Christopher H. Contag

All Publications

• Role of Mitochondrial Complex I in the Proinflammatory Response to Polylactide Implants ACS APPLIED ENGINEERING MATERIALS Maduka, C. V., Makela, A. V., Tundo, A., Ural, E., Stivers, K. B., Alhaj, M., Narayan, R., Goodman, S. B., Ashammakhi, N., Elisseeff, J. H., Hankenson, K. D., Contag, C. H. 2024

  View details for DOI 10.1021/acsaenm.4c00393

  View details for Web of Science ID 001356529200001

• Polylactide Degradation Activates Immune Cells by Metabolic Reprogramming. Advanced science (Weinheim, Baden-Wurttemberg, Germany) Maduka, C. V., Alhaj, M., Ural, E., Habeeb, O. M., Kuhnert, M. M., Smith, K., Makela, A. V., Pope, H., Chen, S., Hix, J. M., Mallett, C. L., Chung, S., Hakun, M., Tundo, A., Zinn, K. R., Hankenson, K. D., Goodman, S. B., Narayan, R., Contag, C. H. 2023: e2304632

  Abstract

  Polylactide (PLA) is the most widely utilized biopolymer in medicine. However, chronic inflammation and excessive fibrosis resulting from its degradation remain significant obstacles to extended clinical use. Immune cell activation has been correlated to the acidity of breakdown products, yet methods to neutralize the pH have not significantly reduced adverse responses. Using a bioenergetic model, delayed cellular changes are observed that are not apparent in the short-term. Amorphous and semi-crystalline PLA degradation products, including monomeric l-lactic acid, mechanistically remodel metabolism in cells leading to a reactive immune microenvironment characterized by elevated proinflammatory cytokines. Selective inhibition of metabolic reprogramming and altered bioenergetics both reduce these undesirable high cytokine levels and stimulate anti-inflammatory signals. The results present a new biocompatibility paradigm by identifying metabolism as a target for immunomodulation to increase tolerance to biomaterials, ensuring safe clinical application of PLA-based implants for soft- and hard-tissue regeneration, and advancing nanomedicine and drug delivery.

  View details for DOI 10.1002/advs.202304632

  View details for PubMedID 37737614

• Glycolytic reprogramming underlies immune cell activation by polyethylene wear particles. Biomaterials advances Maduka, C. V., Habeeb, O. M., Kuhnert, M. M., Hakun, M., Goodman, S. B., Contag, C. H. 2023; 152: 213495

  Abstract

  Primary total joint arthroplasties (TJAs) are widely and successfully applied reconstructive procedures to treat end-stage arthritis. Nearly 50% of TJAs are now performed in young patients, posing a new challenge: performing TJAs which last a lifetime. The urgency is justified because subsequent TJAs are costlier and fraught with higher complication rates, not to mention the toll taken on patients and their families. Polyethylene particles, generated by wear at joint articulations, drive aseptic loosening by inciting insidious inflammation associated with surrounding bone loss. Down modulating polyethylene particle-induced inflammation enhances integration of implants to bone (osseointegration), preventing loosening. A promising immunomodulation strategy could leverage immune cell metabolism, however, the role of immunometabolism in polyethylene particle-induced inflammation is unknown. Our findings reveal that immune cells exposed to sterile or contaminated polyethylene particles show fundamentally altered metabolism, resulting in glycolytic reprogramming. Inhibiting glycolysis controlled inflammation, inducing a pro-regenerative phenotype that could enhance osseointegration.

  View details for DOI 10.1016/j.bioadv.2023.213495

  View details for PubMedID 37301057

• Bioenergetic Profile Of Mesenchymal Stromal Cells And Macrophages In The Presence Of An Inflammatory Stimulus In A 3D Model Teissier, V., Gao, Q., Shen, H., Li, J., Li, X., Huang, E., Kushioka, J., Toya, M., Tsubosaka, M., Hirata, H., Alizadeh, H., Maduka, C., Zhang, N., Contag, C., Yang, Y. P., Goodman, S. B. MARY ANN LIEBERT, INC. 2023

  View details for Web of Science ID 001120356800285

• A protease-activated, near-infrared fluorescent probe for early endoscopic detection of premalignant gastrointestinal lesions. Proceedings of the National Academy of Sciences of the United States of America Yim, J. J., Harmsen, S., Flisikowski, K., Flisikowska, T., Namkoong, H., Garland, M., van den Berg, N. S., Vilches-Moure, J. G., Schnieke, A., Saur, D., Glasl, S., Gorpas, D., Habtezion, A., Ntziachristos, V., Contag, C. H., Gambhir, S. S., Bogyo, M., Rogalla, S. 2021; 118 (1)

  Abstract

  Fluorescence imaging is currently being actively developed for surgical guidance; however, it remains underutilized for diagnostic and endoscopic surveillance of incipient colorectal cancer in high-risk patients. Here we demonstrate the utility and potential for clinical translation of a fluorescently labeled cathepsin-activated chemical probe to highlight gastrointestinal lesions. This probe stays optically dark until it is activated by proteases produced by tumor-associated macrophages and accumulates within the lesions, enabling their detection using an endoscope outfitted with a fluorescence detector. We evaluated the probe in multiple murine models and a human-scale porcine model of gastrointestinal carcinogenesis. The probe provides fluorescence-guided surveillance of gastrointestinal lesions and augments histopathological analysis by highlighting areas of dysplasia as small as 400 m, which were visibly discernible with significant tumor-to-background ratios, even in tissues with a background of severe inflammation and ulceration. Given these results, we anticipate that this probe will enable sensitive fluorescence-guided biopsies, even in the presence of highly inflamed colorectal tissue, which will improve early diagnosis to prevent gastrointestinal cancers.

  View details for DOI 10.1073/pnas.2008072118

  View details for PubMedID 33443161

• Mind Over Magnets - How Magnetic Particle Imaging is Changing the Way We Think About the Future of Neuroscience. Neuroscience V Makela, A., Gaudet, J. M., Murrell, D. H., Mansfield, J. R., Wintermark, M., Contag, C. H. 2020

  Abstract

  Magnetic particle imaging (MPI) is an emerging imaging technique, which has the potential to provide the sensitivity, specificity and temporal resolution necessary for novel imaging advances in neurological applications. MPI relies on the detection of superparamagnetic iron-oxide nanoparticles, which allows for visualization and quantification of iron or iron-labeled cells throughout a subject. The combination of these qualities can be used to image many neurological conditions including cancer, inflammatory processes, vascular-related issues and could even focus on cell therapies and theranostics to treat these problems. This review will provide a basic introduction to MPI, discuss the current use of this technology to image neurological conditions, and touch on future applications including the potential for clinical translation.

  View details for DOI 10.1016/j.neuroscience.2020.10.036

  View details for PubMedID 33197498

• Biodegradable fluorescent nanoparticles for endoscopic detection of colorectal carcinogenesis. Advanced functional materials Rogalla, S., Flisikowski, K., Gorpas, D., Mayer, A. T., Flisikowska, T., Mandella, M. J., Ma, X., Casey, K. M., Felt, S. A., Saur, D., Ntziachristos, V., Schnieke, A., Contag, C. H., Gambhir, S. S., Harmsen, S. 2019; 29 (51)

  Abstract

  Early and comprehensive endoscopic detection of colonic dysplasia - the most clinically significant precursor lesion to colorectal adenocarcinoma - provides an opportunity for timely, minimally-invasive intervention to prevent malignant transformation. Here, the development and evaluation of biodegradable near-infrared fluorescent silica nanoparticles (FSN) is described that have the potential to improve adenoma detection during fluorescence-assisted white-light colonoscopic surveillance in rodent and human-scale models of colorectal carcinogenesis. FSNs are biodegradable (t1/2 of 2.7 weeks), well-tolerated, and enable detection and delineation of adenomas as small as 0.5 mm2 with high tumor-to-background ratios. Furthermore, in the human-scale, APC 1311/+ porcine model, the clinical feasibility and benefit of using FSN-guided detection of colorectal adenomas using video-rate fluorescence-assisted white-light endoscopy is demonstrated. Since nanoparticles of similar size (e.g., 100-150-nm) or composition (i.e., silica, silica/gold hybrid) have already been successfully translated to the clinic, and, clinical fluorescent/white light endoscopy systems are becoming more readily available, there is a viable path towards clinical translation of the proposed strategy for early colorectal cancer detection and prevention in high-risk patients.

  View details for DOI 10.1002/adfm.201904992

  View details for PubMedID 33041743

  View details for PubMedCentralID PMC7546531

• Microvesicle-mediated delivery of minicircle DNA results in effective gene-directed enzyme prodrug cancer therapy. Molecular cancer therapeutics Kanada, M., Kim, B. D., Hardy, J. W., Bachmann, M. H., Bernard, M. P., Perez, G. I., Zarea, A. A., Ge, T. J., Withrow, A., Ibrahim, S. A., Toomajian, V., Gambhir, S. S., Paulmurugan, R., Contag, C. H., Ronald, J. A. 2019

  Abstract

  An emerging approach for cancer treatment employs the use of extracellular vesicles (EVs), specifically exosomes and microvesicles, as delivery vehicles. We previously demonstrated that microvesicles can functionally deliver plasmid DNA to cells and showed that plasmid size and sequence, in part, determine the delivery efficiency. In this study, delivery vehicles comprised of microvesicles loaded with engineered minicircle (MC) DNA that encodes prodrug converting enzymes were developed as a cancer therapy in mammary carcinoma models. We demonstrated that MCs can be loaded into shed microvesicles with greater efficiency than their parental plasmid counterparts and that microvesicle-mediated MC delivery led to significantly higher and more prolonged transgene expression in recipient cells than microvesicles loaded with the parental plasmid. Microvesicles loaded with MCs encoding a thymidine kinase (TK)/nitroreductase (NTR) fusion protein produced prolonged TK-NTR expression in mammary carcinoma cells. In vivo delivery of TK-NTR and administration of prodrugs led to the effective killing of both targeted cells and surrounding tumor cells via TK-NTR-mediated conversion of co-delivered prodrugs into active cytotoxic agents. In vivo evaluation of the bystander effect in mouse models demonstrated that for effective therapy, at least 1% of tumor cells need to be delivered with TK-NTR-encoding MCs. These results suggest that MC delivery via microvesicles can mediate gene transfer to an extent that enables effective prodrug conversion and tumor cell death such that it comprises a promising approach to cancer therapy.

  View details for DOI 10.1158/1535-7163.MCT-19-0299

  View details for PubMedID 31451563

• Detection of Premalignant Gastrointestinal Lesions Using Surface-Enhanced Resonance Raman Scattering-Nanoparticle Endoscopy. ACS nano Harmsen, S., Rogalla, S., Huang, R., Spaliviero, M., Neuschmelting, V., Hayakawa, Y., Lee, Y., Tailor, Y., Toledo-Crow, R., Kang, J. W., Samii, J. M., Karabeber, H., Davis, R. M., White, J. R., van de Rijn, M., Gambhir, S. S., Contag, C. H., Wang, T. C., Kircher, M. F. 2019; 13 (2): 1354–64

  Abstract

  Cancers of the gastrointestinal (GI) tract are among the most frequent and most lethal cancers worldwide. An important reason for this high mortality is that early disease is typically asymptomatic, and patients often present with advanced, incurable disease. Even in high-risk patients who routinely undergo endoscopic screening, lesions can be missed due to their small size or subtle appearance. Thus, current imaging approaches lack the sensitivity and specificity to accurately detect incipient GI tract cancers. Here we report our finding that a single dose of a high-sensitivity surface-enhanced resonance Raman scattering nanoparticle (SERRS-NP) enables reliable detection of precancerous GI lesions in animal models that closely mimic disease development in humans. Some of these animal models have not been used previously to evaluate imaging probes for early cancer detection. The studies were performed using a commercial Raman imaging system, a newly developed mouse Raman endoscope, and finally a clinically applicable Raman endoscope for larger animal studies. We show that this SERRS-NP-based approach enables robust detection of small, premalignant lesions in animal models that faithfully recapitulate human esophageal, gastric, and colorectal tumorigenesis. This method holds promise for much earlier detection of GI cancers than currently possible and could lead therefore to marked reduction of morbidity and mortality of these tumor types.

  View details for PubMedID 30624916

• Charge-altering releasable transporters (CARTs) for the delivery and release of mRNA in living animals PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA McKinlay, C. J., Vargas, J. R., Blake, T. R., Hardy, J. W., Kanada, M., Contag, C. H., Wender, P. A., Waymouth, R. M. 2017; 114 (4): E448-E456

  Abstract

  Functional delivery of mRNA to tissues in the body is key to implementing fundamentally new and potentially transformative strategies for vaccination, protein replacement therapy, and genome editing, collectively affecting approaches for the prevention, detection, and treatment of disease. Broadly applicable tools for the efficient delivery of mRNA into cultured cells would advance many areas of research, and effective and safe in vivo mRNA delivery could fundamentally transform clinical practice. Here we report the step-economical synthesis and evaluation of a tunable and effective class of synthetic biodegradable materials: charge-altering releasable transporters (CARTs) for mRNA delivery into cells. CARTs are structurally unique and operate through an unprecedented mechanism, serving initially as oligo(α-amino ester) cations that complex, protect, and deliver mRNA and then change physical properties through a degradative, charge-neutralizing intramolecular rearrangement, leading to intracellular release of functional mRNA and highly efficient protein translation. With demonstrated utility in both cultured cells and animals, this mRNA delivery technology should be broadly applicable to numerous research and therapeutic applications.

  View details for DOI 10.1073/pnas.1614193114

  View details for Web of Science ID 000392597000002

  View details for PubMedID 28069945

  View details for PubMedCentralID PMC5278438

• Differential fates of biomolecules delivered to target cells via extracellular vesicles PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kanada, M., Bachmann, M. H., Hardy, J. W., Frimannson, D. O., Bronsart, L., Wang, A., Sylvester, M. D., Schmidt, T. L., Kaspar, R. L., Butte, M. J., Matin, A. C., Contag, C. H. 2015; 112 (12): E1433-E1442

  Abstract

  Extracellular vesicles (EVs), specifically exosomes and microvesicles (MVs), are presumed to play key roles in cell-cell communication via transfer of biomolecules between cells. The biogenesis of these two types of EVs differs as they originate from either the endosomal (exosomes) or plasma (MVs) membranes. To elucidate the primary means through which EVs mediate intercellular communication, we characterized their ability to encapsulate and deliver different types of macromolecules from transiently transfected cells. Both EV types encapsulated reporter proteins and mRNA but only MVs transferred the reporter function to recipient cells. De novo reporter protein expression in recipient cells resulted only from plasmid DNA (pDNA) after delivery via MVs. Reporter mRNA was delivered to recipient cells by both EV types, but was rapidly degraded without being translated. MVs also mediated delivery of functional pDNA encoding Cre recombinase in vivo to tissues in transgenic Cre-lox reporter mice. Within the parameters of this study, MVs delivered functional pDNA, but not RNA, whereas exosomes from the same source did not deliver functional nucleic acids. These results have significant implications for understanding the role of EVs in cellular communication and for development of EVs as delivery tools. Moreover, studies using EVs from transiently transfected cells may be confounded by a predominance of pDNA transfer.

  View details for DOI 10.1073/pnas.1418401112

  View details for Web of Science ID 000351477000008

  View details for PubMedID 25713383

  View details for PubMedCentralID PMC4378439

• A real-time clinical endoscopic system for intraluminal, multiplexed imaging of surface-enhanced Raman scattering nanoparticles. PloS one Garai, E., Sensarn, S., Zavaleta, C. L., Loewke, N. O., Rogalla, S., Mandella, M. J., Felt, S. A., Friedland, S., Liu, J. T., Gambhir, S. S., Contag, C. H. 2015; 10 (4)

  Abstract

  The detection of biomarker-targeting surface-enhanced Raman scattering (SERS) nanoparticles (NPs) in the human gastrointestinal tract has the potential to improve early cancer detection; however, a clinically relevant device with rapid Raman-imaging capability has not been described. Here we report the design and in vivo demonstration of a miniature, non-contact, opto-electro-mechanical Raman device as an accessory to clinical endoscopes that can provide multiplexed molecular data via a panel of SERS NPs. This device enables rapid circumferential scanning of topologically complex luminal surfaces of hollow organs (e.g., colon and esophagus) and produces quantitative images of the relative concentrations of SERS NPs that are present. Human and swine studies have demonstrated the speed and simplicity of this technique. This approach also offers unparalleled multiplexing capabilities by simultaneously detecting the unique spectral fingerprints of multiple SERS NPs. Therefore, this new screening strategy has the potential to improve diagnosis and to guide therapy by enabling sensitive quantitative molecular detection of small and otherwise hard-to-detect lesions in the context of white-light endoscopy.

  View details for DOI 10.1371/journal.pone.0123185

  View details for PubMedID 25923788

  View details for PubMedCentralID PMC4414592

• High-sensitivity, real-time, ratiometric imaging of surface-enhanced Raman scattering nanoparticles with a clinically translatable Raman endoscope device. Journal of biomedical optics Garai, E., Sensarn, S., Zavaleta, C. L., Van de Sompel, D., Loewke, N. O., Mandella, M. J., Gambhir, S. S., Contag, C. H. 2013; 18 (9): 096008-?

  Abstract

  ABSTRACT. Topical application and quantification of targeted, surface-enhanced Raman scattering (SERS) nanoparticles offer a new technique that has the potential for early detection of epithelial cancers of hollow organs. Although less toxic than intravenous delivery, the additional washing required to remove unbound nanoparticles cannot necessarily eliminate nonspecific pooling. Therefore, we developed a real-time, ratiometric imaging technique to determine the relative concentrations of at least two spectrally unique nanoparticle types, where one serves as a nontargeted control. This approach improves the specific detection of bound, targeted nanoparticles by adjusting for working distance and for any nonspecific accumulation following washing. We engineered hardware and software to acquire SERS signals and ratios in real time and display them via a graphical user interface. We report quantitative, ratiometric imaging with nanoparticles at pM and sub-pM concentrations and at varying working distances, up to 50 mm. Additionally, we discuss optimization of a Raman endoscope by evaluating the effects of lens material and fiber coating on background noise, and theoretically modeling and simulating collection efficiency at various working distances. This work will enable the development of a clinically translatable, noncontact Raman endoscope capable of rapidly scanning large, topographically complex tissue surfaces for small and otherwise hard to detect lesions.

  View details for DOI 10.1117/1.JBO.18.9.096008

  View details for PubMedID 24008818

  View details for PubMedCentralID PMC3763230

• A Raman-based endoscopic strategy for multiplexed molecular imaging. Proceedings of the National Academy of Sciences of the United States of America Zavaleta, C. L., Garai, E., Liu, J. T., Sensarn, S., Mandella, M. J., Van de Sompel, D., Friedland, S., Van Dam, J., Contag, C. H., Gambhir, S. S. 2013; 110 (25): E2288-97

  View details for DOI 10.1073/pnas.1211309110

  View details for PubMedID 23703909

• Visualizing cellular interactions with a generalized proximity reporter. Proceedings of the National Academy of Sciences of the United States of America Sellmyer, M. A., Bronsart, L., Imoto, H., Contag, C. H., Wandless, T. J., Prescher, J. A. 2013; 110 (21): 8567-8572

  Abstract

  Interactions among neighboring cells underpin many physiological processes ranging from early development to immune responses. When these interactions do not function properly, numerous pathologies, including infection and cancer, can result. Molecular imaging technologies, especially optical imaging, are uniquely suited to illuminate complex cellular interactions within the context of living tissues in the body. However, no tools yet exist that allow the detection of microscopic events, such as two cells coming into close proximity, on a global, whole-animal scale. We report here a broadly applicable, longitudinal strategy for probing interactions among cells in living subjects. This approach relies on the generation of bioluminescent light when two distinct cell populations come into close proximity, with the intensity of the optical signal correlating with relative cellular location. We demonstrate the ability of this reporter strategy to gauge cell-cell proximity in culture models in vitro and then evaluate this approach for imaging tumor-immune cell interactions using a murine breast cancer model. In these studies, our imaging strategy enabled the facile visualization of features that are otherwise difficult to observe with conventional imaging techniques, including detection of micrometastatic lesions and potential sites of tumor immunosurveillance. This proximity reporter will facilitate probing of numerous types of cell-cell interactions and will stimulate the development of similar techniques to detect rare events and pathological processes in live animals.

  View details for DOI 10.1073/pnas.1218336110

  View details for PubMedID 23650381

  View details for PubMedCentralID PMC3666728

• Heme Oxygenase-1 Deletion Affects Stress Erythropoiesis PLOS ONE Cao, Y., Kusy, S., Luong, R., Wong, R. J., Stevenson, D. K., Contag, C. H. 2011; 6 (5)

  Abstract

  Homeostatic erythropoiesis leads to the formation of mature red blood cells under non-stress conditions, and the production of new erythrocytes occurs as the need arises. In response to environmental stimuli, such as bone marrow transplantation, myelosuppression, or anemia, erythroid progenitors proliferate rapidly in a process referred to as stress erythropoiesis. We have previously demonstrated that heme oxygenase-1 (HO-1) deficiency leads to disrupted stress hematopoiesis. Here, we describe the specific effects of HO-1 deficiency on stress erythropoiesis.We used a transplant model to induce stress conditions. In irradiated recipients that received hmox(+/-) or hmox(+/+) bone marrow cells, we evaluated (i) the erythrocyte parameters in the peripheral blood; (ii) the staining intensity of CD71-, Ter119-, and CD49d-specific surface markers during erythroblast differentiation; (iii) the patterns of histological iron staining; and (iv) the number of Mac-1(+)-cells expressing TNF-α. In the spleens of mice that received hmox(+/-) cells, we show (i) decreases in the proerythroblast, basophilic, and polychromatophilic erythroblast populations; (ii) increases in the insoluble iron levels and decreases in the soluble iron levels; (iii) increased numbers of Mac-1(+)-cells expressing TNF-α; and (iv) decreased levels of CD49d expression in the basophilic and polychromatophilic erythroblast populations.As reflected by effects on secreted and cell surface proteins, HO-1 deletion likely affects stress erythropoiesis through the retention of erythroblasts in the erythroblastic islands of the spleen. Thus, HO-1 may serve as a therapeutic target for controlling erythropoiesis, and the dysregulation of HO-1 may be a predisposing condition for hematologic diseases.

