Biomedical subjects

Christopher H Contag

Publications and source records attributed to Christopher H Contag.

At least 55 records · Page 3Linked to original sources

PubMed2004

Extracellular replication of Listeria monocytogenes in the murine gall bladder.

The bacterium Listeria monocytogenes can cause a life-threatening systemic illness in humans. Despite decades of progress in animal models of listeriosis, much remains unknown about the processes of infection and colonization. Here, we report that L. monocytogenes can replicate in the murine gall bladder and provide evidence that its replication there is extracellular and intraluminal. In vivo bioluminescence imaging was employed to determine the location of the infection over time in live animals, revealing strong signals from the gall bladder over a period of several days, in diseased as well as asymptomatic animals. The data suggest that L. monocytogenes may be carried in the human gall bladder.

Animals↗

PubMed2004

p47phox deficiency impairs NF-kappa B activation and host defense in Pseudomonas pneumonia.

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.

Animals↗

PubMed2004

A genetic reporter of thermal stress defines physiologic zones over a defined temperature range.

We define five unique cellular responses to thermal stress using a reporter construct generated using the stress-inducible promoter from the gene encoding a murine 70 kDa heat shock protein (Hsp70A.1) to express luciferase (luc). Thermal stress was delivered over a range of temperatures (42-68 degrees C) for 5 s to 20 min and luciferase activity was measured in live cells using a cooled CCD camera as a measure of reporter gene transcription. Reporter gene expression was assessed every 2 h for 10 h, and at 24 h post-stress. Expression patterns were validated for selected temperatures. A transition zone where cells lose the ability to produce light and beyond which >50% of cells die was observed to occur within a narrow (2.5 degrees C) temperature window. Although luc and hsp70 mRNA levels in this transition zone were high, there were reduced levels of Luc and Hsp70 protein and ATP levels. Cells treated at these temperatures recovered the ability to produce light in response to a secondary stress at 30 h. This Hsp70-luc reporter gene construct may be useful for defining zones of physiologic responses and assessing collateral thermal damage generated during treatment of biological tissue with lasers and other sources of heat.

Animals↗

PubMed2004

Apoptosis in a rodent model of cranial suture fusion: in situ imaging and gene expression analysis.

Craniosynostosis, the premature fusion of cranial sutures, is one of the most common craniofacial anomalies, with a reported incidence of up to one in 2500 live births. Despite its prevalence, the cause of craniosynostosis remains unknown. Previously, apoptosis has been postulated to be a contributing factor in the pathogenesis of craniosynostosis, although the role of programmed cell death in cranial sutures is poorly understood. To address this problem, the authors used an established rodent model of posterior-frontal suture fusion and sagittal suture patency to globally examine apoptosis in cranial sutures. Apoptosis was evaluated by systemically coinjecting Sprague-Dawley rats with both fluorescent and technetium-99m-labeled annexin V at time points before, during, and after the period of predicted posterior-frontal suture fusion to determine the magnitude and time course of overall apoptotic activity in both fusing and patent sutures. Using these novel in situ imaging techniques, the authors observed a significant increase in the overall levels of apoptosis in both the posterior-frontal and sagittal suture complexes during the period of predicted posterior-frontal suture fusion. To further explore this increase in apoptotic activity, they used microarray technology to study apoptosis-related genes within the suture complex. Interestingly, there was activation of distinct apoptotic pathways in the posterior-frontal and sagittal sutures during the period of predicted posterior-frontal suture fusion. Whereas increased transcription of genes associated with the mitochondria-mediated apoptotic pathway occurred in the posterior-frontal suture during fusion, activation of genes associated with the death receptor-mediated apoptotic pathway predominated in the patent sagittal suture during the same time period. These data suggest that although overall apoptotic activity in rat patent and fusing sutures is similar, the pathways mediating apoptosis within each suture are distinct.

Animals↗

PubMed2004

In vivo bioluminescence imaging for integrated studies of infection.

Understanding biological processes in the context of intact organ systems with fine temporal resolution has required the development of imaging strategies that reveal cellular and molecular changes in the living body. Reporter genes that confer optical signatures on a given biological process have been used widely in cell biology and have been used more recently to interrogate biological processes in living animal models of human biology and disease. The use of internal biological sources of light, luciferases, to tag cells, pathogens, and genes has proved to be a versatile tool to provide in vivo indicators that can be detected externally. The application of this technology to the study of animal models of infectious disease has not only provided insights into disease processes, but has also revealed new mechanisms by which pathogens may avoid host defences during infection.

