No abstract

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

For Abstract see ChemInform Abstract in Full Text.

An optical imaging probe was synthesized by attaching a near-infrared carbocyanine fluorophore to an affinity group containing two zinc(II) dipicolylamine (Zn-DPA) units. The probe has a strong and selective affinity for the surfaces of bacteria, and it was used to image infections of Gram-positive S. aureus and Gram-negative E. coli bacteria in living nude mice. After intravenous injection, the probe selectively accumulates at the sites of localized bacterial infections in the thigh muscles of the mice.Bacterial imaging is an emerging technology that has many health and environmental applications. 1 For example, there is an obvious need to develop highly sensitive assays that can detect very small numbers of pathogenic bacterial cells in food, drinking water or biomedical samples. In other situations, the goal is to study in vivo the temporal and spatial distribution of bacteria in live animals.Optical imaging of bacteria in vivo is much less developed than methods such as radioimaging and MRI. One approach is to use bacteria that are genetically encoded to produce luciferase or green fluorescent protein. 2 A second strategy, which is the focus of this study, employs a molecular probe with a fluorescent reporter group. An obvious limitation with a live animal is restricted tissue penetration of the light. However, near-infrared (NIR) dyes with emission wavelengths in the region of 650−900 nm can propagate through two or more centimeters of tissue, and may enable deeper tissue imaging if sensitive detection techniques are employed. Molecular imaging probes can often be deconstructed into two structural components, an affinity ligand and a reporter group. In the case of bacterial targeting, previously reported affinity ligands include antibodies, 5 sugars, 6 bacteria binding peptides, 7 antimicrobial peptides, 8 enzyme substrates, 9 and antibiotic drugs. 10 Recently, we discovered that fluorescent molecular probes containing synthetic zinc(II) dipicolylamine (Zn-DPA) coordination complexes as affinity groups are able to selectively stain the surfaces of bacterial cells 11 and apoptotic animal cells. 12 Zn-DPA affinity ligands bind strongly to the anionic surfaces that are a common characteristic of these two cell-types, whereas affinity for the zwitterionic surfaces of healthy animal cells is weak. These in vitro results have motivated us to pursue in vivo studies, and we report that molecular probe 1, which has a NIR fluorophore attached to an affinity group with two Zn-DPA units, can be used for targeted, fluorescence imaging of bacterial infection in a living whole animal.The bacterial imaging probe 1 (λ max abs: 794 nm, em: 810 nm) was prepared in straightforward fashion using a carbocyanine dye as the NIR fluorophore. 13 Researchers have incorporated this fluorophore into probes for other optical imaging applications. 14 In vitro fluorescence microscopy studies proved that probe 1 can effectively stain the periphery of bacterial cells (Figure 1). In contrast, the cells are not stained when treated un...