  View details for DOI 10.1371/journal.pone.0020634

  View details for PubMedID 21655188

• Synergistic antitumor effects of immune cell-viral biotherapy SCIENCE Thorne, S. H., Negrin, R. S., Contag, C. H. 2006; 311 (5768): 1780-1784

  Abstract

  Targeted biological therapies hold tremendous potential for treatment of cancer, yet their use has been limited by constraints on delivery and effective tumor targeting. We combined an immune effector cell population [cytokine-induced killer (CIK) cells] with an oncolytic viral therapy to achieve directed delivery to, and regression of, tumors in both immunodeficient and immunocompetent mouse models. Preinfection of CIK cells with modified vaccinia virus resulted in a prolonged eclipse phase with the virus remaining hidden until interaction with the tumor. Whole-body imaging revealed that the cells retained their ability to traffic to and to infiltrate the tumor effectively before releasing the virus. These results illustrate the potential of combining biotherapeutics for synergistic effects that more effectively treat cancer.

  View details for DOI 10.1126/science.1121411

  View details for PubMedID 16556847

• MYC inactivation uncovers pluripotent differentiation and tumour dormancy in hepatocellular cancer NATURE Shachaf, C. M., Kopelman, A. M., Arvanitis, C., Karlsson, A., Beer, S., Mandl, S., Bachmann, M. H., Borowsky, A. D., Ruebner, B., Cardiff, R. D., Yang, Q. W., BISHOP, J. M., Contag, C. H., Felsher, D. W. 2004; 431 (7012): 1112-1117

  Abstract

  Hepatocellular carcinoma is generally refractory to clinical treatment. Here, we report that inactivation of the MYC oncogene is sufficient to induce sustained regression of invasive liver cancers. MYC inactivation resulted en masse in tumour cells differentiating into hepatocytes and biliary cells forming bile duct structures, and this was associated with rapid loss of expression of the tumour marker alpha-fetoprotein, the increase in expression of liver cell markers cytokeratin 8 and carcinoembryonic antigen, and in some cells the liver stem cell marker cytokeratin 19. Using in vivo bioluminescence imaging we found that many of these tumour cells remained dormant as long as MYC remain inactivated; however, MYC reactivation immediately restored their neoplastic features. Using array comparative genomic hybridization we confirmed that these dormant liver cells and the restored tumour retained the identical molecular signature and hence were clonally derived from the tumour cells. Our results show how oncogene inactivation may reverse tumorigenesis in the most clinically difficult cancers. Oncogene inactivation uncovers the pluripotent capacity of tumours to differentiate into normal cellular lineages and tissue structures, while retaining their latent potential to become cancerous, and hence existing in a state of tumour dormancy.

  View details for DOI 10.1038/nature03043

  View details for Web of Science ID 000224730800044

  View details for PubMedID 15475948

• Extracellular replication of Listeria monocytogenes in the murine gall bladder SCIENCE Hardy, J., Francis, K. P., deBoer, M., Chu, P., Gibbs, K., Contag, C. H. 2004; 303 (5659): 851-853

  Abstract

  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.

  View details for Web of Science ID 000188753800062

  View details for PubMedID 14764883

• Shifting foci of hematopoiesis during reconstitution from single stem cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Cao, Y. A., Wagers, A. J., Beilhack, A., Dusich, J., Bachmann, M. H., Negrin, R. S., Weissman, I. L., Contag, C. H. 2004; 101 (1): 221-226

  Abstract

  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.

  View details for PubMedID 14688412

• Visualizing gene expression in living mammals using a bioluminescent reporter PHOTOCHEMISTRY AND PHOTOBIOLOGY Contag, C. H., Spilman, S. D., Contag, P. R., Oshiro, M., Eames, B., Dennery, P., Stevenson, D. K., Benaron, D. A. 1997; 66 (4): 523-531

  Abstract

  Control of gene expression often involves an interwoven set of regulatory processes. As information regarding regulatory pathways may be lost in ex vivo analyses, we used bioluminescence to monitor gene expression in living mammals. Viral promoters fused to firefly luciferase as transgenes in mice allowed external monitoring of gene expression both superficially and in deep tissues. In vivo bioluminescence was detectable using either intensified or cooled charge-coupled device cameras, and could be detected following both topical and systemic delivery of substrate. In vivo control of the promoter from the human immunodeficiency virus was demonstrated. As a model for DNA-based therapies and vaccines, in vivo transfection of a luciferase expression vector (SV-40 promoter and enhancer controlling expression) was detected. We conclude that gene regulation, DNA delivery and expression can now be noninvasively monitored in living mammals using a luciferase reporter. Thus, real-time, noninvasive study of gene expression in living animal models for human development and disease is possible.

  View details for PubMedID 9337626

• Photonic detection of bacterial pathogens in living hosts MOLECULAR MICROBIOLOGY Contag, C. H., Contag, P. R., Mullins, J. I., Spilman, S. D., Stevenson, D. K., Benaron, D. A. 1995; 18 (4): 593-603

  Abstract

  The study of pathogenic processes is often limited to ex vivo assays and cell-culture correlates. A greater understanding of infectious diseases would be facilitated by in vivo analyses. Therefore, we have developed a method for detecting bacterial pathogens in a living host and used this method to evaluate disease processes for strains of Salmonella typhimurlum that differ in their virulence for mice. Three strains of Salmonella were marked with bioluminescence through transformation with a plasmid conferring constitutive expression of bacterial luciferase. Detection of photons transmitted through tissues of animals infected with bioluminescent Salmonella allowed localization of the bacteria to specific tissues. In this manner progressive infections were distinguished from those that were persistent or abortive. We observed patterns of bioluminescence that suggested the caecum may play a pivotal role in Salmonella pathogenesis. In vivo efficacy of an antibiotic was monitored using this optical method. This study demonstrates that real time non-invasive analyses of pathogenic events and pharmacological monitoring can be performed in vivo.

  View details for PubMedID 8817482

• Flavinated SDHA underlies the change in intrinsic optical properties of oral cancers. Communications biology Marumo, T., Maduka, C. V., Ural, E., Apu, E. H., Chung, S., Tanabe, K., van den Berg, N. S., Zhou, Q., Martin, B. A., Miura, T., Rosenthal, E. L., Shibahara, T., Contag, C. H. 2023; 6 (1): 1134

  Abstract

  The molecular basis of reduced autofluorescence in oral squamous cell carcinoma (OSCC) cells relative to normal cells has been speculated to be due to lower levels of free flavin adenine dinucleotide (FAD). This speculation, along with differences in the intrinsic optical properties of extracellular collagen, lies at the foundation of the design of currently-used clinical optical detection devices. Here, we report that free FAD levels may not account for differences in autofluorescence of OSCC cells, but that the differences relate to FAD as a co-factor for flavination. Autofluorescence from a 70kDa flavoprotein, succinate dehydrogenase A (SDHA), was found to be responsible for changes in optical properties within the FAD spectral region, with lower levels of flavinated SDHA in OSCC cells. Since flavinated SDHA is required for functional complexation with succinate dehydrogenase B (SDHB), decreased SDHB levels were observed in human OSCC tissue relative to normal tissues. Accordingly, the metabolism of OSCC cells was found to be significantly altered relative to normal cells, revealing vulnerabilities for both diagnosis and targeted therapy. Optimizing non-invasive tools based on optical and metabolic signatures of cancers will enable more precise and early diagnosis leading to improved outcomes in patients.

  View details for DOI 10.1038/s42003-023-05510-w

  View details for PubMedID 37945749

• Glycolytic reprogramming in macrophages and MSCs during inflammation. Frontiers in immunology Li, X., Shen, H., Zhang, M., Teissier, V., Huang, E. E., Gao, Q., Tsubosaka, M., Toya, M., Kushioka, J., Maduka, C. V., Contag, C. H., Chow, S. K., Zhang, N., Goodman, S. B. 2023; 14: 1199751

  Abstract

  Dysregulated inflammation is associated with many skeletal diseases and disorders, such as osteolysis, non-union of fractures, osteonecrosis, osteoarthritis and orthopaedic infections. We previously showed that continuous infusion of lipopolysaccharide (LPS) contaminated polyethylene particles (cPE) caused prolonged inflammation and impaired bone formation. However, the metabolic and bioenergetic processes associated with inflammation of bone are unknown. Mitochondria are highly dynamic organelles that modulate cell metabolism and orchestrate the inflammatory responses that involve both resident and recruited cells. Glycolytic reprogramming, the shift from oxidative phosphorylation (OXPHOS) to glycolysis causes inappropriate cell activation and function, resulting in dysfunctional cellular metabolism. We hypothesized that impaired immunoregulation and bone regeneration from inflammatory states are associated with glycolytic reprogramming and mitochondrial dysfunction in macrophages (Mφ) and mesenchymal stromal cells (MSCs).We used the Seahorse XF96 analyzer and real-time qPCR to study the bioenergetics of Mφ and MSCs exposed to cPE. To understand the oxygen consumption rate (OCR), we used Seahorse XF Cell Mito Stress Test Kit with Seahorse XF96 analyzer. Similarly, Seahorse XF Glycolytic Rate Assay Kit was used to detect the extracellular acidification rate (ECAR) and Seahorse XF Real-Time ATP Rate Assay kit was used to detect the real-time ATP production rates from OXPHOS and glycolysis. Real-time qPCR was performed to analyze the gene expression of key enzymes in glycolysis and mitochondrial biogenesis. We further detected the gene expression of proinflammatory cytokines in Mφ and genes related to cell differentiation in MSC during the challenge of cPE.Our results demonstrated that the oxidative phosphorylation of Mφ exposed to cPE was significantly decreased when compared with the control group. We found reduced basal, maximal and ATP-production coupled respiration rates, and decreased proton leak in Mφ during challenge with cPE. Meanwhile, Mφ showed increased basal glycolysis and proton efflux rates (PER) when exposed to cPE. The percentage (%) of PER from glycolysis was higher in Mφ exposed to cPE, indicating that the contribution of the glycolytic pathway to total extracellular acidification was elevated during the challenge of cPE. In line with the results of OCR and ECAR, we found Mφ during cPE challenge showed higher glycolytic ATP (glycoATP) production rates and lower mitochondrial ATP (mitoATP) production rates which is mainly from OXPHOS. Interestingly, MSCs showed enhanced glycolysis during challenge with cPE, but no significant changes in oxygen consumption rates (OCR). In accordance, seahorse assay of real-time ATP revealed glycoATP rates were elevated while mitoATP rates showed no significant differences in MSC during challenge with cPE. Furthermore, Mφ and MSCs exposed to cPE showed upregulated gene expression levels of glycolytic regulators and Mφ exposed to cPE expressed higher levels of pro-inflammatory cytokines.This study demonstrated the dysfunctional bioenergetic activity of bone marrow-derived Mφ and MSCs exposed to cPE, which could impair the immunoregulatory properties of cells in the bone niche. The underlying molecular defect related to disordered mitochondrial function could represent a potential therapeutic target during the resolution of inflammation.

  View details for DOI 10.3389/fimmu.2023.1199751

  View details for PubMedID 37675119

  View details for PubMedCentralID PMC10477714

• Gla-domain mediated targeting of externalized phosphatidylserine for intracellular delivery. FASEB journal : official publication of the Federation of American Societies for Experimental Biology Hardy, J., Bauzon, M., Chan, C. K., Makela, A. V., Kanada, M., Schneider, D., Blankenberg, F., Contag, C. H., Hermiston, T. 2023; 37 (8): e23113

  Abstract

  Phosphatidylserine (PS) is a negatively charged phospholipid normally localized to the inner leaflet of the plasma membrane of cells but is externalized onto the cell surface during apoptosis as well as in malignant and infected cells. Consequently, PS may comprise an important molecular target in diagnostics, imaging, and targeted delivery of therapeutic agents. While an array of PS-binding molecules exist, their utility has been limited by their inability to internalize diagnostic or therapeutic payloads. We describe the generation, isolation, characterization, and utility of a PS-binding motif comprised of a carboxylated glutamic acid (GLA) residue domain that both recognizes and binds cell surface-exposed PS, and then unlike other PS-binding molecules is internalized into these cells. Internalization is independent of the traditional endosomal-lysosomal pathway, directly entering the cytosol of the target cell rapidly. We demonstrate that this PS recognition extends to stem cells and that GLA-domain-conjugated probes can be detected upon intravenous administration in animal models of infectious disease and cancer. GLA domain binding and internalization offer new opportunities for specifically targeting cells with surface-exposed PS for imaging and delivery of therapeutics.

  View details for DOI 10.1096/fj.202201250RRR

  View details for PubMedID 37486772

• Flavinated SDHA Underlies the Change in Intrinsic Optical Properties of Oral Cancers. bioRxiv : the preprint server for biology Marumo, T., Maduka, C. V., Ural, E., Apu, E. H., Chung, S., van den Berg, N. S., Zhou, Q., Martin, B. A., Rosenthal, E. L., Shibahara, T., Contag, C. H. 2023

  Abstract

  The molecular basis of reduced autofluorescence in oral squamous cell carcinoma (OSCC) cells relative to normal cells has been speculated to be due to lower levels of free flavin adenine dinucleotide (FAD). This speculation, along with differences in the intrinsic optical properties of extracellular collagen, lie at the foundation of the design of currently-used clinical optical detection devices. Here, we report that free FAD levels may not account for differences in autofluorescence of OSCC cells, but that the differences relate to FAD as a co-factor for flavination. Autofluorescence from a 70 kDa flavoprotein, succinate dehydrogenase A (SDHA), was found to be responsible for changes in optical properties within the FAD spectral region with lower levels of flavinated SDHA in OSCC cells. Since flavinated SDHA is required for functional complexation with succinate dehydrogenase B (SDHB), decreased SDHB levels were observed in human OSCC tissue relative to normal tissues. Accordingly, the metabolism of OSCC cells was found to be significantly altered relative to normal cells, revealing vulnerabilities for both diagnosis and targeted therapy. Optimizing non-invasive tools based on optical and metabolic signatures of cancers will enable more precise and early diagnosis leading to improved outcomes in patients.

  View details for DOI 10.1101/2023.07.30.551184

  View details for PubMedID 37577521

• Metabolic profile of mesenchymal stromal cells and macrophages in the presence of polyethylene particles in a 3D model. Stem cell research & therapy Teissier, V., Gao, Q., Shen, H., Li, J., Li, X., Huang, E. E., Kushioka, J., Toya, M., Tsubosaka, M., Hirata, H., Alizadeh, H. V., Maduka, C. V., Contag, C. H., Yang, Y. P., Zhang, N., Goodman, S. B. 2023; 14 (1): 99

  Abstract

  Continuous cross talk between MSCs and macrophages is integral to acute and chronic inflammation resulting from contaminated polyethylene particles (cPE); however, the effect of this inflammatory microenvironment on mitochondrial metabolism has not been fully elucidated. We hypothesized that (a) exposure to cPE leads to impaired mitochondrial metabolism and glycolytic reprogramming and (b) macrophages play a key role in this pathway.We cultured MSCs with/without uncommitted M0 macrophages, with/without cPE in 3-dimensional gelatin methacrylate (3D GelMA) constructs/scaffolds. We evaluated mitochondrial function (membrane potential and reactive oxygen species-ROS production), metabolic pathways for adenosine triphosphate (ATP) production (glycolysis or oxidative phosphorylation) and response to stress mechanisms. We also studied macrophage polarization toward the pro-inflammatory M1 or the anti-inflammatory M2 phenotype and the osteogenic differentiation of MSCs.Exposure to cPE impaired mitochondrial metabolism of MSCs; addition of M0 macrophages restored healthy mitochondrial function. Macrophages exposed to cPE-induced glycolytic reprogramming, but also initiated a response to this stress to restore mitochondrial biogenesis and homeostatic oxidative phosphorylation. Uncommitted M0 macrophages in coculture with MSC polarized to both M1 and M2 phenotypes. Osteogenesis was comparable among groups after 21 days.This work confirmed that cPE exposure triggers impaired mitochondrial metabolism and glycolytic reprogramming in a 3D coculture model of MSCs and macrophages and demonstrated that macrophages cocultured with MSCs undergo metabolic changes to maintain energy production and restore homeostatic metabolism.

  View details for DOI 10.1186/s13287-023-03260-4

  View details for PubMedID 37085909

  View details for PubMedCentralID PMC10122387

• Stereochemistry Determines Immune Cellular Responses to Polylactide Implants. ACS biomaterials science & engineering Maduka, C. V., Alhaj, M., Ural, E., Kuhnert, M. M., Habeeb, O. M., Schilmiller, A. L., Hankenson, K. D., Goodman, S. B., Narayan, R., Contag, C. H. 2023

  Abstract

  Repeating l- and d-chiral configurations determine polylactide (PLA) stereochemistry, which affects its thermal and physicochemical properties, including degradation profiles. Clinically, degradation of implanted PLA biomaterials promotes prolonged inflammation and excessive fibrosis, but the role of PLA stereochemistry is unclear. Additionally, although PLA of varied stereochemistries causes differential immune responses in vivo, this observation has yet to be effectively modeled in vitro. A bioenergetic model was applied to study immune cellular responses to PLA containing >99% l-lactide (PLLA), >99% d-lactide (PDLA), and a 50/50 melt-blend of PLLA and PDLA (stereocomplex PLA). Stereocomplex PLA breakdown products increased IL-1β, TNF-α, and IL-6 protein levels but not MCP-1. Expression of these proinflammatory cytokines is mechanistically driven by increases in glycolysis in primary macrophages. In contrast, PLLA and PDLA degradation products selectively increase MCP-1 protein expression. Although both oxidative phosphorylation and glycolysis are increased with PDLA, only oxidative phosphorylation is increased with PLLA. For each biomaterial, glycolytic inhibition reduces proinflammatory cytokines and markedly increases anti-inflammatory (IL-10) protein levels; differential metabolic changes in fibroblasts were observed. These findings provide mechanistic explanations for the diverse immune responses to PLA of different stereochemistries and underscore the pivotal role of immunometabolism in the biocompatibility of biomaterials applied in medicine.

  View details for DOI 10.1021/acsbiomaterials.2c01279

  View details for PubMedID 36634351

• Efficacy of Cathelicidin-Mimetic Antimicrobial Peptoids against Staphylococcus aureus. Microbiology spectrum Benjamin, A. B., Moule, M. G., Didwania, M. K., Hardy, J., Saenkham-Huntsinger, P., Sule, P., Nielsen, J. E., Lin, J. S., Contag, C. H., Barron, A. E., Cirillo, J. D. 2022: e0053422

  Abstract

  Staphylococcus aureus is one of the most common pathogens associated with infection in wounds. The current standard of care uses a combination of disinfection and drainage followed by conventional antibiotics such as methicillin. Methicillin and vancomycin resistance has rendered these treatments ineffective, often causing the reemergence of infection. This study examines the use of antimicrobial peptoids (sequence-specific poly-N-substituted glycines) designed to mimic naturally occurring cationic, amphipathic host defense peptides, as an alternative to conventional antibiotics. These peptoids also show efficient and fast (<30min) killing of methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) at low micromolar concentrations without having apparent cytotoxic side effects in vivo. Additionally, these novel peptoids show excellent efficacy against biofilm formation and detachment for both MSSA and MRSA. In comparison, conventional antibiotics were unable to detach or prevent formation of biofilms. One cationic 12mer, Peptoid 1, shows great promise, as it could prevent formation of and detach biofilms at concentrations as low as 1.6muM. The use of a bioluminescent S. aureus murine incision wound model demonstrated clearance of infection in peptoid-treated mice within 8days, conveying another advantage these peptoids have over conventional antibiotics. These results provide clear evidence of the potential for antimicrobial peptoids for the treatment of S. aureus wound infections. IMPORTANCE Staphylococcus aureus resistance is a consistent problem with a large impact on the health care system. Infections with resistant S. aureus can cause serious adverse effects and can result in death. These antimicrobial peptoids show efficient killing of bacteria both as a biofilm and as free bacteria, often doing so in less than 30 min. As such, these antimicrobials have the potential to alleviate the burden that Staphylococcus infections have on the health care system and cause better outcomes for infected patients.

  View details for DOI 10.1128/spectrum.00534-22

  View details for PubMedID 35467395

• Molecular Imaging of Infective Endocarditis With 6''-[18F]Fluoromaltotriose Positron Emission Tomography-Computed Tomography. Circulation Wardak, M., Gowrishankar, G., Zhao, X., Liu, Y., Chang, E., Namavari, M., Haywood, T., Gabr, M. T., Neofytou, E., Chour, T., Qin, X., Vilches-Moure, J. G., Hardy, J., Contag, C. H., McConnell, M. V., Wu, J. C., Gambhir, S. S. 2020; 141 (21): 1729–31

  View details for DOI 10.1161/CIRCULATIONAHA.119.043924

  View details for PubMedID 32453662

• Software-Based Phase Control, Video-Rate Imaging, and Real-Time Mosaicing With a Lissajous-Scanned Confocal Microscope IEEE TRANSACTIONS ON MEDICAL IMAGING Loewke, N. O., Qiu, Z., Mandella, M. J., Ertsey, R., Loewke, A., Gunaydin, L. A., Rosenthal, E. L., Contag, C. H., Solgaard, O. 2020; 39 (4): 1127–37

  Abstract

  We present software-based methods for automatic phase control and for mosaicing high-speed, Lissajous-scanned images. To achieve imaging speeds fast enough for mosaicing, we first increase the image update rate tenfold from 3 to 30 Hz, then vertically interpolate each sparse image in real-time to eliminate fixed pattern noise. We validate our methods by imaging fluorescent beads and automatically maintaining phase control over the course of one hour. We then image fixed mouse brain tissues at varying update rates and compare the resulting mosaics. Using reconstructed image data as feedback for phase control eliminates the need for phase sensors and feedback controllers, enabling long-term imaging experiments without additional hardware. Mosaicing subsampled images results in video-rate imaging speeds, nearly fully recovered spatial resolution, and millimeter-scale fields of view.

  View details for DOI 10.1109/TMI.2019.2942552

  View details for Web of Science ID 000525265800029

  View details for PubMedID 31567074

• Nano-immunoimaging. Nanoscale horizons Wang, P. n., Kim, T. n., Harada, M. n., Contag, C. n., Huang, X. n., Smith, B. R. 2020; 5 (4): 628–53

  Abstract

  Immunoimaging is a rapidly growing field stoked in large part by the intriguing triumphs of immunotherapy. On the heels of immunotherapy's successes, there exists a growing need to evaluate tumor response to therapy particularly immunotherapy, stratify patients into responders vs. non-responders, identify inflammation, and better understand the fundamental roles of immune system components to improve both immunoimaging and immunotherapy. Innovative nanomaterials have begun to provide novel opportunities for immunoimaging, in part due to their sensitivity, modularity, capacity for many potentially varied ligands (high avidity), and potential for multifunctionality/multimodality imaging. This review strives to comprehensively summarize the integration of nanotechnology and immunoimaging, and the field's potential for clinical applications.