Animals↗

PubMed2004

Multi-modality imaging identifies key times for annexin V imaging as an early predictor of therapeutic outcome.

Radiolabeled annexin V may provide an early indication of the success or failure of anticancer therapy on a patient-by-patient basis as an in vivo marker of tumor cell killing. An important question that remains is when, after initiation of treatment, should annexin V imaging be performed. To address this issue, we obtained simultaneous in vivo measurements of tumor burden and uptake of radiolabeled annexin V in the syngeneic orthotopic murine BCL1 lymphoma model using in vivo bioluminescence imaging (BLI) and small animal single-photon emission computed tomography (SPECT). BCL1 cells labeled for fluorescence and bioluminescence assays (BCL1-gfp/luc) were injected into mice at a dose that leads to progressive disease within two to three weeks. Tumor response was followed by BLI and SPECT before and after treatment with a single dose of 10 mg/kg doxorubicin. Biodistribution analyses revealed a biphasic increase of annexin V uptake within the tumor-bearing tissues of mice. An early peak occurring before actual tumor cells loss was observed between 1 and 5 hr after treatment, and a second longer sustained rise from 9 to 24 hr after therapy, which heralds the onset of tumor cell loss as confirmed by BLI. Multimodality imaging revealed the temporal patterns of tumor cell loss and annexin V uptake revealing a better understanding of the timing of radiolabeled annexin V uptake for its development as a marker of therapeutic efficacy.

Animals↗

PubMed2004

Characterization of coelenterazine analogs for measurements of Renilla luciferase activity in live cells and living animals.

In vivo imaging of bioluminescent reporters relies on expression of light-emitting enzymes, luciferases, and delivery of chemical substrates to expressing cells. Coelenterazine (CLZN) is the substrate for a group of bioluminescent enzymes obtained from marine organisms. At present, there are more than 10 commercially available CLZN analogs. To determine which analog is most suitable for activity measurements in live cells and living animals, we characterized 10 CLZN analogs using Renilla luciferase (Rluc) as the reporter enzyme. For each analog, we monitored enzyme activity, auto-oxidation, and efficiency of cellular uptake. All CLZN analogs tested showed higher auto-oxidation signals in serum than was observed in phosphate buffer or medium, mainly as a result of auto-oxidation by binding to albumin. CLZN-f, -h, and -e analogs showed 4- to 8-fold greater Rluc activity, relative to CLZN-native, in cells expressing the enzyme from a stable integrant. In studies using living mice expressing Rluc in hepatocytes, administration of CLZN-e and -native produced the highest signal. Furthermore, distinct temporal differences in signal for each analog were revealed following intravenous or intraperitoneal delivery. We conclude that the CLZN analogs that are presently available vary with respect to hRluc utilization in culture and in vivo, and that the effective use of CLZN-utilizing enzymes in living animals depends on the selection of an appropriate substrate.

Animals↗

PubMed2004

Assessment of cellular response to thermal laser injury through bioluminescence imaging of heat shock protein 70.

Assessment of laser-induced tissue damage is not complete without an investigation into the resulting cellular and molecular changes. In the past, tissue damage was quantified macroscopically by visual effects such as tissue mass removal, carbonization and melting. Microscopically, assessment of tissue damage has been typically limited to histological analysis of excised tissue samples. In this research, we used heat shock protein (hsp70) transcription to track cellular response to laser-induced injury. A stable cell line (NIH-3T3) was generated containing the firefly luciferase (luc) reporter gene attached to the hsp promoter (murine hsp70a1). After thermal injury with a pulsed holmium-yttrium aluminum garnet laser (lambda = 2.1 microm, taup = 250 micros, 30 pulses, 3 Hz), luciferase is produced on hsp70 activation and emits broad-spectrum bioluminescence over a range of 500-700 nm, with a peak at 563 nm. The onset of bioluminescence can be seen as early as 2 h after treatment and usually peaks at 8-12 h depending on the severity of heat shock. The luminescence was quantified in live cells using bioluminescence imaging. A minimum pulse energy (65 mJ/pulse [total energy 1.95 J; total radiant exposure = 6 J/cm2]) was needed to activate the hsp70 response, and a higher energy (103 mJ/pulse [total energy 3.09 J; total radiant exposure = 9.6 J/cm2]) was associated with a reduction in hsp70 response and cell death. Bioluminescence levels correlated well with actual hsp70 protein concentrations as determined by enzyme-linked immunosorbent assay. Photon counts were normalized to the percentage of live cells by means of a flow cytometry cell viability assay. Within a relatively small range between a lower activation threshold and an upper threshold that leads to cell death, the hsp70 response followed an Arrhenius relationship when constant-temperature water bath and laser experiments were carried out.

