Journal Article

IntegriSense, Animals; Carcinoma, Squamous Cell/*pathology/surgery; Fluorescent Dyes/*diagnostic use; Humans; Integrin alphaVbeta3/*metabolism; Mice; Mice, Inbred BALB C; Mouth Neoplasms/*pathology/surgery; Spectroscopy, Near-Infrared; *Surgery, Computer-Assisted

BACKGROUND AND OBJECTIVES: Near-infrared (NIR) fluorescence optical imaging is a promising technique to assess the tumor margins during cancer surgery. This technique requires targeting by specific fluorescence agents to differentiate tumor from normal surrounding tissue. We assessed the feasibility of cancer detection using NIR fluorescence agents that target either alphavbeta3 integrins or the enhanced permeability and retention (EPR) effect in an orthotopic mouse model of oral cancer. METHODS: Binding of the integrin-targeted agent to tumor cells was assessed in vitro. Oral cancer was induced in 6 BALB/c nu/nu mice by submucosal inoculation of human OSC19-luc cells into the tongue. Tumor growth was followed with bioluminescence imaging. A combination of agents targeting integrins or EPR effect was injected followed by fluorescence imaging in vivo and ex vivo after resection of the tongues. RESULTS: Oral cancer was clearly demarcated in vitro; in vivo; and on histological analysis with sufficient tumor-to-background ratios of the contrast agents. CONCLUSION: This study demonstrates the feasibility of optical imaging of oral squamous cell carcinoma based on targeting of alphavbeta3 integrins and the EPR effect. Once these NIR fluorescence agents become available for clinical testing, optical image-guided surgery could reduce residual disease after oral cancer surgery.

Journal Article

IntegriSense, Adoptive Transfer; Animals; Biological Markers/metabolism; Cell Death/genetics; Disease Models, Animal; Female; *Hematopoiesis, Extramedullary; Inflammation/immunology/metabolism; Interleukin-1beta/genetics/metabolism; Kinetics; Macrophages/cytology/*physiology; Mice; Mice, Inbred C57BL; Mice, Knockout; Models, Biological; Monocytes/*cytology/*physiology; Myeloid Cells/metabolism; Myocardial Infarction/immunology/pathology/*physiopathology; Signal Transduction; Spleen/physiology; Stroke/immunology/metabolism; Wound Healing/physiology

Monocytes (Mo) and macrophages (MPhi) are emerging therapeutic targets in malignant, cardiovascular, and autoimmune disorders. Targeting of Mo/MPhi and their effector functions without compromising innate immunity's critical defense mechanisms first requires addressing gaps in knowledge about the life cycle of these cells. Here we studied the source, tissue kinetics, and clearance of Mo/MPhi in murine myocardial infarction, a model of acute inflammation after ischemic injury. We found that a) Mo tissue residence time was surprisingly short (20 h); b) Mo recruitment rates were consistently high even days after initiation of inflammation; c) the sustained need of newly made Mo was fostered by extramedullary monocytopoiesis in the spleen; d) splenic monocytopoiesis was regulated by IL-1beta; and e) the balance of cell recruitment and local death shifted during resolution of inflammation. Depending on the experimental approach, we measured a 24 h Mo/MPhi exit rate from infarct tissue between 5 and 13% of the tissue cell population. Exited cells were most numerous in the blood, liver, and spleen. Abrogation of extramedullary monocytopoiesis proved deleterious for infarct healing and accelerated the evolution of heart failure. We also detected rapid Mo kinetics in mice with stroke. These findings expand our knowledge of Mo/MPhi flux in acute inflammation and provide the groundwork for novel anti-inflammatory strategies for treating heart failure.