  View details for DOI 10.1039/c9nh00514e

  View details for PubMedID 32226975

• Biodegradable Fluorescent Nanoparticles for Endoscopic Detection of Colorectal Carcinogenesis ADVANCED FUNCTIONAL MATERIALS Rogalla, S., Flisikowski, K., Gorpas, D., Mayer, A. T., Flisikowska, T., Mandella, M. J., Ma, X., Casey, K. M., Felt, S. A., Saur, D., Ntziachristos, V., Schnieke, A., Contag, C. H., Gambhir, S. S., Harmsen, S. 2019; 29 (51)

  View details for DOI 10.1002/adfm.201904992

  View details for Web of Science ID 000516572400007

• Tunable structured illumination light sheet microscopy for background rejection and imaging depth in minimally processed tissues. Journal of biomedical optics Landry, J. R., Itoh, R., Li, J. M., Hamann, S. S., Mandella, M., Contag, C. H., Solgaard, O. 2019; 24 (4): 1–6

  Abstract

  We demonstrate improved optical sectioning in light sheet fluorescence microscopy using tunable structured illumination (SI) frequencies to optimize image quality in scattering specimens. The SI patterns are generated coherently using a one-dimensional spatial light modulator for maximum pattern contrast, and the pattern spatial frequency is adjustable up to half the incoherent cutoff frequency of our detection objective. At this frequency, we demonstrate background reductions of 2 orders of magnitude.

  View details for PubMedID 30968649

• Tunable structured illumination light sheet microscopy for background rejection and imaging depth in minimally processed tissues JOURNAL OF BIOMEDICAL OPTICS Landry, J. R., Itoh, R., Li, J. M., Hamann, S. S., Mandella, M., Contag, C. H., Solgaard, O. 2019; 24 (4)

  View details for DOI 10.1117/1.JBO.24.4.046501

  View details for Web of Science ID 000481879600009

• Functional DNA Delivery Enabled by Lipid-Modified Charge-Altering Releasable Transporters (CARTs) BIOMACROMOLECULES Benner, N. L., Near, K. E., Bachmann, M. H., Contag, C. H., Waymouth, R. M., Wender, P. A. 2018; 19 (7): 2812–24

  Abstract

  Safe and effective DNA delivery systems are required to enable or enhance clinical strategies and research involving gene therapy and DNA vaccinations. To address this delivery problem, a series of charge-altering releasable transporters (CARTs) with varied lipid content were prepared and evaluated for plasmid DNA (pDNA) delivery into cultured cells. These lipid-modified CART co-oligomers were synthesized in only two steps via sequential organocatalytic ring-opening polymerization of lipid-containing cyclic carbonate monomers and morpholinone monomers. Lipid variations of the CARTs substantially impacted the delivery efficiency of pDNA, with oleyl- and linoleyl-based CARTs showing enhanced performance relative to the commercial transfection agent Lipofectamine 2000 (L2000). The best-performing oleyl CART was carried forward to study stable luciferase transfection with a Sleeping Beauty ( SB) transposon system. The oleyl CART outperformed the L2000 positive control with respect to stable transfection efficiency. CART-pDNA complexes represent a new DNA delivery system for research and clinical applications.

  View details for DOI 10.1021/acs.biomac.8b00401

  View details for Web of Science ID 000438470800044

  View details for PubMedID 29727572

• A Dual-Modality Hybrid Imaging System Harnesses Radioluminescence and Sound to Reveal Molecular Pathology of Atherosclerotic Plaques SCIENTIFIC REPORTS Zaman, R. T., Yousefi, S., Long, S. R., Saito, T., Mandella, M., Qiu, Z., Chen, R., Contag, C. H., Gambhir, S. S., Chin, F. T., Khuri-Yakub, B. T., McConnell, M. V., Shung, K., Xing, L. 2018; 8: 8992

  Abstract

  Atherosclerosis is a progressive inflammatory condition caused by an unstable lesion, called thin-cap fibro atheromata (TCFA) that underlies coronary artery disease (CAD)-one of the leading causes of death worldwide. Therefore, early clinical diagnosis and effective risk stratification is important for CAD management as well as preventing progression to catastrophic events. However, early detection could be difficult due to their small size, motion, obscuring 18F-FDG uptake by adjacent myocardium, and complex morphological/biological features. To overcome these limitations, we developed a catheter-based Circumferential-Intravascular-Radioluminescence-Photoacoustic-Imaging (CIRPI) system that can detect vulnerable plaques in coronary arteries and characterizes them with respect to pathology and biology. Our CIRPI system combined two imaging modalities: Circumferential Radioluminescence Imaging (CRI) and PhotoAcoustic Tomography (PAT) within a novel optical probe. The probe's CaF2:Eu based scintillating imaging window provides a 360° view of human (n = 7) and murine carotid (n = 10) arterial plaques by converting β-particles into visible photons during 18F-FDG decay. A 60× and 63× higher radioluminescent signals were detected from the human and murine plaque inflammations, respectively, compared to the control. The system's photoacoustic imaging provided a comprehensive analysis of the plaque compositions and its morphologic information. These results were further verified with IVIS-200, immunohistochemical analysis, and autoradiography.

  View details for PubMedID 29895966

• Automated Cell Segmentation for Quantitative Phase Microscopy IEEE TRANSACTIONS ON MEDICAL IMAGING Loewke, N. O., Pai, S., Cordeiro, C., Black, D., King, B. L., Contag, C. H., Chen, B., Baer, T. M., Solgaard, O. 2018; 37 (4): 929–40

  Abstract

  Automated cell segmentation and tracking is essential for dynamic studies of cellular morphology, movement, and interactions as well as other cellular behaviors. However, accurate, automated, and easy-to-use cell segmentation remains a challenge, especially in cases of high cell densities, where discrete boundaries are not easily discernable. Here, we present a fully automated segmentation algorithm that iteratively segments cells based on the observed distribution of optical cell volumes measured by quantitative phase microscopy. By fitting these distributions to known probability density functions, we are able to converge on volumetric thresholds that enable valid segmentation cuts. Since each threshold is determined from the observed data itself, virtually no input is needed from the user. We demonstrate the effectiveness of this approach over time using six cell types that display a range of morphologies, and evaluate these cultures over a range of confluencies. Facile dynamic measures of cell mobility and function revealed unique cellular behaviors that relate to tissue origins, state of differentiation, and real-time signaling. These will improve our understanding of multicellular communication and organization.

  View details for DOI 10.1109/TMI.2017.2775604

  View details for Web of Science ID 000428886700010

  View details for PubMedID 29610072

  View details for PubMedCentralID PMC5907807

• Regulatory Aspects of Optical Methods and Exogenous Targets for Cancer Detection (vol 77, pg 2197, 2017) CANCER RESEARCH Tummers, W. S., Warram, J. M., Tipirneni, K. E., Fengler, J., Jacobs, P., Shankar, L., Henderson, L., Ballard, B., Pfefer, T., Pogue, B. W., Weichert, J. P., Bouvet, M., Sorger, J., Contag, C. H., Frangioni, J. V., Tweedle, M. F., Basilion, J. P., Gambhir, S. S., Rosenthal, E. L. 2018; 78 (4): 1123

  View details for DOI 10.1158/0008-5472.CAN-17-3847

  View details for Web of Science ID 000425148400022

• Cell Labeling with Magneto-Endosymbionts and the Dissection of the Subcellular Location, Fate, and Host Cell Interactions MOLECULAR IMAGING AND BIOLOGY Lee, K. R., Wakeel, A., Chakraborty, P., Foote, C. S., Kajiura, L., Barrozo, J. C., Chan, A. C., Bazarov, A. V., Spitler, R., Kutny, P. M., Denegre, J. M., Taft, R. A., Seemann, J., Rice, B. W., Contag, C. H., Rutt, B. K., Bell, C. B. 2018; 20 (1): 55–64

  Abstract

  The purposes of this study are to characterize magneto-endosymbiont (ME) labeling of mammalian cells and to discern the subcellular fate of these living contrast agents. MEs are novel magnetic resonance imaging (MRI) contrast agents that are being used for cell tracking studies. Understanding the fate of MEs in host cells is valuable for designing in vivo cell tracking experiments.The ME's surface epitopes, contrast-producing paramagnetic magnetosomal iron, and genome were studied using immunocytochemistry (ICC), Fe and MRI contrast measurements, and quantitative polymerase chain reaction (qPCR), respectively. These assays, coupled with other common assays, enabled validation of ME cell labeling and dissection of ME subcellular processing.The assays mentioned above provide qualitative and quantitative assessments of cell labeling, the subcellular localization and the fate of MEs. ICC results, with an ME-specific antibody, qualitatively shows homogenous labeling with MEs. The ferrozine assay shows that MEs have an average of 7 fg Fe/ME, ∼30 % of which contributes to MRI contrast and ME-labeled MDA-MB-231 (MDA-231) cells generally have 2.4 pg Fe/cell, implying ∼350 MEs/cell. Adjusting the concentration of Fe in the ME growth media reduces the concentration of non-MRI contrast-producing Fe. Results from the qPCR assay, which quantifies ME genomes in labeled cells, shows that processing of MEs begins within 24 h in MDA-231 cells. ICC results suggest this intracellular digestion of MEs occurs by the lysosomal degradation pathway. MEs coated with listeriolysin O (LLO) are able to escape the primary phagosome, but subsequently co-localize with LC3, an autophagy-associated molecule, and are processed for digestion. In embryos, where autophagy is transiently suppressed, MEs show an increased capacity for survival and even replication. Finally, transmission electron microscopy (TEM) of ME-labeled MDA-231 cells confirms that the magnetosomes (the MRI contrast-producing particles) remain intact and enable in vivo cell tracking.MEs are used to label mammalian cells for the purpose of cell tracking in vivo, with MRI. Various assays described herein (ICC, ferrozine, and qPCR) allow qualitative and quantitative assessments of labeling efficiency and provide a detailed understanding of subcellular processing of MEs. In some cell types, MEs are digested, but the MRI-producing particles remain. Coating with LLO allows MEs to escape the primary phagosome, enhances retention slightly, and confirms that MEs are ultimately processed by autophagy. Numerous intracellular bacteria and all endosymbiotically derived organelles have evolved molecular mechanisms to avoid intracellular clearance, and identification of the specific processes involved in ME clearance provides a framework on which to develop MEs with enhanced retention in mammalian cells.

  View details for PubMedID 28631141

  View details for PubMedCentralID PMC5736464

• Characterization of Magneto-Endosymbionts as MRI Cell Labeling and Tracking Agents MOLECULAR IMAGING AND BIOLOGY Brewer, K. D., Spitler, R., Lee, K. R., Chan, A. C., Barrozo, J. C., Wakeel, A., Foote, C. S., Machtaler, S., Rioux, J., Willmann, J. K., Chakraborty, P., Rice, B. W., Contag, C. H., Bell, C. B., Rutt, B. K. 2018; 20 (1): 65–73

  Abstract

  Magneto-endosymbionts (MEs) show promise as living magnetic resonance imaging (MRI) contrast agents for in vivo cell tracking. Here we characterize the biomedical imaging properties of ME contrast agents, in vitro and in vivo.By adapting and engineering magnetotactic bacteria to the intracellular niche, we are creating magneto-endosymbionts (MEs) that offer advantages relative to passive iron-based contrast agents (superparamagnetic iron oxides, SPIOs) for cell tracking. This work presents a biomedical imaging characterization of MEs including: MRI transverse relaxivity (r 2) for MEs and ME-labeled cells (compared to a commercially available iron oxide nanoparticle); microscopic validation of labeling efficiency and subcellular locations; and in vivo imaging of a MDA-MB-231BR (231BR) human breast cancer cells in a mouse brain.At 7T, r 2 relaxivity of bare MEs was higher (250 s-1 mM-1) than that of conventional SPIO (178 s-1 mM-1). Optimized in vitro loading of MEs into 231BR cells yielded 1-4 pg iron/cell (compared to 5-10 pg iron/cell for conventional SPIO). r 2 relaxivity dropped by a factor of ~3 upon loading into cells, and was on the same order of magnitude for ME-loaded cells compared to SPIO-loaded cells. In vivo, ME-labeled cells exhibited strong MR contrast, allowing as few as 100 cells to be detected in mice using an optimized 3D SPGR gradient-echo sequence.Our results demonstrate the potential of magneto-endosymbionts as living MR contrast agents. They have r 2 relaxivity values comparable to traditional iron oxide nanoparticle contrast agents, and provide strong MR contrast when loaded into cells and implanted in tissue.

  View details for PubMedID 28616842

  View details for PubMedCentralID PMC5730509

• Implantable, insertable and wearable micro-optical devices for early detection of cancer Contag, C. H., Winzer, P., Tsang, H. K., Capmany, J., Yao, J., Fontaine, N., Dutta, N. IEEE. 2018

  View details for Web of Science ID 000460542800140

• Magnetic particle imaging of islet transplantation in the liver and under the kidney capsule in mouse models. Quantitative imaging in medicine and surgery Wang, P. n., Goodwill, P. W., Pandit, P. n., Gaudet, J. n., Ross, A. n., Wang, J. n., Yu, E. n., Hensley, D. W., Doyle, T. C., Contag, C. H., Conolly, S. n., Moore, A. n. 2018; 8 (2): 114–22

  Abstract

  Islet transplantation (Tx) represents the most promising therapy to restore normoglycemia in type 1 diabetes (T1D) patients to date. As significant islet loss has been observed after the procedure, there is an urgent need for developing strategies for monitoring transplanted islet grafts. In this report we describe for the first time the application of magnetic particle imaging (MPI) for monitoring transplanted islets in the liver and under the kidney capsule in experimental animals.Pancreatic islets isolated from Papio hamadryas were labeled with superparamagnetic iron oxides (SPIOs) and used for either islet phantoms or Tx in the liver or under the kidney capsule of NOD scid mice. MPI was used to image and quantify islet phantoms and islet transplanted experimental animals post-mortem at 1 and 14 days after Tx. Magnetic resonance imaging (MRI) was used to confirm the presence of labeled islets in the liver and under the kidney capsule 1 day after Tx.MPI of labeled islet phantoms confirmed linear correlation between the number of islets and the MPI signal (R2=0.988). Post-mortem MPI performed on day 1 after Tx showed high signal contrast in the liver and under the kidney capsule. Quantitation of the signal supports islet loss over time, which is normally observed 2 weeks after Tx. No MPI signal was observed in control animals. In vivo MRI confirmed the presence of labeled islets/islet clusters in liver parenchyma and under the kidney capsule one day after Tx.Here we demonstrate that MPI can be used for quantitative detection of labeled pancreatic islets in the liver and under the kidney capsule of experimental animals. We believe that MPI, a modality with no depth attenuation and zero background tissue signal could be a suitable method for imaging transplanted islet grafts.

  View details for PubMedID 29675353

  View details for PubMedCentralID PMC5891680

• Preparation of Tc99m-Labeled Pseudomonas Bacteriophage without Adversely Impacting Infectivity or Biodistribution BIOCONJUGATE CHEMISTRY Holman, D., Lungren, M. P., Hardy, J., Contag, C., Blankenberg, F. 2017; 28 (11): 2698–2706

  Abstract

  Bacteriophages (phages) are ubiquitous viruses which have adapted to infect and replicate within target bacteria, their only known hosts, in a strain specific fashion with minimal cross infectivity. The recent steep rise in antibiotic resistance throughout the world has renewed interest in adapting phages for the imaging and treatment of bacterial infection in humans. In this article, we describe the current limitations surrounding the radiolabeling of phage for the imaging and treatment of bacterial infection and methods to overcome these difficulties. Specifically, we examined the effects of hydrazinonicotinamide conjugation and removal of bacterial DNA on the infectivity, biodistribution, and radionuclide imaging of a phage lytic for a clinically relevant strain of Pseudomonas aeruginosa, a common Gram-negative bacterial pathogen often resistant to multiple antibiotics. We found that all but the briefest reaction of concentrated phage with hydrazinonicotinamide (≤3 min) resulted in nearly complete loss of infectivity. Furthermore, we determined that digestion and removal of bacterial DNA was needed to avoid high nonspecific uptake of hydrazinonicotinamide-labeled phage within the liver and spleen as well as prolonged circulation in the blood. We also demonstrate the surprisingly wide soft tissue and organ biodistribution and rapid pharmacokinetics of 99mTc-hydrazinonicotinamide-labeled phage in normal mice as well as its imaging characteristics and efficacy in wounded mice infected with bioluminescent Pseudomonas aeruginosa. In conclusion, the preservation of phage infectivity and removal of all bacterial containments including DNA are critical methodologic considerations in the labeling of phages for imaging and therapy.

  View details for PubMedID 29020448

• The tyrosine kinase inhibitor imatinib mesylate suppresses uric acid crystal-induced acute gouty arthritis in mice PLOS ONE Reber, L. L., Starkl, P., Balbino, B., Sibilano, R., Gaudenzio, N., Rogalla, S., Sensarn, S., Kang, D., Raghu, H., Sokolove, J., Robinson, W. H., Contag, C. H., Tsai, M., Galli, S. J. 2017; 12 (10): e0185704

  Abstract

  Gouty arthritis is caused by the deposition of monosodium urate (MSU) crystals in joints. Despite many treatment options for gout, there is a substantial need for alternative treatments for patients unresponsive to current therapies. Tyrosine kinase inhibitors have demonstrated therapeutic benefit in experimental models of antibody-dependent arthritis and in rheumatoid arthritis in humans, but to date, the potential effects of such inhibitors on gouty arthritis has not been evaluated. Here we demonstrate that treatment with the tyrosine kinase inhibitor imatinib mesylate (imatinib) can suppress inflammation induced by injection of MSU crystals into subcutaneous air pouches or into the ankle joint of wild type mice. Moreover, imatinib treatment also largely abolished the lower levels of inflammation which developed in IL-1R1-/- or KitW-sh/W-sh mice, indicating that this drug can inhibit IL-1-independent pathways, as well as mast cell-independent pathways, contributing to pathology in this model. Imatinib treatment not only prevented ankle swelling and synovial inflammation when administered before MSU crystals but also diminished these features when administrated after the injection of MSU crystals, a therapeutic protocol more closely mimicking the clinical situation in which treatment occurs after the development of an acute gout flare. Finally, we also assessed the efficiency of local intra-articular injections of imatinib-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles in this model of acute gout. Treatment with low doses of this long-acting imatinib:PLGA formulation was able to reduce ankle swelling in a therapeutic protocol. Altogether, these results raise the possibility that tyrosine kinase inhibitors might have utility in the treatment of acute gout in humans.

  View details for PubMedID 28982129

• F-Fluoromaltotriose: A Second Generation PET Tracer Targeting the Maltodextrin Transporter in Bacteria. Journal of nuclear medicine Gowrishankar, G., Hardy, J., Wardak, M., Namavari, M., Reeves, R., Neofytou, E., Srinivasan, A., Wu, J., Contag, C., Gambhir, S. 2017

  Abstract

  Purpose: 6"-(18)F-fluoromaltotriose is a novel positron emission tomography (PET) tracer that can potentially be used to image and localize most bacterial infections, much like 2-deoxy-2-(18)F-fluoro-D-glucose ((18)F-FDG) has been used to image and localize many cancers. However, unlike (18)F-FDG, 6"-(18)F-fluoromaltotriose is not taken up by inflammatory lesions and appears to be specific to bacterial infections by targeting the maltodextrin transporter that is expressed in most Gram-positive and Gram-negative strains of bacteria. Materials and Methods: 6"-(18)F-fluoromaltotriose was synthesized with high radiochemical purity and evaluated in several clinically relevant bacterial strains incultures in vitro and in living mice. Results: 6"-(18)F-fluoromaltotriose was taken up in both Gram-positive and Gram-negative bacterial strains. 6"-[(18)F]-fluoromaltotriose was also able to detect Pseudomonas aeruginosa in a clinically relevant mouse model of wound infection. The utility of 6"-(18)F-fluoromaltotriose to help monitor antibiotic therapies was also evaluated in rats. Conclusion: 6"-(18)F-fluoromaltotriose is a promising new tracer that has significant diagnostic utility, with the potential to change the clinical management of patients suffering from infectious diseases of bacterial origin.

  View details for DOI 10.2967/jnumed.117.191452

  View details for PubMedID 28490473

• Regulatory Aspects of Optical Methods and Exogenous Targets for Cancer Detection CANCER RESEARCH Tummers, W. S., Warram, J. M., Tipirneni, K. E., Fengler, J., Jacobs, P., Shankar, L., Henderson, L., Ballard, B., Pogue, B. W., Weichert, J. P., Bouvet, M., Sorger, J., Contag, C. H., Frangioni, J. V., Tweedle, M. F., Basilion, J. P., Gambhir, S. S., Rosenthal, E. L. 2017; 77 (9): 2197-2206

  Abstract

  Considerable advances in cancer-specific optical imaging have improved the precision of tumor resection. In comparison to traditional imaging modalities, this technology is unique in its ability to provide real-time feedback to the operating surgeon. Given the significant clinical implications of optical imaging, there is an urgent need to standardize surgical navigation tools and contrast agents to facilitate swift regulatory approval. Because fluorescence-enhanced surgery requires a combination of both device and drug, each may be developed in conjunction, or separately, which are important considerations in the approval process. This report is the result of a one-day meeting held on May 4, 2016 with officials from the National Cancer Institute, the FDA, members of the American Society of Image-Guided Surgery, and members of the World Molecular Imaging Society, which discussed consensus methods for FDA-directed human testing and approval of investigational optical imaging devices as well as contrast agents for surgical applications. The goal of this workshop was to discuss FDA approval requirements and the expectations for approval of these novel drugs and devices, packaged separately or in combination, within the context of optical surgical navigation. In addition, the workshop acted to provide clarity to the research community on data collection and trial design. Reported here are the specific discussion items and recommendations from this critical and timely meeting. Cancer Res; 77(9); 2197-206. ©2017 AACR.

