imaging is at a crossroads, with molecularly targeted imaging agents expected to broadly expand the capabilities of conventional anatomical imaging methods. Molecular imaging will allow clinicians to not only see where a tumor is located in the body, but also to visualize the expression and activity of specific molecules (e.g., proteases and protein kinases) and biological processes (e.g., apoptosis, angiogenesis, and metastasis) that influence tumor behavior and/or response to therapy. This information is expected to have a major impact on cancer detection, individualized treatment, and drug development, as well as our understanding of how cancer arises.

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Molecular Imaging in Cancer

Granzyme B and Perforin Are Important for Regulatory T Cell-Mediated Suppression of Tumor Clearance

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.

Bioluminescence imaging of myeloperoxidase activity in vivo.

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

The transcription factor NF-κB is a key regulator of cellular activation, proliferation and apoptosis. Defects in the NF-κB pathway contribute to a broad array of malignant, neurodegenerative and chronic inflammatory diseases. IKK-dependent IκBα degradation by the 26S proteasome is a critical NF-κB 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 IκBα–firefly luciferase (IκBα-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 IκBα-FLuc reporter now permits continuous readout of IKK activation in vivo, facilitates development and validation of target-specific therapeutics, and complements conventional NF-κB transcriptional reporters for more complete temporal and regional investigations of the NF-κB signaling pathway in health and disease.

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

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 (LD50=15.3±0.2 μM) and L-02 (LD50=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 ...

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

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 exp...

Shimon Gross

Papers

Bioluminescence imaging of myeloperoxidase activity in vivo.

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

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

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

Spectral unmixing of multicolored bioluminescence emitted from heterogeneous biological sources.