Journal Article

VivoTag, IVIS, Vivotag

Biomarkers are becoming increasingly important in the clinical management of complex diseases, yet our ability to discover new biomarkers remains limited by our dependence on endogenous molecules. Here we describe the development of exogenously administered 'synthetic biomarkers' composed of mass-encoded peptides conjugated to nanoparticles that leverage intrinsic features of human disease and physiology for noninvasive urinary monitoring. These protease-sensitive agents perform three functions in vivo: they target sites of disease, sample dysregulated protease activities and emit mass-encoded reporters into host urine for multiplexed detection by mass spectrometry. Using mouse models of liver fibrosis and cancer, we show that these agents can noninvasively monitor liver fibrosis and resolution without the need for invasive core biopsies and substantially improve early detection of cancer compared with current clinically used blood biomarkers. This approach of engineering synthetic biomarkers for multiplexed urinary monitoring should be broadly amenable to additional pathophysiological processes and point-of-care diagnostics.

Journal Article

VivoTag, IVIS, Vivotag

Recombinant adenovirus-engineered dendritic cells (Ad.DC) are potent vaccines for induction of anti-viral and anti-cancer T cell immunity. The effectiveness of Ad.DC vaccines may depend on the newly described ability of Ad.DC to crosstalk with natural killer (NK) cells via cell-to-cell contact, and to mediate activation, polarization and bridging of innate and adaptive immunity. For this interaction to occur in vivo, Ad.DC must be able to attract NK cells from surrounding tissues or peripheral blood. We developed a novel live mouse imaging system-based NK-cell migration test, and demonstrated for the first time that human Ad.DC induced directional migration of human NK cells across subcutaneous tissues, indicating that Ad.DC-NK cell contact and interaction could occur in vivo. We examined the mechanism of Ad.DC-induced migration of NK cells in vitro and in vivo. Ad.DC produced multiple chemokines previously reported to recruit NK cells, including immunoregulatory CXCL10/IP-10 and proinflammatory CXCL8/IL-8. In vitro chemotaxis experiments utilizing neutralizing antibodies and recombinant human chemokines showed that CXCL10/IP-10 and CXCL8/IL-8 were critical for Ad.DC-mediated recruitment of CD56(hi)CD16(-) and CD56(lo)CD16(+) NK cells, respectively. The importance of CXCL8/IL-8 was further demonstrated in vivo. Pretreatment of mice with the neutralizing anti-CXCL8/IL-8 antibody led to significant inhibition of Ad.DC-induced migration of NK cells in vivo. These data show that Ad.DC can recruit spatially distant NK cells toward a vaccine site via specific chemokines. Therefore, an Ad.DC vaccine can likely induce interaction with endogenous NK cells via transmembrane mediators, and consequently mediate Th1 polarization and amplification of immune functions in vivo.

Journal Article

ReninSense 680 FAST, FMT, Animal Feed/analysis; Animals; Cathepsin D; Cathepsin G; Female; Fluorescent Dyes/*pharmacology; Humans; Mice; Mice, Inbred C57BL; Peptides/*pharmacology; Peptidyl-Dipeptidase A/metabolism; Rats; Renin/*blood/*metabolism; Renin-Angiotensin System/physiology; Sensitivity and Specificity; Sodium, Dietary

The renin-angiotensin system (RAS) is well studied for its regulation of blood pressure and fluid homeostasis, as well as for increased activity associated with a variety of diseases and conditions, including cardiovascular disease, diabetes, and kidney disease. The enzyme renin cleaves angiotensinogen to form angiotensin I (ANG I), which is further cleaved by angiotensin-converting enzyme to produce ANG II. Although ANG II is the main effector molecule of the RAS, renin is the rate-limiting enzyme, thus playing a pivotal role in regulating RAS activity in hypertension and organ injury processes. Our objective was to develop a near-infrared fluorescent (NIRF) renin-imaging agent for noninvasive in vivo detection of renin activity as a measure of tissue RAS and in vitro plasma renin activity. We synthesized a renin-activatable agent, ReninSense 680 FAST (ReninSense), using a NIRF-quenched substrate derived from angiotensinogen that is cleaved specifically by purified mouse and rat renin enzymes to generate a fluorescent signal. This agent was assessed in vitro, in vivo, and ex vivo to detect and quantify increases in plasma and kidney renin activity in sodium-sensitive inbred C57BL/6 mice maintained on a low dietary sodium and diuretic regimen. Noninvasive in vivo fluorescence molecular tomographic imaging of the ReninSense signal in the kidney detected increased renin activity in the kidneys of hyperreninemic C57BL/6 mice. The agent also effectively detected renin activity in ex vivo kidneys, kidney tissue sections, and plasma samples. This approach could provide a new tool for assessing disorders linked to altered tissue and plasma renin activity and to monitor the efficacy of therapeutic treatments.

Journal Article

IVIS Imaging

Spirulina platensis, a water blue-green alga, has been associated with potent biological effects, which might have important relevance in atheroprotection. We investigated whether S. platensis or phycocyanobilin (PCB), its tetrapyrrolic chromophore, can activate atheroprotective heme oxygenase-1 (Hmox1), a key enzyme in the heme catabolic pathway responsible for generation of a potent antioxidant bilirubin, in endothelial cells and in a mouse model of atherosclerosis. In vitro experiments were performed on EA.hy926 endothelial cells exposed to extracts of S. platensis or PCB. In vivo studies were performed on ApoE-deficient mice fed a cholesterol diet and S. platensis. The effect of these treatments on Hmox1, as well as other markers of oxidative stress and endothelial dysfunction, was then investigated. Both S. platensis and PCB markedly upregulated Hmox1 in vitro, and a substantial overexpression of Hmox1 was found in aortic atherosclerotic lesions of ApoE-deficient mice fed S. platensis. In addition, S. platensis treatment led to a significant increase in Hmox1 promoter activity in the spleens of Hmox-luc transgenic mice. Furthermore, both S. platensis and PCB were able to modulate important markers of oxidative stress and endothelial dysfunction, such as eNOS, p22 NADPH oxidase subunit, and/or VCAM-1. Both S. platensis and PCB activate atheroprotective HMOX1 in endothelial cells and S. platensis increased the expression of Hmox1 in aortic atherosclerotic lesions in ApoE-deficient mice, and also in Hmox-luc transgenic mice beyond the lipid lowering effect. Therefore, activation of HMOX1 and the heme catabolic pathway may represent an important mechanism of this food supplement for the reduction of atherosclerotic disease.