  View details for DOI 10.1158/0008-5472.CAN-16-3217

  View details for Web of Science ID 000400270100004

  View details for PubMedID 28428283

• Neutrophil myeloperoxidase diminishes the toxic effects and mortality induced by lipopolysaccharide JOURNAL OF EXPERIMENTAL MEDICINE Reber, L. L., Gillis, C. M., Starkl, P., Joensson, F., Sibilano, R., Marichal, T., Gaudenzio, N., Berard, M., Rogalla, S., Contag, C. H., Bruhns, P., Galli, S. J. 2017; 214 (5): 1249-1258

  Abstract

  Neutrophils have crucial antimicrobial functions but are also thought to contribute to tissue injury upon exposure to bacterial products, such as lipopolysaccharide (LPS). To study the role of neutrophils in LPS-induced endotoxemia, we developed a new mouse model, PMN(DTR) mice, in which injection of diphtheria toxin induces selective neutrophil ablation. Using this model, we found, surprisingly, that neutrophils serve to protect the host from LPS-induced lethal inflammation. This protective role was observed in conventional and germ-free animal facilities, indicating that it does not depend on a particular microbiological environment. Blockade or genetic deletion of myeloperoxidase (MPO), a key neutrophil enzyme, significantly increased mortality after LPS challenge, and adoptive transfer experiments confirmed that neutrophil-derived MPO contributes importantly to protection from endotoxemia. Our findings imply that, in addition to their well-established antimicrobial properties, neutrophils can contribute to optimal host protection by limiting the extent of endotoxin-induced inflammation in an MPO-dependent manner.

  View details for DOI 10.1084/jem.20161238

  View details for Web of Science ID 000400379300006

  View details for PubMedID 28385925

• Imaging of Tumor-Associated Macrophages in a Transgenic Mouse Model of Orthotopic Ovarian Cancer. Molecular imaging and biology He, H., Chiu, A. C., Kanada, M., Schaar, B. T., Krishnan, V., Contag, C. H., Dorigo, O. 2017

  Abstract

  Tumor-associated macrophages (TAMs) are often associated with a poor prognosis in cancer. To gain a better understanding of cellular recruitment and dynamics of TAM biology during cancer progression, we established a novel transgenic mouse model for in vivo imaging of luciferase-expressing macrophages.B6.129P2-Lyz2(tm1(cre)Ifo)/J mice, which express Cre recombinase under the control of the lysozyme M promoter (LysM) were crossed to Cre-lox Luc reporter mice (RLG), to produce LysM-LG mice whose macrophages express luciferase. Cell-type-specific luciferase expression in these mice was verified by flow cytometry, and via in vivo bioluminescence imaging under conditions where macrophages were either stimulated with lipopolysaccharide or depleted with clodronate liposomes. The distribution of activated macrophages was longitudinally imaged in two immunocompetent LysM-LG mouse models with either B16 melanoma or ID8 ovarian cancer cells.In vivo imaging of LysM-LG mice showed luciferase activity was generated by macrophages. Clodronate liposome-mediated depletion of macrophages lowered overall bioluminescence while lipopolysaccharide injection increased macrophage bioluminescence in both the B16 and ID8 models. Tracking macrophages weekly in tumor-bearing animals after intraperitoneal (i.p.) or intraovarian (i.o.) injection resulted in distinct, dynamic patterns of macrophage activity. Animals with metastatic ovarian cancer after i.p. injection exhibited significantly higher peritoneal macrophage activity compared to animals after i.o. injection.The LysM-LG model allows tracking of macrophage recruitment and activation during disease initiation and progression in a noninvasive manner. This model provides a tool to visualize and monitor the benefit of pharmacological interventions targeting macrophages in preclinical models.

  View details for DOI 10.1007/s11307-017-1061-2

  View details for PubMedID 28233218

• Local estrogen axis in the human bone microenvironment regulates estrogen receptor-positive breast cancer cells. Breast cancer research : BCR Amanatullah, D. F., Tamaresis, J. S., Chu, P. n., Bachmann, M. H., Hoang, N. M., Collyar, D. n., Mayer, A. T., West, R. B., Maloney, W. J., Contag, C. H., King, B. L. 2017; 19 (1): 121

  Abstract

  Approximately 70% of all breast cancers express the estrogen receptor, and are regulated by estrogen. While the ovaries are the primary source of estrogen in premenopausal women, most breast cancer is diagnosed following menopause, when systemic levels of this hormone decline. Estrogen production from androgen precursors is catalyzed by the aromatase enzyme. Although aromatase expression and local estrogen production in breast adipose tissue have been implicated in the development of primary breast cancer, the source of estrogen involved in the regulation of estrogen receptor-positive (ER+) metastatic breast cancer progression is less clear.Bone is the most common distant site of breast cancer metastasis, particularly for ER+ breast cancers. We employed a co-culture model using trabecular  bone tissues obtained from total hip replacement (THR) surgery specimens to study ER+ and estrogen receptor-negative (ER-) breast cancer cells within the human bone microenvironment. Luciferase-expressing ER+ (MCF-7, T-47D, ZR-75) and ER- (SK-BR-3, MDA-MB-231, MCF-10A) breast cancer cells were cultured directly on bone tissue fragments or in bone tissue-conditioned media, and monitored over time with bioluminescence imaging (BLI). Bone tissue-conditioned media were generated in the presence vs. absence of aromatase inhibitors, and testosterone. Bone tissue fragments were analyzed for aromatase expression by immunohistochemistry.ER+ breast cancer cells were preferentially sustained in co-cultures with bone tissues and bone tissue-conditioned media relative to ER- cells. Bone fragments analyzed by immunohistochemistry revealed expression of the aromatase enzyme. Bone tissue-conditioned media generated in the presence of testosterone had increased estrogen levels and heightened capacity to stimulate ER+ breast cancer cell proliferation. Pretreatment of cultured bone tissues with aromatase inhibitors, which inhibited estrogen production, reduced the capacity of conditioned media to stimulate ER+ cell proliferation.These results suggest that a local estrogen signaling axis regulates ER+ breast cancer cell viability and proliferation within the bone metastatic niche, and that aromatase inhibitors modulate this axis. Although endocrine therapies are highly effective in the treatment of ER+ breast cancer, resistance to these treatments reduces their efficacy. Characterization of estrogen signaling networks within the bone microenvironment will identify new strategies for combating metastatic progression and endocrine resistance.

  View details for PubMedID 29141657

  View details for PubMedCentralID PMC5688761

• Intracellular biomass flocculation as a key mechanism of rapid bacterial killing by cationic, amphipathic antimicrobial peptides and peptoids. Scientific reports Chongsiriwatana, N. P., Lin, J. S., Kapoor, R. n., Wetzler, M. n., Rea, J. A., Didwania, M. K., Contag, C. H., Barron, A. E. 2017; 7 (1): 16718

  Abstract

  Many organisms rely on antimicrobial peptides (AMPs) as a first line of defense against pathogens. In general, most AMPs are thought to kill bacteria by binding to and disrupting cell membranes. However, certain AMPs instead appear to inhibit biomacromolecule synthesis, while causing less membrane damage. Despite an unclear understanding of mechanism(s), there is considerable interest in mimicking AMPs with stable, synthetic molecules. Antimicrobial N-substituted glycine (peptoid) oligomers ("ampetoids") are structural, functional and mechanistic analogs of helical, cationic AMPs, which offer broad-spectrum antibacterial activity and better therapeutic potential than peptides. Here, we show through quantitative studies of membrane permeabilization, electron microscopy, and soft X-ray tomography that both AMPs and ampetoids trigger extensive and rapid non-specific aggregation of intracellular biomacromolecules that correlates with microbial death. We present data demonstrating that ampetoids are "fast killers", which rapidly aggregate bacterial ribosomes in vitro and in vivo. We suggest intracellular biomass flocculation is a key mechanism of killing for cationic, amphipathic AMPs, which may explain why most AMPs require micromolar concentrations for activity, show significant selectivity for killing bacteria over mammalian cells, and finally, why development of resistance to AMPs is less prevalent than developed resistance to conventional antibiotics.

  View details for PubMedID 29196622

  View details for PubMedCentralID PMC5711933

• A Clinical Wide-Field Fluorescence Endoscopic Device for Molecular Imaging Demonstrating Cathepsin Protease Activity in Colon Cancer. Molecular imaging and biology Sensarn, S., Zavaleta, C. L., Segal, E., Rogalla, S., Lee, W., Gambhir, S. S., Bogyo, M., Contag, C. H. 2016; 18 (6): 820-829

  Abstract

  Early and effective detection of cancers of the gastrointestinal tract will require novel molecular probes and advances in instrumentation that can reveal functional changes in dysplastic and malignant tissues. Here, we describe adaptation of a wide-field clinical fiberscope to perform wide-field fluorescence imaging while preserving its white-light capability for the purpose of providing wide-field fluorescence imaging capability to point-of-care microscopes.We developed and used a fluorescent fiberscope to detect signals from a quenched probe, BMV109, that becomes fluorescent when cleaved by, and covalently bound to, active cathepsin proteases. Cathepsins are expressed in inflammation- and tumor-associated macrophages as well as directly from tumor cells and are a promising target for cancer imaging. The fiberscope has a 1-mm outer diameter enabling validation via endoscopic exams in mice, and therefore we evaluated topically applied BMV109 for the ability to detect colon polyps in an azoxymethane-induced colon tumor model in mice.This wide-field endoscopic imaging device revealed consistent and clear fluorescence signals from BMV109 that specifically localized to the polypoid regions as opposed to the normal adjacent colon tissue (p < 0.004) in the murine colon carcinoma model.The sensitivity of detection of BMV109 with the fluorescence fiberscope suggested utility of these tools for early detection at hard-to-reach sites. The fiberscope was designed to be used in conjunction with miniature, endoscope-compatible fluorescence microscopes for dual wide-field and microscopic cancer detection.

  View details for PubMedID 27154508

• Reactive Oxygen Species Imaging in a Mouse Model of Inflammatory Bowel Disease MOLECULAR IMAGING AND BIOLOGY Bronsart, L., Linh Nguyen, L., Habtezion, A., Contag, C. 2016; 18 (4): 473-478

  Abstract

  Reactive oxygen species (ROS) are important contributors to inflammatory bowel disease (IBD); however, there are insufficient tools for their in vivo evaluation.To determine if a chemiluminescent ROS reporter, coelenterazine, would be a useful tool for the detection of immune cell activation, the macrophage cell line (RAW 264.7) was treated with phorbol myristate acetate (PMA). Additionally, coelenterazine was used to monitor the changes in ROS production over time in a mouse model of IBD.In vitro, coelenterazine enabled the dynamic monitoring of the RAW 264.7 cell oxidative burst. In vivo, there were early, preclinical, changes in the localization and magnitude of coelenterazine chemiluminescent foci.Coelenterazine offers a high-throughput method for assessing immune cell activation in culture and provides a means for the in vivo detection and localization of ROS during IBD disease progression.

  View details for DOI 10.1007/s11307-016-0934-0

  View details for Web of Science ID 000379191800001

  View details for PubMedID 26873653

  View details for PubMedCentralID PMC4927601

• World Molecular Imaging Congress 2016: Imaging Biology-Improving Therapy. Molecular imaging and biology : MIB : the official publication of the Academy of Molecular Imaging Kiessling, F., Contag, C. H. 2016; 18 (3): 313-4

  View details for DOI 10.1007/s11307-016-0958-5

  View details for PubMedID 27080321

• Multimodality Imaging of Cancer Superoxide Anion Using the Small Molecule Coelenterazine. Molecular imaging and biology Bronsart, L. L., Stokes, C., Contag, C. H. 2016; 18 (2): 166-171

  Abstract

  We evaluated the small molecule coelenterazine as a potential reporter of cancer-associated superoxide anion in cell culture and in mice.The superoxide anion concentrations of various cancer cell lines were quantified by coelenterazine chemiluminescence in vitro. Coelenteramide fluorescence was detected via flow cytometry and fluorescent microscopy. Coelenterazine was used for the in vivo detection of cancer-associated superoxide anion using the 4T1 breast adenocarcinoma mouse model.Various cell lines in culture demonstrated different superoxide anion concentrations, with a signal range of 3.15 ± 0.06 to 11.80 ± 0.24 times that of background. In addition to chemiluminescent detection of coelenterazine, we demonstrated fluorescent detection of coelenteramide within the cytoplasm of cells. 4T1 murine mammary adenocarcinoma tumors in mice demonstrated significantly higher 2.13 ± 0.19-fold coelenterazine-based chemiluminescence than that of surrounding normal tissues.Collectively, our results indicate that coelenterazine can be used to assay superoxide anion concentrations in cultured cancer cells and in tumors growing in mice.

  View details for DOI 10.1007/s11307-015-0896-7

  View details for PubMedID 26420297

• Non-Invasive Intravital Imaging of siRNA-Mediated Mutant Keratin Gene Repression in Skin. Molecular imaging and biology Hickerson, R. P., Speaker, T. J., Lara, M. F., González-González, E., Flores, M. A., Contag, C. H., Kaspar, R. L. 2016; 18 (1): 34-42

  Abstract

  Small interfering RNAs (siRNAs) specifically and potently inhibit target gene expression. Pachyonychia congenita (PC) is a skin disorder caused by mutations in genes encoding keratin (K) 6a/b, K16, and K17, resulting in faulty intermediate filaments. A siRNA targeting a single nucleotide, PC-relevant mutation inhibits K6a expression and has been evaluated in the clinic with encouraging results.To better understand the pathophysiology of PC, and develop a model system to study siRNA delivery and visualize efficacy in skin, wild type (WT) and mutant K6a complementary DNAs (cDNAs) were fused to either enhanced green fluorescent protein or tandem tomato fluorescent protein cDNA to allow covisualization of mutant and WT K6a expression in mouse footpad skin using a dual fluorescence in vivo confocal imaging system equipped with 488 and 532 nm lasers.Expression of mutant K6a/reporter resulted in visualization of keratin aggregates, while expression of WT K6a/reporter led to incorporation into filaments. Addition of mutant K6a-specific siRNA resulted in inhibition of mutant, but not WT, K6a/reporter expression.Intravital imaging offers subcellular resolution for tracking functional activity of siRNA in real time and enables detailed analyses of therapeutic effects in individual mice to facilitate development of nucleic acid-based therapeutics for skin disorders.

  View details for DOI 10.1007/s11307-015-0875-z

  View details for PubMedID 26169581

• Signaling by Extracellular Vesicles Advances Cancer Hallmarks. Trends in cancer Kanada, M., Bachmann, M. H., Contag, C. H. 2016; 2 (2): 84-94

  Abstract

  Mammalian cells secrete various extracellular vesicles (EVs; exosomes, microvesicles, and apoptotic bodies) that differ in biogenesis, composition, and function. Each vesicle type can originate from normal or cancerous cells, transfer molecular cargo to both neighboring and distant cells, and modulate cellular behaviors involved in eubiology and pathology, such as tumor development. Here, we review evidence for the role of EVs in the establishment and maintenance of cancer hallmarks, including sustaining proliferative signaling, evading growth suppression, resisting cell death, reprogramming energy metabolism, acquiring genomic instability, and remodeling the tumor microenvironment. We also discuss how EVs are implicated in the induction of angiogenesis, control of cellular invasion, initiation of premetastatic niches, maintenance of inflammation, and evasion of immune surveillance. The deeper understanding of the biology of EVs and their contribution to the development and progression of tumors is leading to new opportunities in the diagnosis and treatment of cancer.

  View details for DOI 10.1016/j.trecan.2015.12.005

  View details for PubMedID 28741553

• Chemiluminescence Imaging of Superoxide Anion Detects Beta-Cell Function and Mass PLOS ONE Bronsart, L. L., Stokes, C., Contag, C. H. 2016; 11 (1)

  View details for DOI 10.1371/journal.pone.0146601

  View details for Web of Science ID 000367888100110

• Chemiluminescence Imaging of Superoxide Anion Detects Beta-Cell Function and Mass. PloS one Bronsart, L. L., Stokes, C., Contag, C. H. 2016; 11 (1): e0146601

  Abstract

  Superoxide anion is produced during normal cellular respiration and plays key roles in cellular physiology with its dysregulation being associated with a variety of diseases. Superoxide anion is a short-lived molecule and, therefore, its homeostatic regulation and role in biology and disease requires dynamic quantification with fine temporal resolution. Here we validated coelenterazine as a reporter of intracellular superoxide anion concentration and used it as a dynamic measure both in vitro and in vivo. Chemiluminescence was dependent upon superoxide anion levels, including those produced during cellular respiration, and concentrations varied both kinetically and temporally in response to physiologically relevant fluctuations in glucose levels. In vivo imaging with coelenterazine revealed that beta cells of the pancreas have increased levels of superoxide anion, which acted as a measure of beta-cell function and mass and could predict the susceptibility of mice to diabetes mellitus. Glucose response and regulation are key elements of cellular physiology and organismal biology, and superoxide anion appears to play a fundamental and dynamic role in both of these processes.

  View details for DOI 10.1371/journal.pone.0146601

  View details for PubMedID 26752052

  View details for PubMedCentralID PMC4709142

• Imaging Functional Nucleic Acid Delivery to Skin. Methods in molecular biology (Clifton, N.J.) Kaspar, R. L., Hickerson, R. P., González-González, E., Flores, M. A., Speaker, T. P., Rogers, F. A., Milstone, L. M., Contag, C. H. 2016; 1372: 1-24

  Abstract

  Monogenic skin diseases arise from well-defined single gene mutations, and in some cases a single point mutation. As the target cells are superficial, these diseases are ideally suited for treatment by nucleic acid-based therapies as well as monitoring through a variety of noninvasive imaging technologies. Despite the accessibility of the skin, there remain formidable barriers for functional delivery of nucleic acids to the target cells within the dermis and epidermis. These barriers include the stratum corneum and the layered structure of the skin, as well as more locally, the cellular, endosomal and nuclear membranes. A wide range of technologies for traversing these barriers has been described and moderate success has been reported for several approaches. The lessons learned from these studies include the need for combinations of approaches to facilitate nucleic acid delivery across these skin barriers and then functional delivery across the cellular and nuclear membranes for expression (e.g., reporter genes, DNA oligonucleotides or shRNA) or into the cytoplasm for regulation (e.g., siRNA, miRNA, antisense oligos). The tools for topical delivery that have been evaluated include chemical, physical and electrical methods, and the development and testing of each of these approaches has been greatly enabled by imaging tools. These techniques allow delivery and real time monitoring of reporter genes, therapeutic nucleic acids and also triplex nucleic acids for gene editing. Optical imaging is comprised of a number of modalities based on properties of light-tissue interaction (e.g., scattering, autofluorescence, and reflectance), the interaction of light with specific molecules (e.g., absorbtion, fluorescence), or enzymatic reactions that produce light (bioluminescence). Optical imaging technologies operate over a range of scales from macroscopic to microscopic and if necessary, nanoscopic, and thus can be used to assess nucleic acid delivery to organs, regions, cells and even subcellular structures. Here we describe the animal models, reporter genes, imaging approaches and general strategies for delivery of nucleic acids to cells in the skin for local expression (e.g., plasmid DNA) or gene silencing (e.g., siRNA) with the intent of developing nucleic acid-based therapies to treat diseases of the skin.

  View details for DOI 10.1007/978-1-4939-3148-4_1

  View details for PubMedID 26530911

• A role of the adaptive immune system in glucose homeostasis. BMJ open diabetes research & care Bronsart, L. L., Contag, C. H. 2016; 4 (1)

  Abstract

  The immune system, including the adaptive immune response, has recently been recognized as having a significant role in diet-induced insulin resistance. In this study, we aimed to determine if the adaptive immune system also functions in maintaining physiological glucose homeostasis in the absence of diet-induced disease.SCID mice and immunocompetent control animals were phenotypically assessed for variations in metabolic parameters and cytokine profiles. Additionally, the glucose tolerance of SCID and immunocompetent control animals was assessed following introduction of a high-fat diet.SCID mice on a normal chow diet were significantly insulin resistant relative to control animals despite having less fat mass. This was associated with a significant increase in the innate immunity-stimulating cytokines granulocyte colony-stimulating factor, monocyte chemoattractant protein 1 (MCP1), and MCP3. Additionally, the SCID mouse phenotype was exacerbated in response to a high-fat diet as evidenced by the further significant progression of glucose intolerance.These results support the notion that the adaptive immune system plays a fundamental biological role in glucose homeostasis, and that the absence of functional B and T cells results in disruption in the concentrations of various cytokines associated with macrophage proliferation and recruitment. Additionally, the absence of functional B and T cells is not protective against diet-induced pathology.

  View details for DOI 10.1136/bmjdrc-2015-000136

  View details for PubMedID 27026807

  View details for PubMedCentralID PMC4800071

• Breast Cancer Cell Colonization of the Human Bone Marrow Adipose Tissue Niche NEOPLASIA Templeton, Z. S., Lie, W., Wang, W., Rosenberg-Hasson, Y., Alluri, R. V., Tamaresis, J. S., Bachmann, M. H., Lee, K., Maloney, W. J., Contag, C. H., King, B. L. 2015; 17 (12): 849-861

  Abstract

  Bone is a preferred site of breast cancer metastasis, suggesting the presence of tissue-specific features that attract and promote the outgrowth of breast cancer cells. We sought to identify parameters of human bone tissue associated with breast cancer cell osteotropism and colonization in the metastatic niche.Migration and colonization patterns of MDA-MB-231-fLuc-EGFP (luciferase-enhanced green fluorescence protein) and MCF-7-fLuc-EGFP breast cancer cells were studied in co-culture with cancellous bone tissue fragments isolated from 14 hip arthroplasties. Breast cancer cell migration into tissues and toward tissue-conditioned medium was measured in Transwell migration chambers using bioluminescence imaging and analyzed as a function of secreted factors measured by multiplex immunoassay. Patterns of breast cancer cell colonization were evaluated with fluorescence microscopy and immunohistochemistry.Enhanced MDA-MB-231-fLuc-EGFP breast cancer cell migration to bone-conditioned versus control medium was observed in 12/14 specimens (P = .0014) and correlated significantly with increasing levels of the adipokines/cytokines leptin (P = .006) and IL-1β (P = .001) in univariate and multivariate regression analyses. Fluorescence microscopy and immunohistochemistry of fragments underscored the extreme adiposity of adult human bone tissues and revealed extensive breast cancer cell colonization within the marrow adipose tissue compartment.Our results show that breast cancer cells migrate to human bone tissue-conditioned medium in association with increasing levels of leptin and IL-1β, and colonize the bone marrow adipose tissue compartment of cultured fragments. Bone marrow adipose tissue and its molecular signals may be important but understudied components of the breast cancer metastatic niche.