3T3 Cells↗

PubMed2003

Shifting foci of hematopoiesis during reconstitution from single stem cells.

To reveal the early events and dynamics of hematopoietic reconstitution in living animals in real-time, we used bioluminescence imaging to monitor engraftment from single luciferase-labeled hematopoietic stem cells (HSC) in irradiated recipients. Transplanted HSC generated discrete foci in the spleen and bone marrow (BM), at a frequency that correlated with BM compartment size. Initially detected foci could expand locally, seed other sites in BM or spleen, and/or recede with different kinetics. These studies reveal dynamic and variable patterns of engraftment from highly purified HSC and indicate that the final overall contribution of individual HSC to hematopoietic chimerism does not depend on the specific site of initial engraftment and expansion.

Animals↗

PubMed2003

Dual-axes confocal microscopy with post-objective scanning and low-coherence heterodyne detection.

We present a dual-axes confocal microscope that employs postobjective scanning and low-coherence heterodyne detection to collect vertical cross-sectional images from biological tissue with high axial resolution, reduced noise from scattered light, deep tissue penetration, and a large dynamic range. This architecture can be scaled down to millimeter dimensions with microelectromechanical systems technology for performance of in vivo optical biopsy.

Biopsy↗

PubMed2003

Gene transfer via reversible plasmid condensation with cysteine-flanked, internally spaced arginine-rich peptides.

Nonviral gene transfer offers biosafety, stability, and expense advantages over viruses; however, it has suffered from poor efficiency. Because arginine-rich peptides facilitate uptake of macromolecules such as proteins, liposomes, and iron nanoparticles, we explored their potential in enhancing plasmid DNA delivery. In their unmodified form, known protein transduction sequences, including hepta-arginine and Tat(47-57), failed to support effective gene delivery. However, by flanking a core of consecutive arginines with amino- and carboxy-terminal cysteines in vitro gene transfer was observed. Furthermore, interspersing arginines with glycine and histidine residues achieved reversible plasmid condensation and dramatically increased transfection levels in a variety of cell types. Unlike most available cationic homopolymers that function only in vitro, these new peptides also increased gene expression in both murine and human tissue in vivo. Thus, cysteine-flanked, internally spaced arginine-rich (CFIS-R) peptides represent a new approach to efficient nonviral plasmid delivery using rationally designed protein transduction domains.

Animals↗

PubMed2003

In vivo activation of the human CYP3A4 promoter in mouse liver and regulation by pregnane X receptors.

Human cytochrome P450 3A4 (CYP3A4) is responsible for the metabolism of numerous xenobiotics in the human liver. We have examined the activation of the human CYP3A4 promoter in mouse liver by using in vivo bioluminescent imaging (BLI). Transcription of the CYP3A4 promoter occurs as a result of a ligand binding to a nuclear orphan receptor, pregnane X receptor (PXR), followed by dimerization with another nuclear receptor, retinoid X receptor (RXR). Since this heterodimer then binds to xenobiotic response elements to activate transcription of CYP3A4, we examined a 13kb promoter region of CYP3A4 for responsiveness to dexamethasone and rifampicin. A reporter vector CYP3A4-luc was constructed consisting of the CYP3A4 promoter driving the firefly luciferase gene. This DNA was injected into the tail veins of mice, and reporter gene expression was monitored in the liver region using BLI. Treatment of transfected mice with dexamethasone resulted in a 188-fold induction of luciferase, whereas treatment with rifampicin resulted in a 68-fold induction. Co-injection with a human PXR expression vector resulted in a dramatic increase in rifampicin-induced activity and a smaller increase of dexamethasone-induced activity. Co-injection of an antisense murine PXR construct with the CYP3A4-luc reduced both the dexamethasone- and rifampicin-induced responses, thus demonstrating that the murine PXR receptor can participate in the regulation of the human CYP3A4 promoter in mice. The approach described here will be of general use in studying the regulation of nuclear receptors in vivo.