Journal Article

IntegriSense

GTP cyclohydrolase (GCH1) is the key-enzyme to produce the essential enzyme cofactor, tetrahydrobiopterin. The byproduct, neopterin is increased in advanced human cancer and used as cancer-biomarker, suggesting that pathologically increased GCH1 activity may promote tumor growth. We found that inhibition or silencing of GCH1 reduced tumor cell proliferation and survival and the tube formation of human umbilical vein endothelial cells, which upon hypoxia increased GCH1 and endothelial NOS expression, the latter prevented by inhibition of GCH1. In nude mice xenografted with HT29-Luc colon cancer cells GCH1 inhibition reduced tumor growth and angiogenesis, determined by in vivo luciferase and near-infrared imaging of newly formed blood vessels. The treatment with the GCH1 inhibitor shifted the phenotype of tumor associated macrophages from the proangiogenic M2 towards M1, accompanied with a shift of plasma chemokine profiles towards tumor-attacking chemokines including CXCL10 and RANTES. GCH1 expression was increased in mouse AOM/DSS-induced colon tumors and in high grade human colon and skin cancer and oppositely, the growth of GCH1-deficient HT29-Luc tumor cells in mice was strongly reduced. The data suggest that GCH1 inhibition reduces tumor growth by (i) direct killing of tumor cells, (ii) by inhibiting angiogenesis, and (iii) by enhancing the antitumoral immune response.

Journal Article

IntegriSense, Animals; Cell Line, Tumor; Diagnostic Imaging/*methods; Fluorescent Dyes/chemistry/metabolism; Humans; Mice; Molecular Probes/*diagnostic use; Nanoparticles/chemistry; Quantum Dots; Spectroscopy, Near-Infrared/*methods

Cellular and tissue imaging in the near-infrared (NIR) wavelengths between 700 and 900 nm is advantageous for in vivo imaging because of the low absorption of biological molecules in this region. This unit presents protocols for small animal imaging using planar and fluorescence lifetime imaging techniques. Included is an overview of NIR fluorescence imaging of cells and small animals using NIR organic fluorophores, nanoparticles, and multimodal imaging probes. The development, advantages, and application of NIR fluorescent probes that have been used for in vivo imaging are also summarized. The use of NIR agents in conjunction with visible dyes and considerations in selecting imaging agents are discussed. We conclude with practical considerations for the use of these dyes in cell and small animal imaging applications.

Journal Article

Animals; Escherichia coli/immunology; Escherichia coli Infections/enzymology/immunology/*prevention & control; Extracellular Space/genetics/*immunology/metabolism; Glutathione Reductase/deficiency/genetics/*physiology; Humans; Mice; Mice, Inbred C3H; Mice, Knockout; Neutrophils/*immunology/*metabolism/microbiology; Oxidative Stress/genetics/*immunology; Phagocytosis/genetics/*immunology; Staphylococcal Infections/enzymology/immunology/*prevention & control; Staphylococcus aureus/immunology

Glutathione reductase (Gsr) catalyzes the reduction of glutathione disulfide to glutathione, which plays an important role in the bactericidal function of phagocytes. Because Gsr has been implicated in the oxidative burst in human neutrophils and is abundantly expressed in the lymphoid system, we hypothesized that Gsr-deficient mice would exhibit marked defects during the immune response against bacterial challenge. We report in this study that Gsr-null mice exhibited enhanced susceptibility to Escherichia coli challenge, indicated by dramatically increased bacterial burden, cytokine storm, striking histological abnormalities, and substantially elevated mortality. Additionally, Gsr-null mice exhibited elevated sensitivity to Staphylococcus aureus. Examination of the bactericidal functions of the neutrophils from Gsr-deficient mice in vitro revealed impaired phagocytosis and defective bacterial killing activities. Although Gsr catalyzes the regeneration of glutathione, a major cellular antioxidant, Gsr-deficient neutrophils paradoxically produced far less reactive oxygen species upon activation both ex vivo and in vivo. Unlike wild-type neutrophils that exhibited a sustained oxidative burst upon stimulation with phorbol ester and fMLP, Gsr-deficient neutrophils displayed a very transient oxidative burst that abruptly ceased shortly after stimulation. Likewise, Gsr-deficient neutrophils also exhibited an attenuated oxidative burst upon encountering E. coli. Biochemical analysis revealed that the hexose monophosphate shunt was compromised in Gsr-deficient neutrophils. Moreover, Gsr-deficient neutrophils displayed a marked impairment in the formation of neutrophil extracellular traps, a bactericidal mechanism that operates after neutrophil death. Thus, Gsr-mediated redox regulation is crucial for bacterial clearance during host defense against massive bacterial challenge.