  View details for DOI 10.1016/j.neo.2015.11.005

  View details for Web of Science ID 000366560300001

  View details for PubMedID 26696367

  View details for PubMedCentralID PMC4688564

• [Tc-99m]Annexin V-128 SPECT Monitoring of Splenic and Disseminated Listeriosis in Mice: a Model of Imaging Sepsis MOLECULAR IMAGING AND BIOLOGY Hardy, J. W., Levashova, Z., Schmidt, T. L., Contag, C. H., Blankenberg, F. G. 2015; 17 (3): 345-354

  Abstract

  Here, we evaluate [(99m)Tc]annexin V-128, an in vivo marker of apoptosis, for single photon emission computed tomography (SPECT) imaging of localization and antibiotic treatment of disseminated bacterial infection, using a well-described mouse model that employs bioluminescent Listeria monocytogenes.Sixteen groups of five mice in six separate experiments were infected with bioluminescent Listeria, and in vivo bioluminescence imaging (BLI) was performed each day, to assess the location and extent of infection and response to antibiotics. [(99m)Tc]annexin V-128 was then injected for SPECT imaging, and the two sets of images were correlated and validated.Signals from BLI and [(99m)Tc]annexin V-128 SPECT co-localized within the spleen and other organs including bone marrow, intestine, nasopharynx, and brain. Decreases in [(99m)Tc]annexin V-128 uptake and BLI signal within the spleen directly reflected the reduction of bacterial infection by ampicillin treatment.Tc-99m-Annexin V-128 uptake as observed by SPECT allowed for the detection of systemic listeriosis and ampicillin treatment in mice. [(99m)Tc]annexin V-128 should be further explored for the assessment of bacterial spread and antibiotic efficacy in patients with disseminated bacterial infection.

  View details for DOI 10.1007/s11307-014-0804-6

  View details for PubMedID 25416406

• Early Cancer Detection at the Epithelial Surface CANCER JOURNAL Rogalla, S., Contag, C. H. 2015; 21 (3): 179-187

  Abstract

  Malignant neoplastic lesions derived from epithelial tissue, carcinomas, account for 80% to 100% of all human cancers including some of the most deadly diseases such as cervical and non-small cell lung cancer. Many of these carcinomas present at readily accessible epithelial surfaces offering unique detection opportunities. Effective clinical management of carcinomas is enabled by early detection, at a time when full surgical resection is possible and before invasion of adjacent tissue or significant intravasation into blood vessels leading to metastasis. Good prognosis with long-term disease-free survival is more likely after early detection when progression is limited. At present, detection of carcinomas at epithelial surfaces largely relies on routine inspection with the naked eye (e.g., skin and oropharynx) or simple white light tools (e.g., cervix and colon). Emerging optical tools based on differential refraction, absorption, reflection, scattering, or fluorescence of carcinomas relative to normal tissues enable label-free visualization of neoplasia. However, the differences in intrinsic optical properties of normal and malignant tissues can be subtle, and relying on these may lead to high miss rates. Enhanced optical contrast offered by molecularly targeted agents can be used to improve early detection; and given that optical imaging and sensing tools can be readily combined, integrated systems that image over a range of scales, or detect multiple parameters, can be developed to aid in early detection. Diagnosis is, at present, made by histologic examination of tissue biopsies after identification of suspicious lesions. Miniature and handheld microscopic imaging tools have recently been developed, and integration of these tools with wide-field optical surveillance devices offers both rapid detection and confirmatory histologic examination at the point-of-care, that can provide guidance for biopsy and/or resection. A wide variety of targeted probe strategies have been described with demonstrated benefit in preclinical models and in a limited number of human studies. Here, we present examples of integrated multimodality optical imaging and sensing tools that use combinations of intrinsic and extrinsic optical contrast for early detection or margin delineation for carcinomas at epithelial surfaces. We will discuss several new technologies that have use in detecting the most common carcinomas that derive from the epithelium of the skin, gastrointestinal and urogenital tracts, and bronchoalveoli.

  View details for DOI 10.1097/PPO.0000000000000122

  View details for Web of Science ID 000355658000008

  View details for PubMedID 26049697

• A Real-Time Clinical Endoscopic System for Intraluminal, Multiplexed Imaging of Surface-Enhanced Raman Scattering Nanoparticles PLOS ONE Garai, E., Sensarn, S., Zavaleta, C. L., Loewke, N. O., Rogalla, S., Mandella, M. J., Felt, S. A., Friedland, S., Liu, J. T., Gambhir, S. S., Contag, C. H. 2015; 10 (4)

  Abstract

  The detection of biomarker-targeting surface-enhanced Raman scattering (SERS) nanoparticles (NPs) in the human gastrointestinal tract has the potential to improve early cancer detection; however, a clinically relevant device with rapid Raman-imaging capability has not been described. Here we report the design and in vivo demonstration of a miniature, non-contact, opto-electro-mechanical Raman device as an accessory to clinical endoscopes that can provide multiplexed molecular data via a panel of SERS NPs. This device enables rapid circumferential scanning of topologically complex luminal surfaces of hollow organs (e.g., colon and esophagus) and produces quantitative images of the relative concentrations of SERS NPs that are present. Human and swine studies have demonstrated the speed and simplicity of this technique. This approach also offers unparalleled multiplexing capabilities by simultaneously detecting the unique spectral fingerprints of multiple SERS NPs. Therefore, this new screening strategy has the potential to improve diagnosis and to guide therapy by enabling sensitive quantitative molecular detection of small and otherwise hard-to-detect lesions in the context of white-light endoscopy.

  View details for DOI 10.1371/journal.pone.0123185

  View details for Web of Science ID 000353711600032

  View details for PubMedID 25923788

  View details for PubMedCentralID PMC4414592

• Differential fates of biomolecules delivered to target cells via extracellular vesicles. Proceedings of the National Academy of Sciences of the United States of America Kanada, M., Bachmann, M. H., Hardy, J. W., Frimannson, D. O., Bronsart, L., Wang, A., Sylvester, M. D., Schmidt, T. L., Kaspar, R. L., Butte, M. J., Matin, A. C., Contag, C. H. 2015; 112 (12): E1433-42

  Abstract

  Extracellular vesicles (EVs), specifically exosomes and microvesicles (MVs), are presumed to play key roles in cell-cell communication via transfer of biomolecules between cells. The biogenesis of these two types of EVs differs as they originate from either the endosomal (exosomes) or plasma (MVs) membranes. To elucidate the primary means through which EVs mediate intercellular communication, we characterized their ability to encapsulate and deliver different types of macromolecules from transiently transfected cells. Both EV types encapsulated reporter proteins and mRNA but only MVs transferred the reporter function to recipient cells. De novo reporter protein expression in recipient cells resulted only from plasmid DNA (pDNA) after delivery via MVs. Reporter mRNA was delivered to recipient cells by both EV types, but was rapidly degraded without being translated. MVs also mediated delivery of functional pDNA encoding Cre recombinase in vivo to tissues in transgenic Cre-lox reporter mice. Within the parameters of this study, MVs delivered functional pDNA, but not RNA, whereas exosomes from the same source did not deliver functional nucleic acids. These results have significant implications for understanding the role of EVs in cellular communication and for development of EVs as delivery tools. Moreover, studies using EVs from transiently transfected cells may be confounded by a predominance of pDNA transfer.

  View details for DOI 10.1073/pnas.1418401112

  View details for PubMedID 25713383

  View details for PubMedCentralID PMC4378439

• Atherosclerotic Plaque Targeting Mechanism of Long-Circulating Nanoparticles Established by Multimodal Imaging ACS NANO Lobatto, M. E., Calcagno, C., Millon, A., Senders, M. L., Fay, F., Robson, P. M., Ramachandran, S., Binderup, T., Paridaans, M. P., Sensarn, S., Rogalla, S., Gordon, R. E., Cardoso, L., Storm, G., Metselaar, J. M., Contag, C. H., Stroes, E. S., Fayad, Z. A., Mulder, W. J. 2015; 9 (2): 1837-1847

  Abstract

  Atherosclerosis is a major cause of global morbidity and mortality that could benefit from novel targeted therapeutics. Recent studies have shown efficient and local drug delivery with nanoparticles, although the nanoparticle targeting mechanism for atherosclerosis has not yet been fully elucidated. Here we used in vivo and ex vivo multimodal imaging to examine permeability of the vessel wall and atherosclerotic plaque accumulation of fluorescently labeled liposomal nanoparticles in a rabbit model. We found a strong correlation between permeability as established by in vivo dynamic contrast enhanced magnetic resonance imaging and nanoparticle plaque accumulation with subsequent nanoparticle distribution throughout the vessel wall. These key observations will enable the development of nanotherapeutic strategies for atherosclerosis.

  View details for DOI 10.1021/nn506750r

  View details for Web of Science ID 000349940500080

  View details for PubMedID 25619964

• Detection of Non-Melanoma Skin Cancer by in vivo Fluorescence Imaging with Fluorocoxib A NEOPLASIA Ra, H., Gonzaez-Gonzaez, E., Uddin, M. J., King, B. L., Lee, A., Ali-Khan, I., Marnett, L. J., Tang, J. Y., Contag, C. H. 2015; 17 (2): 201-207

  Abstract

  Non-melanoma skin cancer (NMSC) is the most common form of cancer in the US and its incidence is increasing. The current standard of care is visual inspection by physicians and/or dermatologists, followed by skin biopsy and pathologic confirmation. We have investigated the use of in vivo fluorescence imaging using fluorocoxib A as a molecular probe for early detection and assessment of skin tumors in mouse models of NMSC. Fluorocoxib A targets the cyclooxygenase-2 (COX-2) enzyme that is preferentially expressed by inflamed and tumor tissue, and therefore has potential to be an effective broadly active molecular biomarker for cancer detection. We tested the sensitivity of fluorocoxib A in a BCC allograft SCID hairless mouse model using a wide-field fluorescence imaging system. Subcutaneous allografts comprised of 1000 BCC cells were detectable above background. These BCC allograft mice were imaged over time and a linear correlation (R(2) = 0.8) between tumor volume and fluorocoxib A signal levels was observed. We also tested fluorocoxib A in a genetically engineered spontaneous BCC mouse model (Ptch1(+/-) K14-Cre-ER2 p53(fl/fl)), where sequential imaging of the same animals over time demonstrated that early, microscopic lesions (100 μm size) developed into visible macroscopic tumor masses over 11 to 17 days. Overall, for macroscopic tumors, the sensitivity was 88% and the specificity was 100%. For microscopic tumors, the sensitivity was 85% and specificity was 56%. These results demonstrate the potential of fluorocoxib A as an in vivo imaging agent for early detection, margin delineation and guided biopsies of NMSCs.

  View details for DOI 10.1016/j.neo.2014.12.009

  View details for Web of Science ID 000350733700007

  View details for PubMedCentralID PMC4351298

• Methods for culturing human femur tissue explants to study breast cancer cell colonization of the metastatic niche. Journal of visualized experiments : JoVE Templeton, Z. S., Bachmann, M. H., Alluri, R. V., Maloney, W. J., Contag, C. H., King, B. L. 2015

  Abstract

  Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little is known about breast cancer cell properties and behaviors within the native microenvironment of human bone tissue.We have developed approaches to track, quantify and modulate human breast cancer cells within the microenvironment of cultured human bone tissue fragments isolated from discarded femoral heads following total hip replacement surgeries. Using breast cancer cells engineered for luciferase and enhanced green fluorescent protein (EGFP) expression, we are able to reproducibly quantitate migration and proliferation patterns using bioluminescence imaging (BLI), track cell interactions within the bone fragments using fluorescence microscopy, and evaluate breast cells after colonization with flow cytometry. The key advantages of this model include: 1) a native, architecturally intact tissue microenvironment that includes relevant human cell types, and 2) direct access to the microenvironment, which facilitates rapid quantitative and qualitative monitoring and perturbation of breast and bone cell properties, behaviors and interactions. A primary limitation, at present, is the finite viability of the tissue fragments, which confines the window of study to short-term culture. Applications of the model system include studying the basic biology of breast cancer and other bone-seeking malignancies within the metastatic niche, and developing therapeutic strategies to effectively target breast cancer cells in bone tissues.

  View details for DOI 10.3791/52656

  View details for PubMedID 25867136

• Non-invasive intravital imaging of cellular differentiation with a bright red-excitable fluorescent protein NATURE METHODS Chu, J., Haynes, R. D., Corbel, S. Y., Li, P., Gonzalez-Gonzalez, E., Burg, J. S., Ataie, N. J., Lam, A. J., Cranfill, P. J., Baird, M. A., Davidson, M. W., Ng, H., Garcia, K. C., Contag, C. H., Shen, K., Blau, H. M., Lin, M. Z. 2014; 11 (5): 572-578

  Abstract

  A method for non-invasive visualization of genetically labeled cells in animal disease models with micrometer-level resolution would greatly facilitate development of cell-based therapies. Imaging of fluorescent proteins (FPs) using red excitation light in the 'optical window' above 600 nm is one potential method for visualizing implanted cells. However, previous efforts to engineer FPs with peak excitation beyond 600 nm have resulted in undesirable reductions in brightness. Here we report three new red-excitable monomeric FPs obtained by structure-guided mutagenesis of mNeptune. Two of these, mNeptune2 and mNeptune2.5, demonstrate improved maturation and brighter fluorescence than mNeptune, whereas the third, mCardinal, has a red-shifted excitation spectrum without reduction in brightness. We show that mCardinal can be used to non-invasively and longitudinally visualize the differentiation of myoblasts into myocytes in living mice with high anatomical detail.

  View details for DOI 10.1038/NMETH.2888

  View details for Web of Science ID 000335873400026

  View details for PubMedID 24633408

  View details for PubMedCentralID PMC4008650

• A killer choice for cancer immunotherapy. Immunologic research Schmidt, T. L., Negrin, R. S., Contag, C. H. 2014; 58 (2-3): 300-306

  Abstract

  The promise of cell-based immunotherapies for the treatment of cancer offers the potential of therapeutic synergy with chemo- and radiotherapies that may overcome current limitations leading to durable responses and prevention of recurrence. There is a wide array of cell-based immunotherapies that are either poised to enter cancer clinical trials or are in clinical trials, and many are showing some success. Yet within this field, there are clear obstacles that need to be overcome, including limited access across tissue barriers, development of antigen tolerance, and the immunosuppressive microenvironment of tumors. Through an understanding of immune cell signaling and trafficking, immune cell populations can be selected for adoptive transfer, and delivery strategies can be developed that circumvent these obstacles to effectively direct populations of cells with robust anti-tumor efficacy to the target. Within the realm of immune cell therapies, cytokine-induced killer (CIK) cells have demonstrated promising trafficking patterns, effective delivery of synergistic therapeutics, and stand-alone efficacy. Here, we discuss the next generation of CIK therapies and their application for the effective treatment of a wide variety of cancers.

  View details for DOI 10.1007/s12026-014-8507-2

  View details for PubMedID 24791943

• Monitoring Dynamic Interactions Between Breast Cancer Cells and Human Bone Tissue in a Co-culture Model. Molecular imaging and biology Contag, C. H., Lie, W., Bammer, M. C., Hardy, J. W., Schmidt, T. L., Maloney, W. J., King, B. L. 2014; 16 (2): 158-166

  Abstract

  Bone is a preferential site of breast cancer metastasis, and models are needed to study this process at the level of the microenvironment. We have used bioluminescence imaging (BLI) and multiplex biomarker immunoassays to monitor dynamic breast cancer cell behaviors in co-culture with human bone tissue.Femur tissue fragments harvested from hip replacement surgeries were co-cultured with luciferase-positive MDA-MB-231-fLuc cells. BLI was performed to quantify breast cell proliferation and track migration relative to bone tissue. Breast cell colonization of bone tissues was assessed with immunohistochemistry. Biomarkers in co-culture supernatants were profiled with MILLIPLEX(®) immunoassays.BLI demonstrated increased MDA-MB-231-fLuc cell proliferation (p < 0.001) in the presence vs. absence of bones and revealed breast cell migration toward bone. Immunohistochemistry illustrated MDA-MB-231-fLuc cell colonization of bone, and MILLIPLEX(®) profiles of culture supernatants suggested breast/bone crosstalk.Breast cell behaviors that facilitate metastasis occur reproducibly in human bone tissue co-cultures and can be monitored and quantified using BLI and multiplex immunoassays.

  View details for DOI 10.1007/s11307-013-0685-0

  View details for PubMedID 24008275

• Reporter gene technologies for imaging cell fates in hematopoiesis. Methods in molecular biology (Clifton, N.J.) Kusy, S., Contag, C. H. 2014; 1109: 1-22

  Abstract

  Advances in noninvasive imaging technologies that allow for in vivo dynamic monitoring of cells and cellular function in living research subjects have revealed new insights into cell biology in the context of intact organs and their native environment. In the field of hematopoiesis and stem cell research, studies of cell trafficking involved in injury repair and hematopoietic engraftment have made great progress using these new tools. Stem cells present unique challenges for imaging since after transplantation, they proliferate dramatically and differentiate. Therefore, the imaging modality used needs to have a large dynamic range, and the genetic regulatory elements used need to be stably expressed during differentiation. Multiple imaging technologies using different modalities are available, and each varies in sensitivity, ease of data acquisition, signal to noise ratios (SNR), substrate availability, and other parameters that affect utility for monitoring cell fates and function. For a given application, there may be several different approaches that can be used. For mouse models, clinically validated technologies such as magnetic resonance imaging (MRI) and positron emission tomography (PET) have been joined by optical imaging techniques such as in vivo bioluminescence imaging (BLI) and fluorescence imaging (FLI), and all have been used to monitor bone marrow and stem cells after transplantation into mice. Photoacoustic imaging that utilizes the sound created by the thermal expansion of absorbed light to generate an image best represents hybrid technologies. Each modality requires that the cells of interest be marked with a genetic reporter that acts as a label making them uniquely visible using that technology. For each modality, there are several labels to choose from. Multiple methods for applying these different labels are available. This chapter provides an overview of the imaging technologies and commonly used labels for each, as well as detailed protocols for gene delivery into hematopoietic cells for the purposes of applying these specific labels to cell trafficking. The goal of this chapter is to provide adequate background information to allow the design and implementation of an experimental system for in vivo imaging in mice.

  View details for DOI 10.1007/978-1-4614-9437-9_1

  View details for PubMedID 24473775

• Monitoring Dynamic Interactions Between Breast Cancer Cells and Human Bone Tissue in a Co-culture Model Molecular Imaging and Biology Contag, C. H., Lie, W., Bammer, M. C., Hardy, J. W., Schmidt, T. L., Maloney, W. J., King, B. L. 2014; 16 (2): 158-166

  Abstract

  Bone is a preferential site of breast cancer metastasis, and models are needed to study this process at the level of the microenvironment. We have used bioluminescence imaging (BLI) and multiplex biomarker immunoassays to monitor dynamic breast cancer cell behaviors in co-culture with human bone tissue.Femur tissue fragments harvested from hip replacement surgeries were co-cultured with luciferase-positive MDA-MB-231-fLuc cells. BLI was performed to quantify breast cell proliferation and track migration relative to bone tissue. Breast cell colonization of bone tissues was assessed with immunohistochemistry. Biomarkers in co-culture supernatants were profiled with MILLIPLEX(®) immunoassays.BLI demonstrated increased MDA-MB-231-fLuc cell proliferation (p < 0.001) in the presence vs. absence of bones and revealed breast cell migration toward bone. Immunohistochemistry illustrated MDA-MB-231-fLuc cell colonization of bone, and MILLIPLEX(®) profiles of culture supernatants suggested breast/bone crosstalk.Breast cell behaviors that facilitate metastasis occur reproducibly in human bone tissue co-cultures and can be monitored and quantified using BLI and multiplex immunoassays.

  View details for DOI 10.1007/s11307-013-0685-0

• Noninvasive imaging of hypoxia-inducible factor-1a gene therapy for myocardial ischemia. Human gene therapy methods Chen, I. Y., Gheysens, O., Li, Z., Rasooly, J. A., Wang, Q., Paulmurugan, R., Rosenberg, J., Rodriguez-Porcel, M., Willmann, J. K., Wang, D. S., Contag, C. H., Robbins, R. C., Wu, J. C., Gambhir, S. S. 2013; 24 (5): 279-288

  Abstract

  Abstract Hypoxia-inducible factor-1 alpha (HIF-1α) gene therapy holds great promise for the treatment of myocardial ischemia. Both preclinical and clinical evaluations of this therapy are underway and can benefit from a vector strategy that allows noninvasive assessment of HIF-1α expression as an objective measure of gene delivery. We have developed a novel bidirectional plasmid vector (pcTnT-HIF-1α-VP2-TSTA-fluc), which employs the cardiac troponin T (cTnT) promoter in conjunction with a two-step transcriptional amplification (TSTA) system to drive the linked expression of a recombinant HIF-1α gene (HIF-1α-VP2) and the firefly luciferase gene (fluc). The firefly luciferase (FLuc) activity serves as a surrogate for HIF-1α-VP2 expression, and can be noninvasively assessed in mice using bioluminescence imaging after vector delivery. Transfection of cultured HL-1 cardiomyocytes with pcTnT-HIF-1α-VP2-TSTA-fluc led to a strong correlation between FLuc and HIF-1α-dependent vascular endothelial growth factor expression (r(2)=0.88). Intramyocardial delivery of pcTnT-HIF-1α-VP2-TSTA-fluc into infarcted mouse myocardium led to persistent HIF-1α-VP2 expression for 4 weeks, even though it improved neither CD31+ microvessel density nor echocardiographically determined left ventricular systolic function. These results lend support to recent findings of suboptimal efficacy associated with plasmid-mediated HIF-1α therapy. The imaging techniques developed herein should be useful for further optimizing HIF-1α-VP2 therapy in preclinical models of myocardial ischemia.

  View details for DOI 10.1089/hgtb.2013.028

  View details for PubMedID 23937265

• Noninvasive imaging of hypoxia-inducible factor-1a gene therapy for myocardial ischemia. Human gene therapy methods Chen, I. Y., Gheysens, O., Li, Z., Rasooly, J. A., Wang, Q., Paulmurugan, R., Rosenberg, J., Rodriguez-Porcel, M., Willmann, J. K., Wang, D. S., Contag, C. H., Robbins, R. C., Wu, J. C., Gambhir, S. S. 2013; 24 (5): 279-288

  View details for DOI 10.1089/hgtb.2013.028

  View details for PubMedID 23937265

• High-sensitivity, real-time, ratiometric imaging of surface-enhanced Raman scattering nanoparticles with a clinically translatable Raman endoscope device. Journal of biomedical optics Garai, E., Sensarn, S., Zavaleta, C. L., Van de Sompel, D., Loewke, N. O., Mandella, M. J., Gambhir, S. S., Contag, C. H. 2013; 18 (9): 96008-?