In vivo optical imaging shows that a fluorescent imaging probe, comprised of a near-infrared fluorophore attached to an affinity group containing two zinc(II)-dipicolylamine (Zn-DPA) units, targets prostate and mammary tumors in two different xenograft animal models. The tumor selectivity is absent with control fluorophores whose structures do not have appended Zn-DPA targeting ligands. Ex vivo biodistribution and histological analyses indicate that the probe is targeting the necrotic regions of the tumors, which is consistent with in vitro microscopy showing selective targeting of the anionic membrane surfaces of dead and dying cells.There is a major ongoing research effort to identify oligonucleotide and protein biomarkers of malignant disease. 1 Phospholipid biomarkers are less common, however, there is increasing evidence that the membrane surfaces of certain cells and particles of biomedical significance, smith.115@nd.edu. Supporting Information Available: Experimental details and additional imaging data. The information is available free of charge via the Internet at http://pubs.acs.org. Synthetic zinc(II)-dipicolylamine (Zn-DPA) coordination complexes are known to associate with multianionic phosphorylated biomolecules, 14 and we have discovered that they can be converted into optical imaging probes that target the outer surfaces of anionic vesicle and cell membranes.15 Fluorescent Zn-DPA probes can distinguish dead and dying mammalian cells from healthy cells in a cell culture,16 and also selectively target bacteria in heterogeneous biological media.17 Furthermore, we have recently demonstrated that the near-IR fluorescent probe 1 can be used to image bacterial infections in living mice, 18 indicating that probe 1 has a notable ability to selectively target anionic cells over other anionic sites in the bloodstream and extracellular matrix. Here, we greatly expand the animal imaging capability of probe 1 by showing that it can also target the anionic dead and dying cells within xenograft tumors in rat and mouse models. The structure of probe 1 includes a near-IR carbocyanine fluorophore whose absorption and emission wavelengths of 794 and 810 nm, respectively, are within the optimal window for maximum penetration through skin and tissue. 19 The high tumor selectivity of 1 is demonstrated by comparison to the less-selective imaging that is achieved by using control near-IR fluorophores 2 and 3 whose structures do not have Zn-DPA targeting ligands. The expected ability of probe 1 to selectively target dead and dying cells with exposed anionic phosphatidylserine was confirmed with in vitro fluorescence microscopy studies of mammalian cells treated with a cytotoxic agent. 16 Specifically, treatment of Jurkat cells (T lymphocytes) with camptothecin induced significant amounts of cell death, and as shown in Figure 1, the near-IR probe 1 stained the same cells as fluorescently labeled Annexin V. Using procedures that were approved by the appropriate institutional animal care and use committee, two tumor...

It's hip to be square: Squaraine rotaxanes have very similar photophysical properties to the commonly used Cy‐5 fluorophore, but are substantially more photostable and resist self‐quenching upon aggregation. Molecular probes containing squaraine rotaxanes (see structure) are shown to be versatile, high‐performance NIR fluorescence stains for in vitro fluorescence imaging of cells (middle) and in vivo whole‐body imaging of living mice (right).

Optical molecular imaging employs relatively harmless, low-energy light and technically straightforward instrumentation. Self-illuminating, chemiluminescent systems are especially attractive since they have inherently high signal contrast due to the lack of background emission. Currently, chemiluminescence imaging involves short-lived molecular species that are not stored but instead generated in situ, and they typically emit visible light, which does not penetrate far through heterogeneous biological media. Here, we describe a new paradigm for optical molecular imaging using squaraine rotaxane endoperoxides (SREPs), interlocked fluorescent and chemiluminescent dye molecules that have a squaraine chromophore encapsulated inside a macrocycle endoperoxide. SREPs can be stored indefinitely at temperatures below −20 °C, but upon warming to body temperature they undergo a unimolecular chemical reaction and emit near infrared light that can pass through a living mouse. Dye-stained microparticles are easily prepared for in vivo near-infrared optical imaging using commercial imaging stations.

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy, with high mortality attributable to widespread intra-peritoneal (i.p.) metastases. Recent meta-analyses report an association between obesity, ovarian cancer incidence, and ovarian cancer survival, but the effect of obesity on metastasis has not been evaluated. The objective of this study was to use an integrative approach combining in vitro, ex vivo, and in vivo studies to test the hypothesis that obesity contributes to ovarian cancer metastatic success. Initial in vitro studies using three-dimensional meso-mimetic cultures showed enhanced cell-cell adhesion to the lipid-loaded mesothelium. Furthermore, in an ex vivo colonization assay, ovarian cancer cells exhibited increased adhesion to mesothelial explants excised from mice modeling diet-induced obesity (DIO), in which they were fed a "Western" diet. Examination of mesothelial ultrastructure revealed a substantial increase in the density of microvilli in DIO mice. Moreover, enhanced i.p. tumor burden was observed in overweight or obese animals in three distinct in vivo models. Further histological analyses suggested that alterations in lipid regulatory factors, enhanced vascularity, and decreased M1/M2 macrophage ratios may account for the enhanced tumorigenicity. Together, these findings show that obesity potently impacts ovarian cancer metastatic success, which likely contributes to the negative correlation between obesity and ovarian cancer survival.