Animals↗

PubMed2003

Optical monitoring and treatment of potentially lethal wound infections in vivo.

We report on the use of optical techniques to monitor and treat Pseudomonas aeruginosa wound infections in mice. Bioluminescent bacteria transduced with a plasmid containing a bacterial lux gene operon allow the infection in excisional mouse wounds to be imaged by use of a sensitive charge-coupled device camera. Photodynamic therapy (PDT) targeted bacteria, by use of a polycationic photosensitizer conjugate, which is designed to penetrate the gram-negative cell wall and was topically applied to the wound and was followed by red-light illumination. There was a rapid light dose-dependent loss of luminescence, as measured by image analysis, in the wounds treated with conjugate and light, a loss that was not seen in untreated wounds, wounds treated with light alone, or wounds treated with conjugate alone. P. aeruginosa was invasive in our mouse model, and all 3 groups of control mice died within 5 days; in contrast, 90% of PDT-treated mice survived. PDT-treated wounds healed significantly faster than did silver nitrate-treated wounds, and this was not due to either inhibition of healing by silver nitrate or stimulation of healing by PDT.

Animals↗

PubMed2003

Dual-axis confocal microscope for high-resolution in vivo imaging.

We describe a novel confocal microscope that uses separate low-numerical-aperture objectives with the illumination and collection axes crossed at angle theta from the midline. This architecture collects images in scattering media with high transverse and axial resolution, long working distance, large field of view, and reduced noise from scattered light. We measured transverse and axial (FWHM) resolution of 1.3 and 2.1 microm, respectively, in free space, and confirm subcellular resolution in excised esophageal mucosa. The optics may be scaled to millimeter dimensions and fiber coupled for collection of high-resolution images in vivo.

Humans↗

PubMed2003

Animal models of bone metastasis.

BACKGROUND: Animal models are important tools to investigate the pathogenesis and develop treatment strategies for bone metastases in humans. However, there are few spontaneous models of bone metastasis despite the fact that rodents (rats and mice) and other animals (dogs and cats) often spontaneously develop cancer. Therefore, most experimental models of bone metastasis in rodents require injection or implantation of neoplastic cells into orthotopic locations, bones, or the left ventricle of the heart. METHODS: The current study reviews the natural incidence and clinical manifestation of bone metastases of mammary and prostate carcinoma in animals, as well as the experimental models developed in mice using animal and human-derived neoplasms. RESULTS: Rats, mice, dogs, and cats often develop spontaneous mammary carcinoma, but bone metastases are rare. Intact and neutered dogs develop prostate carcinoma that is usually androgen independent and may be associated with regional bone invasion or distant bone metastasis. Normal dog prostate tissue induces new bone formation in vivo and can serve as a model of osteoblastic metastasis without concurrent bone destruction. Experimental models of osteolytic, osteoblastic, and mixed osteolytic/osteoblastic bone metastases include syngeneic rodent neoplasms or human xenografts implanted at orthotopic sites (e.g., breast or prostate glands) in immunodeficient mice, injection of cancer cells into the left ventricle of the heart, or direct injection into bones. New transgenic mouse models of cancer have a low incidence of spontaneous bone metastasis, but cell lines derived from these tumors can be selected in vivo for increased incidence of bone metastasis. It is essential to validate and correctly interpret the lesions in models of bone metastasis to accurately correlate the data from animal models to human disease. Animal models have provided support for the "seed and soil" hypothesis of bone metastasis. However, the roles of vascular patterns in the metaphyses of long bones and rapid bone turnover in young animals in the pathogenesis of metastasis in experimental models are uncertain. Improvements in the imaging of experimental animals in vivo using fluorescent markers or light emitted from luciferase have led to increased sensitivity of detection and more accurate quantification of bone metastases. For example, imaging of human prostate carcinoma PC-3M cells transfected with luciferase, following injection into the left ventricle, has demonstrated that there is rapid localization of tumor cells to bones and other organs, such as the kidneys and lungs. CONCLUSIONS: Animal models of metastasis have supported drug development and have been useful for identification of metastasis suppressor and promoter genes as novel targets for the development of novel therapies. Further refinement of these models will involve spatiotemporal analysis of the metastatic process by imaging and use of image data to stage disease and guide tissue sampling for gene expression profiling via gene array technology. In the future, integrated analyses of these models will be needed to understand the complexities of this important disease process.