  View details for DOI 10.1117/1.JBO.18.9.096008

  View details for PubMedID 24008818

• Minimal-length Synthetic shRNAs Formulated with Lipid Nanoparticles are Potent Inhibitors of Hepatitis C Virus IRES-linked Gene Expression in Mice MOLECULAR THERAPY-NUCLEIC ACIDS Dallas, A., Ilves, H., Shorenstein, J., Judge, A., Spitler, R., Contag, C., Wong, S. P., Harbottle, R. P., MacLachlan, I., Johnston, B. H. 2013; 2

  Abstract

  We previously identified short synthetic shRNAs (sshRNAs) that target a conserved hepatitis C virus (HCV) sequence within the internal ribosome entry site (IRES) of HCV and potently inhibit HCV IRES-linked gene expression. To assess in vivo liver delivery and activity, the HCV-directed sshRNA SG220 was formulated into lipid nanoparticles (LNP) and injected i.v. into mice whose livers supported stable HCV IRES-luciferase expression from a liver-specific promoter. After a single injection, RNase protection assays for the sshRNA and (3)H labeling of a lipid component of the nanoparticles showed efficient liver uptake of both components and long-lasting survival of a significant fraction of the sshRNA in the liver. In vivo imaging showed a dose-dependent inhibition of luciferase expression (>90% 1 day after injection of 2.5 mg/kg sshRNA) with t1/2 for recovery of about 3 weeks. These results demonstrate the ability of moderate levels of i.v.-injected, LNP-formulated sshRNAs to be taken up by liver hepatocytes at a level sufficient to substantially suppress gene expression. Suppression is rapid and durable, suggesting that sshRNAs may have promise as therapeutic agents for liver indications.Molecular Therapy-Nucleic Acids (2013) 2, e123; doi:10.1038/mtna.2013.50; published online 17 September 2013.

  View details for DOI 10.1038/mtna.2013.50

  View details for Web of Science ID 000332467500006

  View details for PubMedID 24045712

• A Raman-based endoscopic strategy for multiplexed molecular imaging. Proceedings of the National Academy of Sciences of the United States of America Zavaleta, C. L., Garai, E., Liu, J. T., Sensarn, S., Mandella, M. J., Van de Sompel, D., Friedland, S., Van Dam, J., Contag, C. H., Gambhir, S. S. 2013; 110 (25): E2288-97

  Abstract

  Endoscopic imaging is an invaluable diagnostic tool allowing minimally invasive access to tissues deep within the body. It has played a key role in screening colon cancer and is credited with preventing deaths through the detection and removal of precancerous polyps. However, conventional white-light endoscopy offers physicians structural information without the biochemical information that would be advantageous for early detection and is essential for molecular typing. To address this unmet need, we have developed a unique accessory, noncontact, fiber optic-based Raman spectroscopy device that has the potential to provide real-time, multiplexed functional information during routine endoscopy. This device is ideally suited for detection of functionalized surface-enhanced Raman scattering (SERS) nanoparticles as molecular imaging contrast agents. This device was designed for insertion through a clinical endoscope and has the potential to detect and quantify the presence of a multiplexed panel of tumor-targeting SERS nanoparticles. Characterization of the Raman instrument was performed with SERS particles on excised human tissue samples, and it has shown unsurpassed sensitivity and multiplexing capabilities, detecting 326-fM concentrations of SERS nanoparticles and unmixing 10 variations of colocalized SERS nanoparticles. Another unique feature of our noncontact Raman endoscope is that it has been designed for efficient use over a wide range of working distances from 1 to 10 mm. This is necessary to accommodate for imperfect centering during endoscopy and the nonuniform surface topology of human tissue. Using this endoscope as a key part of a multiplexed detection approach could allow endoscopists to distinguish between normal and precancerous tissues rapidly and to identify flat lesions that are otherwise missed.

  View details for DOI 10.1073/pnas.1211309110

  View details for PubMedID 23703909

  View details for PubMedCentralID PMC3690865

• Non-Invasive High-Resolution Imaging of Muscle Regeneration with a New Red-Absorbing Fluorescent Protein 16th Annual Meeting of the American-Society-of-Gene-and-Cell-Therapy (ASGCT) Chu, J., Haynes, R. D., Corbel, S. Y., Li, P., Gonzalez-Gonzalez, E., Cranfill, P. J., Baird, M., Davidson, M. W., Contag, C. H., Shen, K., Blau, H. M., Lin, M. Z. NATURE PUBLISHING GROUP. 2013: S97–S97

  View details for Web of Science ID 000319858400254

• Gene silencing following siRNA delivery to skin via coated steel microneedles: In vitro and in vivo proof-of-concept JOURNAL OF CONTROLLED RELEASE Chong, R. H., Gonzalez-Gonzalez, E., Lara, M. F., Speaker, T. J., Contag, C. H., Kaspar, R. L., Coulman, S. A., Hargest, R., Birchall, J. C. 2013; 166 (3): 211-219

  Abstract

  The development of siRNA-based gene silencing therapies has significant potential for effectively treating debilitating genetic, hyper-proliferative or malignant skin conditions caused by aberrant gene expression. To be efficacious and widely accepted by physicians and patients, therapeutic siRNAs must access the viable skin layers in a stable and functional form, preferably without painful administration. In this study we explore the use of minimally-invasive steel microneedle devices to effectively deliver siRNA into skin. A simple, yet precise microneedle coating method permitted reproducible loading of siRNA onto individual microneedles. Following recovery from the microneedle surface, lamin A/C siRNA retained full activity, as demonstrated by significant reduction in lamin A/C mRNA levels and reduced lamin A/C protein in HaCaT keratinocyte cells. However, lamin A/C siRNA pre-complexed with a commercial lipid-based transfection reagent (siRNA lipoplex) was less functional following microneedle coating. As Accell-modified "self-delivery" siRNA targeted against CD44 also retained functionality after microneedle coating, this form of siRNA was used in subsequent in vivo studies, where gene silencing was determined in a transgenic reporter mouse skin model. Self-delivery siRNA targeting the reporter (luciferase/GFP) gene was coated onto microneedles and delivered to mouse footpad. Quantification of reporter mRNA and intravital imaging of reporter expression in the outer skin layers confirmed functional in vivo gene silencing following microneedle delivery of siRNA. The use of coated metal microneedles represents a new, simple, minimally-invasive, patient-friendly and potentially self-administrable method for the delivery of therapeutic nucleic acids to the skin.

  View details for DOI 10.1016/j.jconrel.2012.12.030

  View details for Web of Science ID 000316676900003

  View details for PubMedID 23313112

  View details for PubMedCentralID PMC3594125

• Advancing the translation of optical imaging agents for clinical imaging BIOMEDICAL OPTICS EXPRESS Sevick-Muraca, E. M., Akers, W. J., Joshi, B. P., Luker, G. D., Cutler, C. S., Marnett, L. J., Contag, C. H., Wang, T. D., Azhdarinia, A. 2013; 4 (1): 160-170

  Abstract

  Despite the development of a large number of promising candidates, few contrast agents for established medical imaging modalities have successfully been translated over the past decade. The emergence of new imaging contrast agents that employ biomedical optics is further complicated by the relative infancy of the field and the lack of approved imaging devices compared to more established clinical modalities such as nuclear medicine. Herein, we propose a navigational approach (as opposed to a fixed "roadmap") for translation of optical imaging agents that is (i) proposed through consensus by four academic research programs that are part of the cooperative U54 NCI Network for Translational Research, (ii) developed through early experiences for translating optical imaging agents in order to meet distinctly varied needs in cancer diagnostics, and (iii) adaptable to the rapidly changing environment of academic medicine. We describe the pathways by which optical imaging agents are synthesized, qualified, and validated for preclinical testing, and ultimately translated for "first-in-humans" studies using investigational optical imaging devices. By identifying and adopting consensus approaches for seemingly disparate optical imaging modalities and clinical indications, we seek to establish a systematic method for navigating the ever-changing "roadmap" to most efficiently arrive at the destination of clinical adoption and improved outcome and survivorship for cancer patients.

  View details for Web of Science ID 000312710100013

  View details for PubMedID 23304655

• Gene Silencing in Skin After Deposition of Self-Delivery siRNA With a Motorized Microneedle Array Device. Molecular therapy. Nucleic acids Hickerson, R. P., Wey, W. C., Rimm, D. L., Speaker, T., Suh, S., Flores, M. A., Gonzalez-Gonzalez, E., Leake, D., Contag, C. H., Kaspar, R. L. 2013; 2

  Abstract

  Despite the development of potent siRNAs that effectively target genes responsible for skin disorders, translation to the clinic has been hampered by inefficient delivery through the stratum corneum barrier and into the live cells of the epidermis. Although hypodermic needles can be used to transport siRNA through the stratum corneum, this approach is limited by pain caused by the injection and the small volume of tissue that can be accessed by each injection. The use of microneedle arrays is a less painful method for siRNA delivery, but restricted payload capacity limits this approach to highly potent molecules. To address these challenges, a commercially available motorized microneedle array skin delivery device was evaluated. This device combines the positive elements of both hypodermic needles and microneedle array technologies with little or no pain to the patient. Application of fluorescently tagged self-delivery (sd)-siRNA to both human and murine skin resulted in distribution throughout the treated skin. In addition, efficient silencing (78% average reduction) of reporter gene expression was achieved in a transgenic fluorescent reporter mouse skin model. These results indicate that this device effectively delivers functional sd-siRNA with an efficiency that predicts successful clinical translation.Molecular Therapy-Nucleic Acids (2013) 2, e129; doi:10.1038/mtna.2013.56; published online 22 October 2013.

  View details for DOI 10.1038/mtna.2013.56

  View details for PubMedID 24150576

• Real-time pathology through in vivo microscopy. Studies in health technology and informatics Liu, J. T., Loewke, N. O., Mandella, M. J., Leigh, S. Y., Levenson, R. M., Crawford, J. M., Contag, C. H. 2013; 185: 235-264

  Abstract

  Miniature microscopes are being developed to examine tissue in situ for early anatomic and molecular indicators of disease, in real time, and at cellular resolution. These new devices will lead to a shift from the current diagnostic paradigm of biopsy followed by histopathology and recommended therapy, to one of non-invasive point-of-care diagnosis with the possibility of treatment in the same session. This potential revolution in disease management may have a major impact on the training of future physicians to include the use and interpretation of real-time in vivo microscopic data, and will also affect the emerging fields of telepathology and telemedicine. Implementation of new technologies into clinical practice is a complex process that requires multidisciplinary communication and collaboration among clinicians, engineers and scientists. As such, our aim is to provide a forward-looking view of the critical issues facing the development of new technologies and directing clinical education. Here, we focus on the use of in vivo microscopy for detection of malignant and pre-malignant lesions as well as for guiding therapy. We will highlight some of the areas in which in vivo microscopy could address unmet clinical needs, and then review the technological challenges that are being addressed, or need to be addressed, for in vivo microscopy to become an effective clinical tool.

  View details for PubMedID 23542938

• Femtosecond plasma mediated laser ablation has advantages over mechanical osteotomy of cranial bone LASERS IN SURGERY AND MEDICINE Lo, D. D., Mackanos, M. A., Chung, M. T., Hyun, J. S., Montoro, D. T., Grova, M., Liu, C., Wang, J., Palanker, D., Connolly, A. J., Longaker, M. T., Contag, C. H., Wan, D. C. 2012; 44 (10): 805-814

  Abstract

  Although mechanical osteotomies are frequently made on the craniofacial skeleton, collateral thermal, and mechanical trauma to adjacent bone tissue causes cell death and may delay healing. The present study evaluated the use of plasma-mediated laser ablation using a femtosecond laser to circumvent thermal damage and improve bone regeneration.Critical-size circular calvarial defects were created with a trephine drill bit or with a Ti:Sapphire femtosecond pulsed laser. Healing was followed using micro-CT scans for 8 weeks. Calvaria were also harvested at various time points for histological analysis. Finally, scanning electron microscopy was used to analyze the microstructure of bone tissue treated with the Ti:Sapphire laser, and compared to that treated with the trephine bur.Laser-created defects healed significantly faster than those created mechanically at 2, 4, and 6 weeks post-surgery. However, at 8 weeks post-surgery, there was no significant difference. In the drill osteotomy treatment group, empty osteocyte lacunae were seen to extend 699 ± 27 µm away from the edge of the defect. In marked contrast, empty osteocyte lacunae were seen to extend only 182 ± 22 µm away from the edge of the laser-created craters. Significantly less ossification and formation of irregular woven bone was noted on histological analysis for drill defects.We demonstrate accelerated bone healing after femtosecond laser ablation in a calvarial defect model compared to traditional mechanical drilling techniques. Improved rates of early regeneration make plasma-mediated ablation of the craniofacial skeleton advantageous for applications to osteotomy.

  View details for DOI 10.1002/lsm.22098

  View details for Web of Science ID 000312941600004

  View details for PubMedID 23184427

• Intravital Fluorescence Imaging of Small Interfering RNA-Mediated Gene Repression in a Dual Reporter Melanoma Xenograft Model NUCLEIC ACID THERAPEUTICS Hickerson, R. P., Gonzalez-Gonzalez, E., Vlassov, A. V., Li, M., Lara, M. F., Contag, C. H., Kaspar, R. L. 2012; 22 (6): 438-443

  Abstract

  Development of RNA interference (RNAi)-based therapeutics has been hampered by the lack of effective and efficient means of delivery. Reliable model systems for screening and optimizing delivery of RNAi-based agents in vivo are crucial for preclinical research aimed at advancing nucleic acid-based therapies. We describe here a dual fluorescent reporter xenograft melanoma model prepared by intradermal injection of human A375 melanoma cells expressing tandem tomato fluorescent protein (tdTFP) containing a small interfering RNA (siRNA) target site as well as enhanced green fluorescent protein (EGFP), which is used as a normalization control. Intratumoral injection of a siRNA specific to the incorporated siRNA target site, complexed with a cationic lipid that has been optimized for in vivo delivery, resulted in 65%±11% knockdown of tdTFP relative to EGFP quantified by in vivo imaging and 68%±10% by reverse transcription-quantitative polymerase chain reaction. No effect was observed with nonspecific control siRNA treatment. This model provides a platform on which siRNA delivery technologies can be screened and optimized in vivo.

  View details for DOI 10.1089/nat.2012.0364

  View details for Web of Science ID 000311832900054

  View details for PubMedID 23098239

  View details for PubMedCentralID PMC3507521

• Microscopic Delineation of Medulloblastoma Margins in a Transgenic Mouse Model Using a Topically Applied VEGFR-1 Probe TRANSLATIONAL ONCOLOGY Wang, D., Chen, Y., Leigh, S. Y., Haeberle, H., Contag, C. H., Liu, J. T. 2012; 5 (6): 408-414

  Abstract

  The unambiguous demarcation of tumor margins is critical at the final stages in the surgical treatment of brain tumors because patient outcomes have been shown to correlate with the extent of resection. Real-time high-resolution imaging with the aid of a tumor-targeting fluorescent contrast agent has the potential to enable intraoperative differentiation of tumor versus normal tissues with accuracy approaching the current gold standard of histopathology. In this study, a monoclonal antibody targeting the vascular endothelial growth factor receptor 1 (VEGFR-1) was conjugated to fluorophores and evaluated as a tumor contrast agent in a transgenic mouse model of medulloblastoma. The probe was administered topically, and its efficacy as an imaging agent was evaluated in vitro using flow cytometry, as well as ex vivo on fixed and fresh tissues through immunohistochemistry and dual-axis confocal microscopy, respectively. Results show a preferential binding to tumor versus normal tissue, suggesting that a topically applied VEGFR-1 probe can potentially be used with real-time intraoperative optical sectioning microscopy to guide brain tumor resections.

  View details for DOI 10.1593/tlo.12277

  View details for Web of Science ID 000313359800003

  View details for PubMedID 23323155

  View details for PubMedCentralID PMC3542836

• Comparing an optical parametric oscillator (OPO) as a viable alternative for mid-infrared tissue ablation with a free electron laser (FEL) LASERS IN MEDICAL SCIENCE Mackanos, M. A., Simanovskii, D. M., Contag, C. H., Kozub, J. A., Jansen, E. D. 2012; 27 (6): 1213-1223

  Abstract

  Beneficial medical laser ablation removes material efficiently with minimal collateral damage. A Mark-III free electron laser (FEL), at a wavelength of 6.45 μm has demonstrated minimal damage and high ablation yield in ocular and neural tissues. While this wavelength has shown promise for surgical applications, further advances are limited by the high overhead for FEL use. Alternative mid-infrared sources are needed for further development. We compared the FEL with a 5-μs pulse duration with a Q-switched ZGP-OPO with a 100-ns pulse duration at mid-infrared wavelengths. There were no differences in the ablation threshold of water and mouse dermis with these two sources in spite of the difference in their pulse structures. There was a significant difference in crater depth between the ZGP:OPO and the FEL. At 6.1 μm, the OPO craters are eight times the depth of the FEL craters. The OPO craters at 6.45 and 6.73 μm were six and five times the depth of the FEL craters, respectively. Bright-field (pump-probe) images showed the classic ablation mechanism from formation of a plume through collapse and recoil. The crater formation, ejection, and collapse phases occurred on a faster time-scale with the OPO than with the FEL. This research showed that a ZGP-OPO laser could be a viable alternative to FEL for clinical applications.

  View details for DOI 10.1007/s10103-011-1048-1

  View details for Web of Science ID 000309346600012

  View details for PubMedID 22278348

• Interrogation of Inhibitor of Nuclear Factor kappa B alpha/Nuclear Factor kappa B (I kappa B alpha/NF-kappa B) Negative Feedback Loop Dynamics FROM SINGLE CELLS TO LIVE ANIMALS IN VIVO JOURNAL OF BIOLOGICAL CHEMISTRY Moss, B. L., Elhammali, A., Fowlkes, T., Gross, S., Vinjamoori, A., Contag, C. H., Piwnica-Worms, D. 2012; 287 (37): 31359-31370

  View details for DOI 10.1074/jbc.M112.364018

  View details for Web of Science ID 000308791300047

• Interrogation of inhibitor of nuclear factor ?B a/nuclear factor ?B (I?Ba/NF-?B) negative feedback loop dynamics: from single cells to live animals in vivo. journal of biological chemistry Moss, B. L., Elhammali, A., Fowlkes, T., Gross, S., Vinjamoori, A., Contag, C. H., Piwnica-Worms, D. 2012; 287 (37): 31359-31370

  Abstract

  Full understanding of the biological significance of negative feedback processes requires interrogation at multiple scales as follows: in single cells, cell populations, and live animals in vivo. The transcriptionally coupled IκBα/NF-κB negative feedback loop, a pivotal regulatory node of innate immunity and inflammation, represents a model system for multiscalar reporters. Using a κB(5)→IκBα-FLuc bioluminescent reporter, we rigorously evaluated the dynamics of ΙκBα degradation and subsequent NF-κB transcriptional activity in response to diverse modes of TNFα stimulation. Modulating TNFα concentration or pulse duration yielded complex, reproducible, and differential ΙκBα dynamics in both cell populations and live single cells. Tremendous heterogeneity in the transcriptional amplitudes of individual responding cells was observed, which was greater than the heterogeneity in the transcriptional kinetics of responsive cells. Furthermore, administration of various TNFα doses in vivo generated ΙκBα dynamic profiles in the liver resembling those observed in single cells and populations of cells stimulated with TNFα pulses. This suggested that dose modulation of circulating TNFα was perceived by hepatocytes in vivo as pulses of increasing duration. Thus, a robust bioluminescent reporter strategy enabled rigorous quantitation of NF-κB/ΙκBα dynamics in both live single cells and cell populations and furthermore, revealed reproducible behaviors that informed interpretation of in vivo studies.

  View details for DOI 10.1074/jbc.M112.364018

  View details for PubMedID 22807442

• Inhibition of CD44 Gene Expression in Human Skin Models, Using Self-Delivery Short Interfering RNA Administered by Dissolvable Microneedle Arrays HUMAN GENE THERAPY Lara, M. F., Gonzalez-Gonzalez, E., Speaker, T. J., Nickerson, R. P., Leake, D., Milstone, L. M., Contag, C. H., Kaspar, R. L. 2012; 23 (8): 816-823

  View details for DOI 10.1089/hum.2011.211

  View details for Web of Science ID 000307684500005

• Inhibition of CD44 gene expression in human skin models, using self-delivery short interfering RNA administered by dissolvable microneedle arrays. Human gene therapy Lara, M. F., González-González, E., Speaker, T. J., Hickerson, R. P., Leake, D., Milstone, L. M., Contag, C. H., Kaspar, R. L. 2012; 23 (8): 816-823

  Abstract

  Treatment of skin disorders with short interfering RNA (siRNA)-based therapeutics requires the development of effective delivery methodologies that reach target cells in affected tissues. Successful delivery of functional siRNA to the epidermis requires (1) crossing the stratum corneum, (2) transfer across the keratinocyte membrane, followed by (3) incorporation into the RNA-induced silencing complex. We have previously demonstrated that treatment with microneedle arrays loaded with self-delivery siRNA (sd-siRNA) can achieve inhibition of reporter gene expression in a transgenic mouse model. Furthermore, treatment of human cultured epidermal equivalents with sd-siRNA resulted in inhibition of target gene expression. Here, we demonstrate inhibition of CD44, a gene that is uniformly expressed throughout the epidermis, by sd-siRNA both in vitro (cultured human epidermal skin equivalents) and in vivo (full-thickness human skin equivalents xenografted on immunocompromised mice). Treatment of human skin equivalents with CD44 sd-siRNA markedly decreased CD44 mRNA levels, which led to a reduction of the target protein as confirmed by immunodetection in epidermal equivalent sections with a CD44-specific antibody. Taken together, these results demonstrate that sd-siRNA, delivered by microneedle arrays, can reduce expression of a targeted endogenous gene in a human skin xenograft model.

