Drug-induced liver injury (DILI) is an important cause of acute liver failure, with limited therapeutic options. During DILI, oncotic necrosis with concomitant release and recognition of intracellular content amplifies liver inflammation and injury. Among these molecules, self-DNA has been widely shown to trigger inflammatory and autoimmune diseases; however, whether DNA released from damaged hepatocytes accumulates into necrotic liver and the impact of its recognition by the immune system remains elusive. Here we show that treatment with two different hepatotoxic compounds (acetaminophen and thioacetamide) caused DNA release into the hepatocyte cytoplasm, which occurred in parallel with cell death in vitro. Administration of these compounds in vivo caused massive DNA deposition within liver necrotic areas, together with an intravascular DNA coating. Using confocal intravital microscopy, we revealed that liver injury due to acetaminophen overdose led to a directional migration of neutrophils to DNA-rich areas, where they exhibit an active patrolling behavior. DNA removal by intravenous DNASE1 injection or ablation of Toll-like receptor 9 (TLR9)-mediated sensing significantly reduced systemic inflammation, liver neutrophil recruitment, and hepatotoxicity. Analysis of liver leukocytes by flow cytometry revealed that emigrated neutrophils up-regulated TLR9 expression during acetaminophen-mediated necrosis, and these cells sensed and reacted to extracellular DNA by activating the TLR9/NF-jB pathway. Likewise, adoptive transfer of wild-type neutrophils to TLR9 2/2 mice reversed the hepatoprotective phenotype otherwise observed in TLR9 absence. Conclusion: Hepatic DNA accumulation is a novel feature of DILI pathogenesis. Blockage of DNA recognition by the innate immune system may constitute a promising therapeutic venue. (HEPATOLOGY 2015;61:348-360) See Editorial on Page 35 D rug-induced liver injury (DILI) is a serious condition, with a high mortality rate and limited therapeutic alternatives.1 Among these

Metastasis by cancer cells relies upon the acquisition of the ability to evade anoikis, a cell death process elicited by detachment from extracellular matrix (ECM). The molecular mechanisms that ECM-detached cancer cells use to survive are not understood. Striking increases in reactive oxygen species (ROS) occur in ECM-detached mammary epithelial cells, threatening cell viability by inhibiting ATP production, suggesting that ROS must be neutralized if cells are to survive ECM-detachment. Here, we report the discovery of a prominent role for antioxidant enzymes, including catalase and superoxide dismutase, in facilitating the survival of breast cancer cells after ECM-detachment. Enhanced expression of antioxidant enzymes in nonmalignant mammary epithelial cells detached from ECM resulted in ATP elevation and survival in the luminal space of mammary acini. Conversely, silencing antioxidant enzyme expression in multiple breast cancer cell lines caused ATP reduction and compromised anchorage-independent growth. Notably, antioxidant enzyme-deficient cancer cells were compromised in their ability to form tumors in mice. In aggregate, our results reveal a vital role for antioxidant enzyme activity in maintaining metabolic activity and anchorage-independent growth in breast cancer cells. Furthermore, these findings imply that eliminating antioxidant enzyme activity may be an effective strategy to enhance susceptibility to cell death in cancer cells that may otherwise survive ECM-detachment. Cancer Res; 73(12); 3704-15. Ó2013 AACR.

