Journal Article

Antibiotics -- Therapeutic use; Bacteriocins; Bioware; Dissertations -- Microbiology; Drug resistance in microorganisms; Nisin; Respiratory infections -- Treatment; Skin -- Infections -- Treatment; Staphylococcus aureus; Theses -- Microbiology; Xen29

Multidrug resistant strains of Staphylococcus aureus is presenting an increasing threat, especially immune compromised individuals. Many of these strains have developed resistance to newly approved drugs such as quinupristin-dalfopristin, linezolid and daptomycin. The search for alternative treatment, including bacteriocins (ribosomally synthesized antimicrobial peptides) of lactic acid bacteria is increasing . Lactococcus lactis subsp. lactis F10, isolated from freshwater catfish, produced a new nisin variant active against clinical strains of S. aureus. The operon encoding nisin F is located on a plasmid and the structural gene has been sequenced. The lantibiotic is closely related to nisin Z, except at position 30 where valine replaced isoleucine. The antimicrobial activity of nisin F against S. aureus was tested in the respiratory tract of Wistar rats. Non-immunosuppressed and immunosuppressed rats were intranasally infected with S. aureus K and then treated with either nisin F or sterile physiological saline. Nisin F protected immunosuppressed rats against S. aureus, as symptoms of an infection were only detected in the trachea and lungs of immunosuppressed rats treated with saline. The safety of intranasally administered nisin F was also evaluated and proved to have no adverse side effects. The potential of nisin F as an antimicrobial agent to treat subcutaneous skin infections was evaluated by infecting C57BL/6 mice with a bioluminescent strain of S. aureus (Xen 36). Immunosuppressed mice were treated with either nisin F or sterile physiological saline 24 h and 48 h after infection with subcutaneously injected S. aureus Xen 36. Histology and bioluminescence flux measurements revealed that nisin F was ineffective in the treatment of deep dermal staphylococcal infections. Non-infected and infected mice treated with nisin F had an influx of polymorphonuclear cells in the deep stroma of the skin tissue. This suggested that nisin F, when injected subcutaneously, may have modulated the immune system. Nisin F proved an effective antimicrobial agent against S. aureus-related infections in the respiratory tract, but not against subcutaneous infections. The outcome of nisin F treatment thus depends on the route of administration and site of infection.

Journal Article

in vivo imaging; Cat K FAST; Cat B FAST; MMPSense

New imaging methods are urgently needed to identify high-risk atherosclerotic lesions prior to the onset of myocardial infarction, stroke, and ischemic limbs. Molecular imaging offers a new approach to visualize key biological features that characterize high-risk plaques associated with cardiovascular events. While substantial progress has been realized in clinical molecular imaging of plaques in larger arterial vessels (carotid, aorta, iliac), there remains a compelling, unmet need to develop molecular imaging strategies targeted to high-risk plaques in human coronary arteries. We present recent developments in intravascular near-IR fluorescence catheter-based strategies for in vivo detection of plaque inflammation in coronary-sized arteries. In particular, the biological, light transmission, imaging agent, and engineering principles that underlie a new intravascular near-IR fluorescence sensing method are discussed. Intravascular near-IR fluorescence catheters appear highly translatable to the cardiac catheterization laboratory, and thus may offer a new in vivo method to detect high-risk coronary plaques and to assess novel atherosclerosis biologics.

Journal Article

Bioware; SKOV3-luc-D3 cells

Peritoneal dissemination is a common feature of human ovarian carcinoma. While this can be mimicked in preclinical models by intraperitoneal injection of human ovarian tumor cells into immunocompromised mice, the resulting tumor burden is difficult to monitor and quantify. Intraperitoneal tumor growth is typically evaluated indirectly by measured changes in mouse abdominal girth and body weight or, directly, by macroscopic and histological examination at the endpoint of the study. In order to establish a model system that allows continuous and accurate assessment of ovarian cancer growth and spread over time we transfected SKOV-3 cells with the firefly luciferase gene. The resulting cell line, SKOV3-luc-D3, expresses stable levels of luciferase in vitro and emits a strong luminescent signal when exposed to luciferin. Xenograft tumors established with this cell line can be tracked and quantified non-invasively by bioluminescent imaging using a highly sensitive, cooled CCD camera (IVIS(R) Imaging System, Xenogen Corp). In addition to providing a direct measure of primary tumor burden and growth, the SKOV3-luc-D3 cell line also allows for real-time evaluation of tumor response to various therapeutic agents, as well as enhanced detection of distal metastases.

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

Molecular imaging; Abdominal aortic aneurysm; Optical imaging; Pre-clinical; Endovascular imaging; Matrix metalloproteinase; in vivo imaging; MMPSense

Objectives: We present a method to quantify the inflammatory processes that drive abdominal aortic aneurysm (AAA) development that may help predict the rate of growth and thus guide medical and surgical management. We use an in vivo optical molecular imaging approach to quantify protease activity within the walls of AAAs in a rodent model.

Methods: AAAs were generated in mice by topical application of calcium chloride, followed by the administration of the MMP inhibitor doxycycline for 3 months. After this time period, an enzyme-activatable optical molecular imaging agent sensitive to MMP activity was administered, and MMP proteolytic activity was measured in vivo. Histology and in situ zymography were performed for validation. AAAs were also generated in rats, and MMP activity within the walls of the AAAs was also quantified endovascularly.

Results: A dose-dependent response of AAA growth rate to doxycycline administration was demonstrated, with high doses of the drug resulting in nearly complete suppression of aneurysm formation. There was a direct relationship between the rate of aneurysmal growth and measured MMP activity, with a linear best-fit well approximating the relationship. We additionally performed endovascular imaging of AAAs in rats and demonstrated a similar suppression of intramural MMP activity following doxycycline administration.

Conclusions: We present an in vivo evaluation of MMP activity within the walls of AAAs in rodents and show a direct, linear relationship between proteolytic activity and aneurysmal growth. We also illustrate that this functional imaging method can be performed endovascularly, demonstrating potential pre-clinical and clinical applications.