  View details for DOI 10.1089/hum.2011.211

  View details for PubMedID 22480249

• Identification of Cell Surface Targets through Meta-analysis of Microarray Data NEOPLASIA Haeberle, H., Dudley, J. T., Liu, J. T., Butte, A. J., Contag, C. H. 2012; 14 (7): 666-669

  Abstract

  High-resolution image guidance for resection of residual tumor cells would enable more precise and complete excision for more effective treatment of cancers, such as medulloblastoma, the most common pediatric brain cancer. Numerous studies have shown that brain tumor patient outcomes correlate with the precision of resection. To enable guided resection with molecular specificity and cellular resolution, molecular probes that effectively delineate brain tumor boundaries are essential. Therefore, we developed a bioinformatics approach to analyze micro-array datasets for the identification of transcripts that encode candidate cell surface biomarkers that are highly enriched in medulloblastoma. The results identified 380 genes with greater than a two-fold increase in the expression in the medulloblastoma compared with that in the normal cerebellum. To enrich for targets with accessibility for extracellular molecular probes, we further refined this list by filtering it with gene ontology to identify genes with protein localization on, or within, the plasma membrane. To validate this meta-analysis, the top 10 candidates were evaluated with immunohistochemistry. We identified two targets, fibrillin 2 and EphA3, which specifically stain medulloblastoma. These results demonstrate a novel bioinformatics approach that successfully identified cell surface and extracellular candidate markers enriched in medulloblastoma versus adjacent cerebellum. These two proteins are high-value targets for the development of tumor-specific probes in medulloblastoma. This bioinformatics method has broad utility for the identification of accessible molecular targets in a variety of cancers and will enable probe development for guided resection.

  View details for DOI 10.1593/neo.12634

  View details for Web of Science ID 000308489500010

  View details for PubMedID 22904683

  View details for PubMedCentralID PMC3421962

• Pulse-Duration-Dependent Mid-Infrared Laser Ablation for Biological Applications IEEE JOURNAL OF SELECTED TOPICS IN QUANTUM ELECTRONICS Mackanos, M. A., Simanovskii, D. M., Schriver, K. E., Hutson, M. S., Contag, C. H., Kozub, J. A., Jansen, E. D. 2012; 18 (4): 1514-1522

  View details for DOI 10.1109/JSTQE.2012.2188501

  View details for Web of Science ID 000308113800026

• Generic and Personalized RNAi-Based Therapeutics for a Dominant-Negative Epidermal Fragility Disorder JOURNAL OF INVESTIGATIVE DERMATOLOGY Pedrioli, D. M., Fu, D. J., Gonzalez-Gonzalez, E., Contag, C. H., Kaspar, R. L., Smith, F. J., McLean, W. H. 2012; 132 (6): 1627-1635

  View details for DOI 10.1038/jid.2012.28

  View details for Web of Science ID 000304035000015

• SU-E-J-198: Bioluminescence Monitoring of Metastatic Breast Cancer: Quantitative Assessment of Radiation Treatment Effects and Tracking of Tumor Cells. Medical physics Park, J., Schmidt, T., Graves, E., Bazalova, M., Lee, J., Contag, C., Suh, T. 2012; 39 (6Part9): 3698

  Abstract

  To evaluate radiation treatment effects on mammary carcinoma cells, quantitative photon radiance were monitored to track light-emitting cancer cells and metastasis using in vivo bioluminescence imaging.Eight female BALB/c mice aged 8 weeks were orthotopically injected with 5×104/cc 4T1 tumor cells into the abdominal mammary gland. The firefly luciferase-based bioluminescence images were acquired every 2-3 days for 1 month. Bioluminescent intensity was analyzed in average surface radiance (photons/sec/cm2 /sr) taken in 3-dimensional bioluminescence tomography (BLT). After 1 week, single-radiation dose of 20 Gy was delivered by orthovoltage X-rays. Variation of detected bioluminescence signals emitted from molecular cancer cells was depicted on BLT images. To delineate tumor volumes according to bioluminescence intensity on anatomical images for radiation therapy, BLT images were registered with the micro computed tomography (CT) images using surface-constrained warping.Multispectral BLT images elaborated on early detection of cancer cells, characteristics of tumor growth, and metastasis for more accurate determination of internal bioluminescent sources. The radiation-treated mice having only primary tumor volumes showed 67% decrease in bioluminescent signals, while the mice with metastatic cancer cells suggested 88% reduction, as compared to the control group. Registration of BLT with CT images guided molecular cancer cells on anatomical coordinates.The BLT imaging was a useful tool to localize cancer cells and to quantify radiation response. Application of BLT led to more accurate definition of tumor volumes including molecular probe-based microscopic cancer cells. Monitoring of bioluminescence signals enables to diagnose real-time metastatic behavior of cancer cells and determine optimal radiation treatment strategies adapted to tumor characteristics.

  View details for DOI 10.1118/1.4735039

  View details for PubMedID 28519066

• Infection of pregnant mice with Listeria monocytogenes induces fetal bradycardia PEDIATRIC RESEARCH Hardy, J., Kirkendoll, B., Zhao, H., Pisani, L., Luong, R., Switzer, A., McConnell, M. V., Contag, C. H. 2012; 71 (5): 539-545

  Abstract

  Listeriosis is one of the most lethal bacterial diseases for fetuses and infants. However, pregnant women who get infected with Listeria may experience only mild symptoms, making the diagnosis difficult, even when the fetus is fatally infected.To reveal features of this infection, we conducted a multimodality imaging study of Listeria-induced miscarriage, using a pregnant mouse model. In this model, fetal morbidity and mortality can be observed in utero, noninvasively, and the timing and extent of infection can be carefully controlled. By employing in vivo bioluminescence imaging (BLI), perinatal infections were localized over time such that a correlation of infection to outcome could be determined without the need to kill the animal subject. The morbidity and viability of fetuses were assessed with ultrasound, and fetal morphology was imaged using magnetic resonance imaging (MRI).The ultrasound revealed sustained fetal bradycardia, the slowing of the fetal heartbeat, in infected fetuses, with an association between slowed fetal heart rate and strong bioluminescent signal.Uninfected fetuses showing no bioluminescent signal in the same uterine horn exhibited normal heartbeats. Thus, fetal bradycardia during infection was localized to the infected fetus and was not systemic or disseminated.

  View details for DOI 10.1038/pr.2012.2

  View details for PubMedID 22314663

• Development of B Cells and Erythrocytes Is Specifically Impaired by the Drug Celastrol in Mice PLOS ONE Kusy, S., Ghosn, E. E., Herzenberg, L. A., Contag, C. H. 2012; 7 (4)

  Abstract

  Celastrol, an active compound extracted from the root of the Chinese medicine "Thunder of God Vine" (Tripterygium wilfordii), exhibits anticancer, antioxidant and anti-inflammatory activities, and interest in the therapeutic potential of celastrol is increasing. However, described side effects following treatment are significant and require investigation prior to initiating clinical trials. Here, we investigated the effects of celastrol on the adult murine hematopoietic system.Animals were treated daily with celastrol over a four-day period and peripheral blood, bone marrow, spleen, and peritoneal cavity were harvested for cell phenotyping. Treated mice showed specific impairment of the development of B cells and erythrocytes in all tested organs. In bone marrow, these alterations were accompanied by decreases in populations of common lymphoid progenitors (CLP), common myeloid progenitors (CMP) and megakaryocyte-erythrocyte progenitors (MEP).These results indicate that celastrol acts through regulators of adult hematopoiesis and could be used as a modulator of the hematopoietic system. These observations provide valuable information for further assessment prior to clinical trials.

  View details for DOI 10.1371/journal.pone.0035733

  View details for Web of Science ID 000305343200053

  View details for PubMedID 22545133

  View details for PubMedCentralID PMC3335785

• Seeing it through: translational validation of new medical imaging modalities BIOMEDICAL OPTICS EXPRESS Aldrich, M. B., Marshall, M. V., Sevick-Muraca, E. M., Lanza, G., Kotyk, J., Culver, J., Wang, L. V., Uddin, J., Crews, B. C., Marnett, L. J., Liao, J. C., Contag, C., Crawford, J. M., Wang, K., Reisdorph, B., Appelman, H., Turgeon, D. K., Meyer, C., Wang, T. 2012; 3 (4): 764-776

  Abstract

  Medical imaging is an invaluable tool for diagnosis, surgical guidance, and assessment of treatment efficacy. The Network for Translational Research (NTR) for Optical Imaging consists of four research groups working to "bridge the gap" between lab discovery and clinical use of fluorescence- and photoacoustic-based imaging devices used with imaging biomarkers. While the groups are using different modalities, all the groups face similar challenges when attempting to validate these systems for FDA approval and, ultimately, clinical use. Validation steps taken, as well as future needs, are described here. The group hopes to provide translational validation guidance for itself, as well as other researchers.

  View details for Web of Science ID 000302788200009

  View details for PubMedID 22574264

  View details for PubMedCentralID PMC3345805

• In vivo near-infrared dual-axis confocal microendoscopy in the human lower gastrointestinal tract JOURNAL OF BIOMEDICAL OPTICS Piyawattanametha, W., Ra, H., Qiu, Z., Friedland, S., Liu, J. T., Loewke, K., Kino, G. S., Solgaard, O., Wang, T. D., Mandella, M. J., Contag, C. H. 2012; 17 (2)

  Abstract

  Near-infrared confocal microendoscopy is a promising technique for deep in vivo imaging of tissues and can generate high-resolution cross-sectional images at the micron-scale. We demonstrate the use of a dual-axis confocal (DAC) near-infrared fluorescence microendoscope with a 5.5-mm outer diameter for obtaining clinical images of human colorectal mucosa. High-speed two-dimensional en face scanning was achieved through a microelectromechanical systems (MEMS) scanner while a micromotor was used for adjusting the axial focus. In vivo images of human patients are collected at 5 frames/sec with a field of view of 362×212 μm(2) and a maximum imaging depth of 140 μm. During routine endoscopy, indocyanine green (ICG) was topically applied a nonspecific optical contrasting agent to regions of the human colon. The DAC microendoscope was then used to obtain microanatomic images of the mucosa by detecting near-infrared fluorescence from ICG. These results suggest that DAC microendoscopy may have utility for visualizing the anatomical and, perhaps, functional changes associated with colorectal pathology for the early detection of colorectal cancer.

  View details for DOI 10.1117/1.JBO.17.2.021102

  View details for Web of Science ID 000303033600004

  View details for PubMedID 22463020

  View details for PubMedCentralID PMC3380818

• Interdigitated Annular CMUT Arrays for Ultrasound Assisted Delivery of Fluorescent Contrast Agents IEEE International Ultrasonics Symposium (IUS) Cristman, P., Oralkan, O., Mandella, M., Solgaard, O., Contag, C., Khuri-Yakub, B. T. IEEE. 2012: 96–99

  View details for Web of Science ID 000309918400024

• In Vivo Bioluminescence Imaging of Inducible Nitric Oxide Synthase Gene Expression in Vascular Inflammation MOLECULAR IMAGING AND BIOLOGY Terashima, M., Ehara, S., Yang, E., Kosuge, H., Tsao, P. S., Quertermous, T., Contag, C. H., McConnell, M. V. 2011; 13 (6): 1061-1066

  Abstract

  Inflammation plays a critical role in atherosclerosis and is associated with upregulation of inducible nitric oxide synthase (iNOS). We studied bioluminescence imaging (BLI) to track iNOS gene expression in a murine model of vascular inflammation.Macrophage-rich vascular lesions were induced by carotid ligation plus high-fat diet and streptozotocin-induced diabetes in 18 iNOS-luc reporter mice. In vivo iNOS expression was imaged serially by BLI over 14 days, followed by in situ BLI and histology.BLI signal from ligated carotids increased over 14 days (9.7 ± 4.4 × 10(3 ) vs. 4.4 ± 1.7 × 10(3) photons/s/cm(2)/sr at baseline, p < 0.001 vs. baseline, p < 0.05 vs. sham controls). Histology confirmed substantial macrophage infiltration, with iNOS and luciferase expression, only in ligated left carotid arteries and not controls.BLI allows in vivo detection of iNOS expression in murine carotid lesions and may provide a valuable approach for monitoring vascular gene expression and inflammation in small animal models.

  View details for DOI 10.1007/s11307-010-0451-5

  View details for PubMedID 21057879

• In Vivo Sustained Release of siRNA from Solid Lipid Nanoparticles ACS NANO Lobovkina, T., Jacobson, G. B., Gonzalez-Gonzalez, E., Hickerson, R. P., Leake, D., Kaspar, R. L., Contag, C. H., Zare, R. N. 2011; 5 (12): 9977-9983

  Abstract

  Small interfering RNA (siRNA) is a highly potent drug in gene-based therapy with a challenge of being delivered in a sustained manner. Nanoparticle drug delivery systems allow for incorporating and controlled release of therapeutic payloads. We demonstrate that solid lipid nanoparticles can incorporate and provide sustained release of siRNA. Tristearin solid lipid nanoparticles, made by nanoprecipitation, were loaded with siRNA (4.4-5.5 wt % loading ratio) using a hydrophobic ion pairing approach that employs the cationic lipid DOTAP. Intradermal injection of these nanocarriers in mouse footpads resulted in prolonged siRNA release over a period of 10-13 days. In vitro cell studies showed that the released siRNA retained its activity. Nanoparticles developed in this study offer an alternative approach to polymeric nanoparticles for encapsulation and sustained delivery of siRNA with the advantage of being prepared from physiologically well-tolerated materials.

  View details for DOI 10.1021/nn203745n

  View details for Web of Science ID 000298316700073

  View details for PubMedID 22077198

  View details for PubMedCentralID PMC3246574

• Visualization of plasmid delivery to keratinocytes in mouse and human epidermis SCIENTIFIC REPORTS Gonzalez-Gonzalez, E., Kim, Y., Speaker, T. J., Hickerson, R. P., Spitler, R., Birchall, J. C., Lara, M. F., Hu, R., Liang, Y., Kirkiles-Smith, N., Prausnitz, M. R., Milstone, L. M., Contag, C. H., Kaspar, R. L. 2011; 1

  Abstract

  The accessibility of skin makes it an ideal target organ for nucleic acid-based therapeutics; however, effective patient-friendly delivery remains a major obstacle to clinical utility. A variety of limited and inefficient methods of delivering nucleic acids to keratinocytes have been demonstrated; further advances will require well-characterized reagents, rapid noninvasive assays of delivery, and well-developed skin model systems. Using intravital fluorescence and bioluminescence imaging and a standard set of reporter plasmids we demonstrate transfection of cells in mouse and human xenograft skin using intradermal injection and two microneedle array delivery systems. Reporter gene expression could be detected in individual keratinocytes, in real-time, in both mouse skin as well as human skin xenografts. These studies revealed that non-invasive intravital imaging can be used as a guide for developing gene delivery tools, establishing a benchmark for comparative testing of nucleic acid skin delivery technologies.

  View details for DOI 10.1038/srep00158

  View details for Web of Science ID 000300557200002

  View details for PubMedID 22355673

  View details for PubMedCentralID PMC3240989

• Molecular Imaging of Inflammation and Carcinogenesis CANCER PREVENTION RESEARCH Ostrov, D. A., Contag, C. H. 2011; 4 (10): 1523-1526

  Abstract

  Development of imaging agents that can be used broadly for early detection of neoplasia at various tissue sites and at various stages of disease and that also can assess states of minimal residual disease would have tremendous utility in the diagnosis and management of cancer. In a series of articles culminating with a report in this issue of the journal (beginning on page 1536), Uddin and colleagues show their ability to systemically target the enzyme COX-2 with imaging probes that will serve as agents for early detection, risk assessment, prognosis, and intervention outcome measures. These probes will enable the detection and localization of regions of inflammation and a wide variety of premalignant lesions and cancers, with utility in monitoring the effects of cancer prevention and therapy.

  View details for DOI 10.1158/1940-6207.CAPR-11-0418

  View details for Web of Science ID 000295620000001

  View details for PubMedID 21972077

• Pulse duration determines levels of Hsp70 induction in tissues following laser irradiation JOURNAL OF BIOMEDICAL OPTICS Mackanos, M. A., Contag, C. H. 2011; 16 (7)

  Abstract

  Induction of heat shock protein (Hsp) expression correlates with cytoprotection, reduced tissue damage, and accelerated healing in animal models. Since Hsps are transcriptionally activated in response to stress, they can act as stress indicators in burn injury or surgical procedures that produce heat and thermal change. A fast in vivo readout for induction of Hsp transcription in tissues would allow for the study of these proteins as therapeutic effect mediators and reporters of thermal stress∕damage. We used a transgenic reporter mouse in which a luciferase expression is controlled by the regulatory region of the inducible 70 kilodalton (kDa) Hsp as a rapid readout of cellular responses to laser-mediated thermal stress∕injury in mouse skin. We assessed the pulse duration dependence of the Hsp70 expression after irradiation with a CO(2) laser at 10.6 μm in wavelength over a range of 1000 to 1 ms. Hsp70 induction varied with changes in laser pulse durations and radiant exposures, which defined the ranges at which thermal activation of Hsp70 can be used to protect cells from subsequent stress, and reveals the window of thermal stress that tissues can endure.

  View details for DOI 10.1117/1.3600013

  View details for Web of Science ID 000294453800052

  View details for PubMedID 21806294

• Efficacy of Antimicrobial Peptoids against Mycobacterium tuberculosis ANTIMICROBIAL AGENTS AND CHEMOTHERAPY Kapoor, R., Eimerman, P. R., Hardy, J. W., Cirillo, J. D., Contag, C. H., Barron, A. E. 2011; 55 (6): 3058-3062

  Abstract

  Tuberculosis is a leading cause of death worldwide. Resistance of Mycobacterium to antibiotics can make treatments less effective in some cases. We tested selected oligopeptoids--previously reported as mimics of natural host defense peptides--for activity against Mycobacterium tuberculosis and assessed their cytotoxicity. A tetrameric, alkylated, cationic peptoid (1-C13(4mer)) was most potent against M. tuberculosis and least cytotoxic, whereas an unalkylated analogue, peptoid 1(4mer), was inactive. Peptoid 1-C13(4mer) thus merits further study as a potential antituberculosis drug.

  View details for DOI 10.1128/AAC.01667-10

  View details for Web of Science ID 000290713400091

  View details for PubMedID 21464254

  View details for PubMedCentralID PMC3101442

• Image-guided genomic analysis of tissue response to laser-induced thermal stress JOURNAL OF BIOMEDICAL OPTICS Mackanos, M. A., Helms, M., Kalish, F., Contag, C. H. 2011; 16 (5)

  Abstract

  The cytoprotective response to thermal injury is characterized by transcriptional activation of "heat shock proteins" (hsp) and proinflammatory proteins. Expression of these proteins may predict cellular survival. Microarray analyses were performed to identify spatially distinct gene expression patterns responding to thermal injury. Laser injury zones were identified by expression of a transgene reporter comprised of the 70 kD hsp gene and the firefly luciferase coding sequence. Zones included the laser spot, the surrounding region where hsp70-luc expression was increased, and a region adjacent to the surrounding region. A total of 145 genes were up-regulated in the laser irradiated region, while 69 were up-regulated in the adjacent region. At 7 hours the chemokine Cxcl3 was the highest expressed gene in the laser spot (24 fold) and adjacent region (32 fold). Chemokines were the most common up-regulated genes identified. Microarray gene expression was successfully validated using qRT- polymerase chain reaction for selected genes of interest. The early response genes are likely involved in cytoprotection and initiation of the healing response. Their regulatory elements will benefit creating the next generation reporter mice and controlling expression of therapeutic proteins. The identified genes serve as drug development targets that may prevent acute tissue damage and accelerate healing.

  View details for DOI 10.1117/1.3573387

  View details for Web of Science ID 000291170200029

  View details for PubMedID 21639585

  View details for PubMedCentralID PMC3107838

• Use of Self-Delivery siRNAs to Inhibit Gene Expression in an Organotypic Pachyonychia Congenita Model JOURNAL OF INVESTIGATIVE DERMATOLOGY Hickerson, R. P., Flores, M. A., Leake, D., Lara, M. F., Contag, C. H., Leachman, S. A., Kaspar, R. L. 2011; 131 (5): 1037-1044

  Abstract

  Although RNA interference offers therapeutic potential for treating skin disorders, delivery hurdles have hampered clinical translation. We have recently demonstrated that high pressure, resulting from intradermal injection of large liquid volumes, facilitated nucleic acid uptake by keratinocytes in mouse skin. Furthermore, similar intradermal injections of small interfering RNA (siRNA; TD101) into pachyonychia congenita (PC) patient foot lesions resulted in improvement. Unfortunately, the intense pain associated with hypodermic needle administration to PC lesions precludes this as a viable delivery option for this disorder. To investigate siRNA uptake by keratinocytes, an organotypic epidermal model, in which pre-existing endogenous gene or reporter gene expression can be readily monitored, was used to evaluate the effectiveness of "self-delivery" siRNA (i.e., siRNA chemically modified to enhance cellular uptake). In this model system, self-delivery siRNA treatment resulted in reduction of pre-existing fluorescent reporter gene expression under conditions in which unmodified controls had little or no effect. Additionally, treatment of PC epidermal equivalents with self-delivery "TD101" siRNA resulted in marked reduction of mutant keratin 6a mRNA with little or no effect on wild-type expression. These results indicate that chemical modification of siRNA may overcome certain limitations to transdermal delivery (specifically keratinocyte uptake) and may have clinical utility for inhibition of gene expression in the skin.

  View details for DOI 10.1038/jid.2010.426

  View details for Web of Science ID 000289789900011

  View details for PubMedID 21248764

• Development of Quantitative Molecular Clinical End Points for siRNA Clinical Trials JOURNAL OF INVESTIGATIVE DERMATOLOGY Hickerson, R. P., Leachman, S. A., Pho, L. N., Gonzalez-Gonzalez, E., Smith, F. J., McLean, W. H., Contag, C. H., Leake, D., Milstone, L. M., Kaspar, R. L. 2011; 131 (5): 1029-1036

  Abstract

  RNA interference (RNAi) is an evolutionarily conserved mechanism that results in specific gene inhibition at the mRNA level. The discovery that short interfering RNAs (siRNAs) are selective, potent, and can largely avoid immune surveillance has resulted in keen interest to develop these inhibitors as therapeutics. A single nucleotide-specific siRNA (K6a_513a.12, also known as TD101) was recently evaluated in a phase 1b clinical trial for the rare skin disorder, pachyonychia congenita (PC). To develop a clinical trial molecular end point for this type of trial, methods were developed to: (1) isolate total RNA containing amplifiable mRNA from human skin and callus material; (2) quantitatively distinguish the single-nucleotide mutant mRNA from wild-type K6a mRNA in both patient-derived keratinocytes and patient callus; and (3) demonstrate that repeated siRNA treatment results in sustained inhibition of mutant K6a mRNA in patient-derived keratinocyte cultures. These methods allow noninvasive sampling and monitoring of gene expression from patient-collected shavings and may be useful in evaluating the effectiveness of RNAi-based therapeutics, including inhibitors that specifically target single-nucleotide mutations.