Abstract:Purpose: We have developed a trimodal PET/SPECT/CT scanner for small animal imaging. The gamma ray sub-systems are based on monolithic crystals coupled to multi-anode photomultiplier tubes (MA-PMTs), while CT comprises a commercially available micro-focus X-ray tube and a CsI scintillator 2D pixelated flat panel X-ray detector. In this study we will report on the design and performance evaluation of the multimodal system. Methods: X-ray transmission measurements are performed based on cone-beam geometry. Individual projections were acquired by rotating the X-ray tube and the 2D flat panel detector, thus making possible a transaxial FOV of roughly 80 mm in diameter and an axial FOV of 65 mm for the CT system. The SPECT component has a dual head detector geometry mounted on a rotating gantry. The distance between the SPECT module detectors can be varied in order to optimize specific user requirements, including variable FOV. The PET system is made up of eight compact modules forming an octagon with an axial Field Of View (FOV) of 40 mm and a transaxial FOV of 80 mm in diameter. The main CT image quality parameters (spatial resolution and uniformity) have been determined. In the case of the SPECT, the tomographic spatial resolution and system sensitivity have been evaluated with a 99m Tc solution using single-pinhole and multipinhole collimators. PET and SPECT images were reconstructed using threedimensional (3D) Maximum Likelihood and Ordered Subset Expectation Maximization (MLEM and OSEM)) algorithms developed by the authors, whereas the CT images were obtained using a 3D based FBP algorithm.Results: CT spatial resolution was 85 μm while a uniformity of 2.7% was obtained for a water filled phantom at 45 kV. The SPECT spatial resolution was better than 0.8 mm measured with a Derenzo-like phantom for a FOV of 20 mm using a 1-mm pinhole aperture collimator. The full width at half-maximum (FWHM) PET radial spatial resolution at the center of the field of view was 1.55 mm. The SPECT system sensitivity for a FOV of 20 mm and 15% energy window was 700 cps/MBq (7.8x10 -2 %) using a multi-pinhole equipped with 5 apertures 1 mm in diameter, whereas the PET absolute sensitivity was 2% for a 350-650 keV energy window and a 5 ns timing window. Several animal images are also presented. Conclusions:The new small animal PET/SPECT/CT proposed here exhibits high performance, producing high-quality images suitable for studies with small animals. Monolithic design for PET and SPECT scintillator crystals reduces cost and complexity without significant performance degradation.

Inhibitor of DNA binding 2 (ID2) is a helix-loop-helix transcriptional repressor rhythmically expressed in many adult tissues. Our earlier studies have demonstrated a role for ID2 in the input pathway, core clock function and output pathways of the mouse circadian system. We have also reported that Id2 null (Id2−/−) mice are lean with low gonadal white adipose tissue deposits and lower lipid content in the liver. These results coincided with altered or disrupted circadian expression profiles of liver genes including those involved in lipid metabolism. In the present phenotypic study we intended to decipher, on a sex-specific basis, the role of ID2 in glucose metabolism and in the circadian regulation of activity, important components of energy balance. We find that Id2−/− mice exhibited altered daily and circadian rhythms of feeding and locomotor activity; activity profiles extended further into the late night/dark phase of the 24-hr cycle, despite mice showing reduced total locomotor activity. Also, male Id2−/− mice consumed a greater amount of food relative to body mass, and displayed less weight gain. Id2−/− females had smaller adipocytes, suggesting sexual-dimorphic programing of adipogenesis. We observed increased glucose tolerance and insulin sensitivity in male Id2−/− mice, which was exacerbated in older animals. FDG-PET analysis revealed increased glucose uptake by skeletal muscle and brown adipose tissue of male Id2−/− mice, suggesting increased glucose metabolism and thermogenesis in these tissues. Reductions in intramuscular triacylglycerol and diacylglycerol were detected in male Id2−/− mice, highlighting its possible mechanistic role in enhanced insulin sensitivity in these mice. Our findings indicate a role for ID2 as a regulator of glucose and lipid metabolism, and in the circadian control of feeding/locomotor behavior; and contribute to the understanding of the development of obesity and diabetes, particularly in shift work personnel among whom incidence of such metabolic disorders is elevated.

Cell death is a fundamental biological process that is present in numerous disease pathologies. Fluorescent probes that detect cell death have been developed for a myriad of research applications ranging from microscopy to in vivo imaging. Here we describe a synthetic near infrared conjugate of zinc(II)-dipicolylamine (Zn2+-DPA) for in vivo imaging of cell death. Chemically induced in vivo models of myopathy were established using an ionphore, ethanol, or ketamine as chemical cytotoxins. The Zn2+-DPA fluorescent probe or corresponding control was subsequently injected and whole animal fluorescence imaging demonstrated probe uptake at the site of muscle damage, which was confirmed by ex vivo and histological analyses. Further, a comparative study with a near-infrared fluorescent conjugate Annexin V showed less intense uptake at the site of muscle damage and high accumulation in the bladder. The results indicate that the fluorescent Zn2+-DPA conjugate is an effective probe for in vivo cell death detection and in some cases may be an appropriate alternative to fluorescent Annexin V conjugates.