Journal Article

IVIS, Xen29, Xen 29, Staphylococcus aureus Xen29, Animals; Blood Glucose/metabolism; Cell Adhesion/drug effects; Cell Count; Cell Movement/drug effects; Chemokine CXCL2/pharmacology; Diabetes Mellitus, Type 1/blood/complications/*immunology/microbiology; Diabetes Mellitus, Type 2/blood/complications/*immunology/microbiology; Diet, High-Fat/adverse effects; Disease Models, Animal; Hyperglycemia/chemically induced/complications; Inflammation/blood/complications/immunology/microbiology; Leukocytes/cytology/drug effects/*immunology/microbiology; Male; Mice; Mice, Inbred C57BL; Phagocytes/cytology/drug effects/microbiology; Staphylococcus aureus/physiology

BACKGROUND: Patients suffering from diabetes show defective bacterial clearance. This study investigates the effects of elevated plasma glucose levels during diabetes on leukocyte recruitment and function in established models of inflammation. METHODOLOGY/PRINCIPAL FINDINGS: Diabetes was induced in C57Bl/6 mice by intravenous alloxan (causing severe hyperglycemia), or by high fat diet (moderate hyperglycemia). Leukocyte recruitment was studied in anaesthetized mice using intravital microscopy of exposed cremaster muscles, where numbers of rolling, adherent and emigrated leukocytes were quantified before and during exposure to the inflammatory chemokine MIP-2 (0.5 nM). During basal conditions, prior to addition of chemokine, the adherent and emigrated leukocytes were increased in both alloxan- (62+/-18% and 85+/-21%, respectively) and high fat diet-induced (77+/-25% and 86+/-17%, respectively) diabetes compared to control mice. MIP-2 induced leukocyte emigration in all groups, albeit significantly more cells emigrated in alloxan-treated mice (15.3+/-1.0) compared to control (8.0+/-1.1) mice. Bacterial clearance was followed for 10 days after subcutaneous injection of bioluminescent S. aureus using non-invasive IVIS imaging, and the inflammatory response was assessed by Myeloperoxidase-ELISA and confocal imaging. The phagocytic ability of leukocytes was assessed using LPS-coated fluorescent beads and flow cytometry. Despite efficient leukocyte recruitment, alloxan-treated mice demonstrated an impaired ability to clear bacterial infection, which we found correlated to a 50% decreased phagocytic ability of leukocytes in diabetic mice. CONCLUSIONS/SIGNIFICANCE: These results indicate that reduced ability to clear bacterial infections observed during experimentally induced diabetes is not due to reduced leukocyte recruitment since sustained hyperglycemia results in increased levels of adherent and emigrated leukocytes in mouse models of type 1 and type 2 diabetes. Instead, decreased phagocytic ability observed for leukocytes isolated from diabetic mice might account for the impaired bacterial clearance.

Journal Article

Bioware; PC-3M-luc; hVEGF-luc-PC3M

Background: Preclinical cancer studies increasingly utilize non-invasive imaging modalities. In the current study we have monitored tumor growth and vascular changes using two in vivo imaging tools: surface bioluminescence (BLI) and ultrasound biomicroscopy (UBM). BLI permits visualization of tumor location in the context of the whole body, including metastases localization. UBM imaging then permits high resolution 3D volumetric tumor measurements as well as blood flow estimates down to 200 microns/s. Measurements obtained from these complementary modalities were analyzed and compared to conventional, biochemical markers. Methods: Human prostate cancer cells expressing Firefly Luciferase constitutively (PC-3M-luc-C6) or under the control of hVEGF promoter (hVEGF-luc/PC3M) were implanted into male nude mice via an intradermal or subcutaneous injection. Tumor-bearing mice were subsequently imaged every week for nine weeks starting at week 2, by UBM to measure tumor burden using 3D volumetric analysis, or to estimate blood flow using speckle-variance flow processing. Surface bioluminescence was also acquired 10 minutes post i.p. injection of D-luciferin. In a longitudinal drug intervention study anti-hVEGF antibody (Bevacizumab, 200 ug) was injected i.p. into nude mice with subcutaneous xenografts of PC-3M-luc-C6 or hVEGF-luc/PC-3M twice per week for three weeks, starting at 14 days post-xenograft. UBM and surface BLI imaging were conducted every week. In order to study the correlation between VEGF expression in hVEGF-luc/PC3M xenografts (estimated by BLI) to tumor hypoxia level, mice were injected with pimonidazole hydrochloride (60 mg/kg i.v.) after three weeks of treatment and tumors were harvested for immunostaining analysis. Results: Surface BLI outputs (photons/s) from subcutaneous PC-3M-luc-C6 xenografts were highly correlated to tumor volumes measured using 3D UBM for small tumors (<100 mm3, r=0.92, n=8), yet poorly correlated to tumors of large size (>100 mm3, r=0.079, n=8). BLI signals in subcutaneous hVEGF-luc/PC3M xenografts showed an inverse trend to tumor blood flow. PC-3M-luc-C6 tumors treated with Bevacizumab showed growth inhibition by day 28 as demonstrated by 3D UBM (control vs treated = 67.27 vs 48.54 mm3). Moreover, control xenografts showed increased average BLI output over time, whereas treated tumors showed variation in BLI output. Necrosis, hypoxia and blood flow estimates were also investigated. Conclusions: Surface bioluminescence imaging demonstrated high correlations to accurate 3D UBM volumetric measurements of small tumor volumes, suggesting its usefulness in tracking early tumor growth quantitatively in drug intervention studies. A complementary imaging modality, like ultrasound biomicroscopy, is recommended to monitor tumor burden in advanced stages.

