Citation Details

Journal Article

Animals, Diagnostic Imaging, Disease Models, Animal, Female, Luminescent Measurements/methods, Meningitis, Pneumococcal/drug therapy/microbiology/ radiography, Mice, Mice, Inbred BALB C, Streptococcus pneumoniae/drug effects IVIS, Xenogen, Xen10

Noninvasive real-time in vivo bioluminescent imaging was used to assess the spread of Streptococcus pneumoniae throughout the spinal cord and brain during the acute stages of bacterial meningitis. A mouse model was established by lumbar (LP) or intracisternal (IC) injection of bioluminescent S. pneumoniae into the subarachnoid space. Bacteria replicated initially at the site of inoculation and spread progressively from the spinal cord to the brain or from the brain down to the cervical part of the spinal column and to the lower vertebral levels. After 24 hr, animals showed strong bioluminescent signals throughout the spinal canal, indicating acute meningitis of the intracranial and intraspinal meninges. A decline in bacterial cell viability, as judged by a reduction in the bioluminescent signal, was observed over time in animals treated with ceftriaxone, but not in untreated groups. Mice treated with the antibiotic survived infection, whereas all mice in untreated groups became moribund, first in the IC group then in the LP group. No untreated animal survived beyond 48 hr after induction of infection. Colony counts of infected cerebrospinal fluid (CSF) correlated positively with bioluminescent signals. This methodology is especially appealing because it allows detecting infected mice as early as 3 hr after inoculation, provide temporal, sequential, and spatial distribution of bacteria within the brain and spinal cord throughout the entire disease process and the rapid monitoring of treatment efficacy in a nondestructive manner. Moreover, it avoids the need to sacrifice the animals for CSF sampling and the potential manipulative damage that can occur with other conventional methods.

Journal Article

IVIS, Xenogen; Bioware; Xen29

Therapeutic options for invasive Staphylococcus aureus infections have become limited due to rising antimicrobial resistance, making relevant animal model testing of new candidate agents more crucial than ever. In the present studies, a rat model of aortic infective endocarditis (IE) caused by a bioluminescently engineered, biofilm-positive S. aureus strain was used to evaluate real-time antibiotic efficacy directly. This strain was vancomycin and cefazolin susceptible but gentamicin resistant. Bioluminescence was detected and quantified daily in antibiotic-treated and control animals with IE, using a highly sensitive in vivo imaging system (IVIS). Persistent and increasing cardiac bioluminescent signals (BLS) were observed in untreated animals. Three days of vancomycin therapy caused significant reductions in both cardiac BLS (>10-fold versus control) and S. aureus densities in cardiac vegetations (P < 0.005 versus control). However, 3 days after discontinuation of vancomycin therapy, a greater than threefold increase in cardiac BLS was observed, indicating relapsing IE (which was confirmed by quantitative culture). Cefazolin resulted in modest decreases in cardiac BLS and bacterial densities. These microbiologic and cardiac BLS differences during therapy correlated with a longer time-above-MIC for vancomycin (>12 h) than for cefazolin (?4 h). Gentamicin caused neither a reduction in cardiac S. aureus densities nor a reduction in BLS. There were significant correlations between cardiac BLS and S. aureus densities in vegetations in all treatment groups. These data suggest that bioluminescent imaging provides a substantial advance in the real-time monitoring of the efficacy of therapy of invasive S. aureus infections in live animals.

Journal Article

IVIS, Xenogen, Xen7, Xen35

Streptococcus pneumoniae is a leading cause of invasive bacterial disease. This is the first study to examine the expression of S. pneumoniae genes in vivo by using whole-genome microarrays available from The Institute for Genomic Research. Total RNA was collected from pneumococci isolated from infected blood, infected cerebrospinal fluid, and bacteria attached to a pharyngeal epithelial cell line in vitro. Microarray analysis of pneumococcal genes expressed in these models identified body site-specific patterns of expression for virulence factors, transporters, transcription factors, translation-associated proteins, metabolism, and genes with unknown function. Contributions to virulence predicted for several unknown genes with enhanced expression in vivo were confirmed by insertion duplication mutagenesis and challenge of mice with the mutants. Finally, we cross-referenced our results with previous studies that used signature-tagged mutagenesis and differential fluorescence induction to identify genes that are potentially required by a broad range of pneumococcal strains for invasive disease.