Animals↗

PubMed2003

Evaluation of effector cell fate and function by in vivo bioluminescence imaging.

The effector functions of immune cells have typically been examined using assays that require sampling of tissues or cells to reveal specific aspects of an immune response (e.g., antigen-specificity, cytokine expression or killing of target cells). The outcome of an immune response in vivo, however, is not solely determined by a single effector function of a specific cell population, but is the result of numerous cellular and molecular interactions that occur in the complex environment of intact organ systems. These interactions influence survival, migration, and activation, as well as final effector function of a given population of cells. Efforts to reveal the cellular and molecular basis of biological processes have resulted in a number of technologies that combine molecular biology and imaging sciences that are collectively termed as Molecular Imaging. This emerging field has developed to reveal functional aspects of cells, genes, and proteins in real time in living animals and humans and embraces multiple modalities, including established clinical imaging methods such as magnetic resonance imaging, single photon emission computed tomography, and positron emission tomography, as well as novel methodologies specifically designed for research animals. Here, we highlight one of the newer modalities, in vivo bioluminescence imaging, as a method for evaluating effector T cell proliferation, migration, and function in model systems of malignant and non-malignant diseases.

Animals↗

PubMed2003

Comparison of gene expression after intraperitoneal delivery of AAV2 or AAV5 in utero.

Correction of diseases may be achieved by delivery of genes to stem cells and developing organ systems. Our previous studies demonstrated life-long expression after in utero injection of adeno-associated virus (AAV) serotype 2 in mice. In the present studies, we compared levels of expression using the elongation factor 1alpha (EF1alpha) or the CMV promoter in AAV2 and AAV5 linked to luciferase via intraperitoneal injection in day 15 fetuses in utero. An additional AAV construct also contained the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). The level and distribution of luciferase expression were assessed by in vivo bioluminescence and luminometric assays. All mice exhibited luciferase expression for >15 months. In vivo, luciferase expression from AAV5 was greater than that produced from AAV2. Vectors containing the CMV promoter produced higher levels of gene expression in all tissues examined compared to EF1alpha-directed vectors. The WPRE increased expression in vitro fourfold and in vivo eightfold. These studies demonstrate that by modifying the promoter and serotype, increases in the efficiency of AAV-directed expression may be achieved. The efficacy of rAAV-mediated gene delivery in utero supports the potential of these vectors for future therapies.

Animals↗

PubMed2003

A potent and specific morpholino antisense inhibitor of hepatitis C translation in mice.

Hepatitis C virus (HCV) is an RNA virus infecting one in every 40 people worldwide. Current treatments are ineffective and HCV is the leading cause of liver failure leading to transplantation in the United States and Europe. Translational control of HCV is a prime therapeutic target. We assessed the inhibitory potential of morpholino phosphoramidate antisense oligonucleotides (morpholinos) on HCV translation by codelivering them with reporter plasmids expressing firefly luciferase under the translational control of the HCV internal ribosome entry site (IRES) into the livers of mice. Real-time imaging of HCV IRES luciferase reporter messenger RNA (mRNA) translation in living mice showed that a 20-mer complementary to nucleotides 345-365 of the IRES inhibited translation by greater than 95% for at least 6 days and showed mismatch specificity. No significant nonspecific inhibition of a cap-dependent luciferase or encephalomyocarditis virus (EMCV) IRES luciferase reporter translation was observed. Inhibition by the 20-mer morpholino was dose dependent, with 1 nmol/mouse giving the highest inhibition. In conclusion, morpholino antisense oligonucleotides are potent inhibitors of HCV IRES translation in a preclinical mouse model; morpholinos have potential as molecular therapeutics for treating HCV and other viral infections. The in vivo model described is a broadly applicable, straightforward, and rapid readout for inhibitor efficacy. As such, it will greatly facilitate the development of novel therapeutic strategies for viral hepatitis. Notably, the level of antisense inhibition observed in this in vivo model is similar to the maximal inhibition we have obtained previously with RNA interference in mice.

Animals↗