Journal Article

BALB/c CrSlc mice; enteral nutrition; Ex vivo; Gastrosense 750; Hine® E-gel; in vivo; IVIS® Spectrum; Nev11121; pectin

BACKGROUND: Semi-solidification by gelation or increased viscosity could slow the influx of liquid enteral nutrition (EN) into the small intestine. A liquid EN formula containing pectin that gels under acidic conditions such as those found in the stomach has been developed. A new near-infrared fluorescent imaging reagent was used to non-invasively acquire real time images of gastric emptying in a murine model in vivo. We postulated that the EN formula delays gastric emptying and tested this hypothesis using imaging in vivo.
METHODS: Male BALB/c mice were given an oral bolus injection of a liquid EN containing the fluorescence reagent GastroSense750 with or without pectin. The EN in the stomach was visualized in vivo at various intervals using a non-invasive live imaging system and fluorescent signals were monitored from the stomach, which was removed at 60 min after EN ingestion.
RESULTS: The fluorescence intensity of signals in stomachs in vivo and in resected stomachs was lower and attenuated over time in mice given EN without, than with pectin.
CONCLUSIONS: Adding a gelling agent such as pectin delayed the transit of liquid EN from the stomach. Fluorescence imaging can visualize the delayed gastric emptying of EN containing pectin.

Journal Article

In vivo imaging; MMPSense; atherosclerotic carotid plaque

BACKGROUND: There is increasing evidence that plaque vulnerability, rather than the degree of stenosis, is important in predicting the occurrence of subsequent cerebral ischemic events in patients with carotid artery stenosis. The many imaging modalities currently available have different properties with regard to the visualization of the extent of vulnerability in carotid plaque formation.

METHODS: Original published studies were identified using the MEDLINE database (January 1966 to March 2008). Manual cross-referencing was also performed.

RESULTS: There is no single imaging modality that can produce definitive information about the state of vulnerability of an atherosclerotic plaque. Each has its own specific drawbacks, which may be the use of ionizing radiation or nephrotoxic contrast agents, an invasive character, low patient tolerability, or simply the paucity of information obtained on plaque vulnerability. Functional molecular imaging techniques such as positron emission tomography (PET), single photon emission-computed tomography (SPECT) and near infra-red spectroscopy (NIRS) do seem able accurately to visualize and even quantify features of plaque vulnerability and its pathophysiologic processes. Promising new techniques like near infra-red fluorescence imaging are being developed and may be beneficial in this field.

CONCLUSION: There is a promising role for functional molecular imaging modalities like PET, SPECT, or NIRS related to improvement of selection criteria for carotid intervention, especially when combined with CT or MRI to add further anatomical details to molecular information. Further information will be needed to define whether and where this functional molecular imaging will fit into a clinical strategy.

Journal Article

IntegriSense, Animals; Cell Line, Tumor; *Diagnostic Imaging; Female; Fluorescence; Fluorescent Dyes/*diagnostic use; Humans; Integrin alphaVbeta3/*metabolism; Luciferases/metabolism; Mammary Neoplasms, Experimental/*diagnosis/metabolism; Mice; Mice, Nude; Spectroscopy, Near-Infrared

BACKGROUND: This study was designed to improve the surgical procedure and outcome of cancer surgery by means of real-time molecular imaging feedback of tumor spread and margin delineation using targeted near-infrared fluorescent probes with specificity to tumor biomarkers. Surgical excision of cancer often is confronted with difficulties in the identification of cancer spread and the accurate delineation of tumor margins. Currently, the assessment of tumor borders is afforded by postoperative pathology or, less reliably, intraoperative frozen sectioning. Fluorescence imaging is a natural modality for intraoperative use by directly relating to the surgeon's vision and offers highly attractive characteristics, such as high-resolution, sensitivity, and portability. Via the use of targeted probes it also becomes highly tumor-specific and can lead to significant improvements in surgical procedures and outcome. METHODS: Mice bearing xenograft human tumors were injected with alphavbeta3-integrin receptor-targeted fluorescent probe and in vivo visualized by using a novel, real-time, multispectral fluorescence imaging system. Confirmatory ex vivo imaging, bioluminescence imaging, and histopathology were used to validate the in vivo findings. RESULTS: Fluorescence images were all in good correspondence with the confirming bioluminescence images in respect to signal colocalization. Fluorescence imaging detected all tumors and successfully guided total tumor excision by effectively detecting small tumor residuals, which occasionally were missed by the surgeon. Tumor tissue exhibited target-to-background ratio of ~4.0, which was significantly higher compared with white-light images representing the visual contrast. Histopathology confirmed the capability of the method to identify tumor negative margins with high specificity and better prediction rate compared with visual inspection. CONCLUSIONS: Real-time multispectral fluorescence imaging using tumor specific molecular probes is a promising modality for tumor excision by offering real time feedback to the surgeon in the operating room.