Journal Article

Animals; Cells, Cultured; Dose-Response Relationship, Immunologic; Immunity, Innate; Lung; Macrophages; Membrane Glycoproteins; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Inbred DBA; Mice, Transgenic; NADPH Oxidase; Neutrophil Infiltration; NF-kappa B; Phosphoproteins; Pneumonia, Bacterial; Pseudomonas Infections; Receptors, Cell Surface; Signal Transduction; Toll-Like Receptors; Xen5

We examined the role of redox signaling generated by NADPH oxidase in activation of NF-kappaB and host defense against Pseudomonas aeruginosa pneumonia. Using mice with an NF-kappaB-driven luciferase reporter construct (HIV-LTR/luciferase (HLL)), we found that intratracheal administration of P. aeruginosa resulted in a dose-dependent neutrophilic influx and activation of NF-kappaB. To determine the effects of reactive oxygen species generated by the NADPH oxidase system on activation of NF-kappaB, we crossbred mice deficient in p47(phox) with NF-kappaB reporter mice (p47(phox-/-)HLL). These p47(phox-/-)HLL mice were unable to activate NF-kappaB to the same degree as HLL mice with intact NADPH oxidase following P. aeruginosa infection. In addition, lung TNF-alpha levels were significantly lower in p47(phox-/-)HLL mice compared with HLL mice. Bacterial clearance was impaired in p47(phox-/-)HLL mice. In vitro studies using bone marrow-derived macrophages showed that Toll-like receptor 4 was necessary for NF-kappaB activation following treatment with P. aeruginosa. Additional studies with macrophages from p47(phox-/-) mice confirmed that redox signaling was necessary for maximal Toll-like receptor 4-dependent NF-kappaB activation in this model. These data indicate that the NADPH oxidase-dependent respiratory burst stimulated by Pseudomonas infection contributes to host defense by modulating redox-dependent signaling through the NF-kappaB pathway.

Journal Article

A549-luc-C8; Animals; Bioware; Cell Line, Tumor; Colonic Neoplasms; Fluorouracil; HT-29-luc-D6 cells; Humans; Image Interpretation, Computer-Assisted; Longitudinal Studies; Luciferases; Luminescent Measurements; Lung Neoplasms; Lymphatic Metastasis; Male; Mice; Mice, SCID; Mitomycin; Models, Biological; Neoplasm Transplantation; PC-3M-luc; Prostatic Neoplasms

Bioluminescent imaging (BLI) permits sensitive in vivo detection and quantification of cells specifically engineered to emit visible light. Three stable human tumor cell lines engineered to express luciferase were assessed for their tumorigenicity in subcutaneous, intravenous and spontaneous metastasis models. Bioluminescent PC-3M-luc-C6 human prostate cancer cells were implanted subcutaneously into SCID-beige mice and were monitored for tumor growth and response to 5-FU and mitomycin C treatments. Progressive tumor development and inhibition/regression following drug treatment were observed and quantified in vivo using BLI. Imaging data correlated to standard external caliper measurements of tumor volume, but bioluminescent data permitted earlier detection of tumor growth. In a lung colonization model, bioluminescent A549-luc-C8 human lung cancer cells were injected intravenously and lung metastases were monitored in vivo by whole animal imaging. Anesthetized mice were imaged weekly allowing a temporal assessment of in vivo lung tumor growth. This longitudinal study design permitted an accurate, real-time evaluation of tumor burden in the same animals over time. End-point bioluminescence measured in vivo correlated to total lung weight at necropsy. For a spontaneous metastatic tumor model, bioluminescent HT-29-luc-D6 human colon cancer cells implanted subcutaneously produced metastases to lung and lymph nodes in SCID-beige mice. Both primary tumors and micrometastases were detected by BLI in vivo. Ex vivo imaging of excised lung lobes and lymph nodes confirmed the in vivo signals and indicated a slightly higher frequency of metastasis in some mice. Levels of bioluminescence from in vivo and ex vivo images corresponded to the frequency and size of metastatic lesions in lungs and lymph nodes as subsequently confirmed by histology. In summary, BLI provided rapid, non-invasive monitoring of tumor growth and regression in animals. Its application to traditional oncology animal models offers quantitative and sensitive analysis of tumor growth and metastasis. The ability to temporally assess tumor development and responses to drug therapies in vivo also improves upon current standard animal models that are based on single end point data.