  View details for DOI 10.1038/jid.2010.372

  View details for Web of Science ID 000289789900010

  View details for PubMedID 21191405

• In Vivo Imaging of Human and Mouse Skin with a Handheld Dual-Axis Confocal Fluorescence Microscope JOURNAL OF INVESTIGATIVE DERMATOLOGY Ra, H., Piyawattanametha, W., Gonzalez-Gonzalez, E., Mandella, M. J., Kino, G. S., Solgaard, O., Leake, D., Kaspar, R. L., Oro, A., Contag, C. H. 2011; 131 (5): 1061-1066

  Abstract

  Advancing molecular therapies for the treatment of skin diseases will require the development of new tools that can reveal spatiotemporal changes in the microanatomy of the skin and associate these changes with the presence of the therapeutic agent. For this purpose, we evaluated a handheld dual-axis confocal (DAC) microscope that is capable of in vivo fluorescence imaging of skin, using both mouse models and human skin. Individual keratinocytes in the epidermis were observed in three-dimensional image stacks after topical administration of near-infrared (NIR) dyes as contrast agents. This suggested that the DAC microscope may have utility in assessing the clinical effects of a small interfering RNA (siRNA)-based therapeutic (TD101) that targets the causative mutation in pachyonychia congenita (PC) patients. The data indicated that (1) formulated indocyanine green (ICG) readily penetrated hyperkeratotic PC skin and normal callused regions compared with nonaffected areas, and (2) TD101-treated PC skin revealed changes in tissue morphology, consistent with reversion to nonaffected skin compared with vehicle-treated skin. In addition, siRNA was conjugated to NIR dye and shown to penetrate through the stratum corneum barrier when topically applied to mouse skin. These results suggest that in vivo confocal microscopy may provide an informative clinical end point to evaluate the efficacy of experimental molecular therapeutics.

  View details for DOI 10.1038/jid.2010.401

  View details for Web of Science ID 000289789900014

  View details for PubMedID 21191407

• Visualization of functional nucleic acid delivery to mouse and human epidermis 71st Annual Meeting of the Society-for-Investigative-Dermatology Gonzalez-Gonzalez, E., Kim, Y., Speacker, T. J., Hickerson, R. P., Spitler, R., Birchall, J., Lara, M., Hu, R., Liang, Y., Kirkiles-Smith, N., Prausnitz, M. R., Milstone, L. M., Contag, C. H., Kaspar, R. L. NATURE PUBLISHING GROUP. 2011: S69–S69

  View details for Web of Science ID 000289035600414

• Components of a Curriculum for Molecular Imaging Scientists JOURNAL OF NUCLEAR MEDICINE Zinn, K. R., Anderson, C. J., Bradbury, M., Cutler, C. S., Peterson, T. E., Morgan, D. E., Price, J. C., Graham, M. M., Contag, C. H., Wittstrom, K., Norenberg, J. P. 2011; 52 (4): 650-656

  Abstract

  Molecular imaging is the visualization, characterization, and measurement of biologic processes at the molecular and cellular levels in humans and other living systems (1). It comprises an emerging set of technologies that builds on advances in imaging procedures (e.g., PET, SPECT, MRI, ultrasound, optical, and photoacoustic), improved understanding of biology, and the development of molecularly targeted agents. These continuously expanding sets of imaging methods are often used in combination, and advances in data acquisition and analyses facilitate a more complete understanding of biology. Molecular imaging aims to improve our understanding of mammalian biology and lead to advances in patient care by providing targeted therapies that will enable personalized medicine and the imaging tools to assess outcome. Implementation of these new technologies in clinical care has many educational, technical, and regulatory challenges that must be overcome before molecular imaging reaches its full potential. The impact of molecular imaging has been significant in several disciplines, because it represents a paradigm shift in how scientists and clinicians can observe biology in real time and in a relatively noninvasive manner to enable the power of repeated measures in living organisms.

  View details for DOI 10.2967/jnumed.110.087064

  View details for Web of Science ID 000288804500027

  View details for PubMedID 21421709

• Non-damaging Retinal Phototherapy: Dynamic Range of Heat Shock Protein Expression INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE Sramek, C., Mackanos, M., Spitler, R., Leung, L., Nomoto, H., Contag, C. H., Palanker, D. 2011; 52 (3): 1780-1787

  Abstract

  Subthreshold retinal phototherapy demonstrated clinical efficacy for the treatment of diabetic macular edema without visible signs of retinal damage. To assess the range of cellular responses to sublethal hyperthermia, expression of the gene encoding a 70 kDa heat shock protein (HSP70) was evaluated after laser irradiation using a transgenic reporter mouse.One hundred millisecond, 532 nm laser exposures with 400 μm beam diameter were applied to the retina surrounding the optic nerve in 32 mice. Transcription from the HSP70 promoter was assessed relative to the control eye using a bioluminescence assay at 7 hours after laser application. The retinal pigmented epithelium (RPE) viability threshold was determined with a fluorescence assay. A computational model was developed to estimate temperature and the extent of cell damage.A significant increase in HSP70 transcription was found at exposures over 20 mW, half the threshold power for RPE cell death. Computational modeling estimated peak temperature T = 49°C at HSP70 expression threshold. At RPE viability threshold, T = 57°C. Similar temperatures and damage indices were calculated for clinical subvisible retinal treatment parameters.Beneficial effects of laser therapy have been previously shown to extend beyond those resulting from destruction of tissue. One hundred millisecond laser exposures at approximately half the threshold power of RPE damage induced transcription of HSP70, an indication of cellular response to sublethal thermal stress. A computational model of retinal hyperthermia can guide further optimization of laser parameters for nondamaging phototherapy.

  View details for DOI 10.1167/iovs.10-5917

  View details for Web of Science ID 000288965300070

  View details for PubMedID 21087969

• In Vivo Micro-Image Mosaicing IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING Loewke, K. E., Camarillo, D. B., Piyawattanametha, W., Mandella, M. J., Contag, C. H., Thrun, S., Salisbury, J. K. 2011; 58 (1): 159-171

  Abstract

  Recent advances in optical imaging have led to the development of miniature microscopes that can be brought to the patient for visualizing tissue structures in vivo. These devices have the potential to revolutionize health care by replacing tissue biopsy with in vivo pathology. One of the primary limitations of these microscopes, however, is that the constrained field of view can make image interpretation and navigation difficult. In this paper, we show that image mosaicing can be a powerful tool for widening the field of view and creating image maps of microanatomical structures. First, we present an efficient algorithm for pairwise image mosaicing that can be implemented in real time. Then, we address two of the main challenges associated with image mosaicing in medical applications: cumulative image registration errors and scene deformation. To deal with cumulative errors, we present a global alignment algorithm that draws upon techniques commonly used in probabilistic robotics. To accommodate scene deformation, we present a local alignment algorithm that incorporates deformable surface models into the mosaicing framework. These algorithms are demonstrated on image sequences acquired in vivo with various imaging devices including a hand-held dual-axes confocal microscope, a miniature two-photon microscope, and a commercially available confocal microendoscope.

  View details for DOI 10.1109/TBME.2010.2085082

  View details for Web of Science ID 000285515500020

  View details for PubMedID 20934939

• 3-D MEMS Scanning System For Dual-Axis Confocal Microendoscopy 16th International Conference on Optical MEMS and Nanophotonics (OMN) Jeong, J., Mandella, M. J., Kino, G. S., Contag, C. H., Solgaard, O. IEEE. 2011: 71–72

  View details for Web of Science ID 000297850100029

• Point-of-care pathology with miniature microscopes ANALYTICAL CELLULAR PATHOLOGY Liu, J. T., Loewke, N. O., Mandella, M. J., Levenson, R. M., Crawford, J. M., Contag, C. H. 2011; 34 (3): 81-98

  Abstract

  Advances in optical designs are enabling the development of miniature microscopes that can examine tissue in situ for early anatomic and molecular indicators of disease, in real time, and at cellular resolution. These new devices will lead to major changes in how diseases are detected and managed, driving a shift from today's diagnostic paradigm of biopsy followed by histopathology and recommended therapy, to non-invasive point-of-care diagnosis with possible same-session definitive treatment. This shift may have major implications for the training requirements of future physicians to enable them to interpret real-time in vivo microscopic data, and will also shape the emerging fields of telepathology and telemedicine. Implementation of new technologies into clinical practice is a complex process that requires bridging gaps between clinicians, engineers and scientists. This article provides a forward-looking discussion of these issues, with a focus on malignant and pre-malignant lesions, by first highlighting some of the clinical areas where point-of-care in vivo microscopy could address unmet needs, and then by reviewing the technological challenges that are being addressed, or need to be addressed, for in vivo microscopy to become a standard clinical tool.

  View details for DOI 10.3233/ACP-2011-0011

  View details for Web of Science ID 000293107200001

  View details for PubMedID 21673433

  View details for PubMedCentralID PMC3166958

• Longitudinal, Noninvasive Imaging of T-Cell Effector Function and Proliferation in Living Subjects CANCER RESEARCH Patel, M. R., Chang, Y., Chen, I. Y., Bachmann, M. H., Yan, X., Contag, C. H., Gambhir, S. S. 2010; 70 (24): 10141-10149

  Abstract

  Adoptive immunotherapy is evolving to assume an increasing role in treating cancer. Most imaging studies in adoptive immunotherapy to date have focused primarily on locating tumor-specific T cells rather than understanding their effector functions. In this study, we report the development of a noninvasive imaging strategy to monitor T-cell activation in living subjects by linking a reporter gene to the Granzyme B promoter (pGB), whose transcriptional activity is known to increase during T-cell activation. Because pGB is relatively weak and does not lead to sufficient reporter gene expression for noninvasive imaging, we specifically employed 2 signal amplification strategies, namely the Two Step Transcription Amplification (TSTA) strategy and the cytomegalovirus enhancer (CMVe) strategy, to maximize firefly luciferase reporter gene expression. Although both amplification strategies were capable of increasing pGB activity in activated primary murine splenocytes, only the level of bioluminescence activity achieved with the CMVe strategy was adequate for noninvasive imaging in mice. Using T cells transduced with a reporter vector containing the hybrid pGB-CMVe promoter, we were able to optically image T-cell effector function longitudinally in response to tumor antigens in living mice. This methodology has the potential to accelerate the study of adoptive immunotherapy in preclinical cancer models.

  View details for DOI 10.1158/0008-5472.CAN-10-1843

  View details for Web of Science ID 000285334200016

  View details for PubMedID 21159636

  View details for PubMedCentralID PMC3057959

• Definition of an Enhanced Immune Cell Therapy in Mice That Can Target Stem-Like Lymphoma Cells CANCER RESEARCH Contag, C. H., Sikorski, R., Negrin, R. S., Schmidt, T., Fan, A. C., Bachireddy, P., Felsher, D. W., Thorne, S. H. 2010; 70 (23): 9837-9845

  Abstract

  Current treatments of high-grade lymphoma often have curative potential, but unfortunately many patients relapse and develop therapeutic resistance. Thus, there remains a need for novel therapeutics that can target the residual cancer cells whose phenotypes are distinct from the bulk tumor and that are capable of reforming tumors from very few cells. Oncolytic viruses offer an approach to destroy tumors by multiple mechanisms, but they cannot effectively reach residual disease or micrometastases, especially within the lymphatic system. To address these limitations, we have generated immune cells infected with oncolytic viruses as a therapeutic strategy that can combine effective cellular delivery with synergistic tumor killing. In this study, we tested this approach against minimal disease states of lymphomas characterized by the persistence of cancer cells that display stem cell-like properties and resistance to conventional therapies. We found that the immune cells were capable of trafficking to and targeting residual cancer cells. The combination biotherapy used prevented relapse by creating a long-term, disease-free state, with acquired immunity to the tumor functioning as an essential mediator of this effect. Immune components necessary for this acquired immunity were identified. We further demonstrated that the dual biotherapy could be applied before or after conventional therapy. Our approach offers a potentially powerful new way to clear residual cancer cells, showing how restoring immune surveillance is critical for maintenance of a disease-free state.

  View details for DOI 10.1158/0008-5472.CAN-10-2650

  View details for PubMedID 20935221

• Microarray Analysis of Cellular Thermotolerance LASERS IN SURGERY AND MEDICINE Beckham, J. T., Wilmink, G. J., Opalenik, S. R., Mackanos, M. A., Abraham, A. A., Takahashi, K., Contag, C. H., Takahashi, T., Jansen, E. D. 2010; 42 (10): 752-765

  Abstract

  Previously, we have shown that a 43°C pretreatment can provide thermotolerance to a following, more severe, thermal stress at 45°C. Using cells that lack the Hsp70 gene, we have also shown that there is still some thermotolerance in the absence of HSP70 protein. The purpose of this study was to determine which genes play a role in thermotolerance by measuring viability and proliferation of the cells at 2 days after heating. Specifically, we wanted to understand which pathways may be responsible for protecting cells in the absence of HSP70.Murine embryonic fibroblast cells with and without Hsp70 (MEF(+/+) and MEF(-/-), respectively) were exposed to a mild heat shock of 43°C for 30 minutes in a constant temperature water bath. After 3 hours of recovery, RNA was harvested from three heated samples alongside three untreated controls using a MicroRNeasy kit with DNAse treatment. RNA quality was verified by an Agilent Bioanalyzer. The RNA was then converted to cDNA and hybridized to Affymetrix gene expression DNA microarrays. The genes that showed a twofold change (up or down) relative to unheated controls were filtered by t-test for significance at a threshold of P < 0.05 using Genespring software. Data were verified by qRT-PCR. Genes were then categorized based upon their ontology.While many genes were similarly upregulated, the main difference between cell types was an increase in transcription factors and nucleic acid binding proteins. Several genes known to be involved in the heat response were upregulated more than twofold (Hsp70, Hsp40, Hsp110, Hsp25, Atf3), however, another well studied heat responsive gene Hsp90 only increased by 1.5-fold under these conditions despite its role in thermotolerance.The data herein presents genetic pathways which are candidates for further study of pretreatment protocols in laser irradiation.

  View details for DOI 10.1002/lsm.20983

  View details for Web of Science ID 000286440100008

  View details for PubMedID 21246580

• Cancer stem cells from human breast tumors are involved in spontaneous metastases in orthotopic mouse models PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Liu, H., Patel, M. R., Prescher, J. A., Patsialou, A., Qian, D., Lin, J., Wen, S., Chang, Y., Bachmann, M. H., Shimono, Y., Dalerba, P., Adorno, M., Lobo, N., Bueno, J., Dirbas, F. M., Goswami, S., Somlo, G., Condeelis, J., Contag, C. H., Gambhir, S. S., Clarke, M. F. 2010; 107 (42): 18115-18120

  Abstract

  To examine the role of breast cancer stem cells (BCSCs) in metastasis, we generated human-in-mouse breast cancer orthotopic models using patient tumor specimens, labeled with optical reporter fusion genes. These models recapitulate human cancer features not captured with previous models, including spontaneous metastasis in particular, and provide a useful platform for studies of breast tumor initiation and progression. With noninvasive imaging approaches, as few as 10 cells of stably labeled BCSCs could be tracked in vivo, enabling studies of early tumor growth and spontaneous metastasis. These advances in BCSC imaging revealed that CD44(+) cells from both primary tumors and lung metastases are highly enriched for tumor-initiating cells. Our metastatic cancer models, combined with noninvasive imaging techniques, constitute an integrated approach that could be applied to dissect the molecular mechanisms underlying the dissemination of metastatic CSCs (MCSCs) and to explore therapeutic strategies targeting MCSCs in general or to evaluate individual patient tumor cells and predict response to therapy.

  View details for DOI 10.1073/pnas.1006732107

  View details for Web of Science ID 000283184800050

  View details for PubMedID 20921380

  View details for PubMedCentralID PMC2964232

• Increased interstitial pressure improves nucleic acid delivery to skin enabling a comparative analysis of constitutive promoters GENE THERAPY Gonzalez-Gonzalez, E., Ra, H., Spitler, R., Hickerson, R. P., Contag, C. H., Kaspar, R. L. 2010; 17 (10): 1270-1278

  Abstract

  Nucleic acid-based therapies hold great promise for treatment of skin disorders if delivery challenges can be overcome. To investigate one mechanism of nucleic acid delivery to keratinocytes, a fixed mass of expression plasmid was intradermally injected into mouse footpads in different volumes, and reporter expression was monitored by intravital imaging or skin sectioning. Reporter gene expression increased with higher delivery volumes, suggesting that pressure drives nucleic acid uptake into cells after intradermal injections similar to previously published studies for muscle and liver. For spatiotemporal analysis of reporter gene expression, a dual-axis confocal (DAC) fluorescence microscope was used for intravital imaging following intradermal injections. Individual keratinocytes expressing hMGFP were readily visualized in vivo and initially appeared to preferentially express in the stratum granulosum and subsequently migrate to the stratum corneum over time. Fluorescence microscopy of frozen skin sections confirmed the patterns observed by intravital imaging. Intravital imaging with the DAC microscope is a noninvasive method for probing spatiotemporal control of gene expression and should facilitate development and testing of new nucleic acid delivery technologies.

  View details for DOI 10.1038/gt.2010.74

  View details for Web of Science ID 000282948600011

  View details for PubMedID 20463756

• Biodegradable Nanoparticles With Sustained Release of Functional siRNA in Skin JOURNAL OF PHARMACEUTICAL SCIENCES Jacobson, G. B., Gonzalez-Gonzalez, E., Spitler, R., Shinde, R., Leake, D., Kaspar, R. L., Contag, C. H., Zare, R. N. 2010; 99 (10): 4261-4266

  Abstract

  A key challenge in developing RNAi-based therapeutics is efficient delivery of functional short interfering RNA (siRNA) to target cells. To address this need, we have used a supercritical CO(2) process to incorporate siRNA in biodegradable polymer nanoparticles (NPs) for in vivo sustained release. By this means we have obtained complete encapsulation of the siRNA with minimal initial burst effect from the surface of the NPs. The slow release of a fluorescently labeled siRNA mimic (siGLO Red) was observed for up to 80 days in vivo after intradermal injection into mouse footpads. In vivo gene silencing experiments were also performed, showing reduction of GFP signal in the epidermis of a reporter transgenic mouse model, which demonstrates that the siRNA retained activity following release from the polymer NPs.

  View details for DOI 10.1002/jps.22147

  View details for Web of Science ID 000282473400012

  View details for PubMedID 20737633

• Silencing of Reporter Gene Expression in Skin Using siRNAs and Expression of Plasmid DNA Delivered by a Soluble Protrusion Array Device (PAD) MOLECULAR THERAPY Gonzalez-Gonzalez, E., Speaker, T. J., Hickerson, R. P., Spitler, R., Flores, M. A., Leake, D., Contag, C. H., Kaspar, R. L. 2010; 18 (9): 1667-1674

  Abstract

  Despite rapid progress in the development of potent and selective small interfering RNA (siRNA) agents for skin disorders, translation to the clinic has been hampered by the lack of effective, patient-friendly delivery technologies. The stratum corneum poses a formidable barrier to efficient delivery of large and/or charged macromolecules including siRNAs. Intradermal siRNA injection results in effective knockdown of targeted gene expression but is painful and the effects are localized to the injection site. The use of microneedle arrays represents a less painful delivery method and may have utility for the delivery of nucleic acids, including siRNAs. For this purpose, we developed a loadable, dissolvable protrusion array device (PAD) that allows skin barrier penetration. The PAD tips dissolve upon insertion, forming a gel-like plug that releases functional cargo. PAD-mediated delivery of siRNA (modified for enhanced stability and cellular uptake) resulted in effective silencing of reporter gene expression in a transgenic reporter mouse model. PAD delivery of luciferase reporter plasmids resulted in expression in cells of the ear, back, and footpad skin as assayed by intravital bioluminescence imaging. These results support the use of PADs for delivery of functional nucleic acids to cells in the skin with an efficiency that may support clinical translation.

  View details for DOI 10.1038/mt.2010.126

  View details for Web of Science ID 000281502300013

  View details for PubMedID 20571543

  View details for PubMedCentralID PMC2956931

• Targeting Localized Immune Suppression Within the Tumor Through Repeat Cycles of Immune Cell-oncolytic Virus Combination Therapy MOLECULAR THERAPY Thorne, S. H., Liang, W., Sampath, P., Schmidt, T., Sikorski, R., Beilhack, A., Contag, C. H. 2010; 18 (9): 1698-1705

  Abstract

  A major limitation to the use of immunotherapy in the treatment of cancer has been the localized immune suppressive environment within the tumor. Although there is evidence that tumor-selective (oncolytic) viruses may help to overcome this immune suppression, a primary limitation to their use has been limited systemic delivery potential, especially in the face of antiviral immunity. We recently demonstrated that tumor-trafficking immune cells can efficiently deliver oncolytic viral therapies to their tumor targets. These cells act as both a therapeutic agent and also a carrier vehicle for the oncolytic virus. Here, we demonstrate that such delivery is also possible in the face of pre-existing antiviral immunity, so overcoming the limited systemic delivery of naked, cell-free virus. It was also found that treatment of previously immunized mice or repeat treatments leading to immunization resulted in a switch from a primarily oncolytic to an immunotherapeutic mechanism of action. Furthermore, repeat cycles of treatment with combination immune cell-viral therapy resulted in increased tumor infiltration of effector T-cells and a general reduction in the levels of known immune suppressive lymphocyte populations. This therefore represents a novel and effective means to overcome localized immune suppression within the tumor microenvironment.

  View details for DOI 10.1038/mt.2010.140

  View details for Web of Science ID 000281502300017

  View details for PubMedID 20606649

  View details for PubMedCentralID PMC2956933

• IL-12 enhances efficacy and shortens enrichment time in cytokine-induced killer cell immunotherapy CANCER IMMUNOLOGY IMMUNOTHERAPY Helms, M. W., Prescher, J. A., Cao, Y., Schaffert, S., Contag, C. H. 2010; 59 (9): 1325-1334

  Abstract

  Cytokine-induced killer (CIK) cells are T cell derived ex vivo expanded cells with both NK and T cell properties. They exhibit potent anti-tumor efficacy against various malignancies in preclinical models and have proven safe and effective in clinical studies. We combined CIK cell adoptive immunotherapy with IL-12 cytokine immunotherapy in an immunocompetent preclinical breast cancer model. Combining CIK cells with IL-12 increased anti-tumor efficacy in vivo compared to either therapy alone. Combination led to full tumor remission and long-term protection in 75% of animals. IL-12 treatment sharply increased the anti-tumor efficacy of short-term cultured CIK cells that exhibited no therapeutic effect alone. Bioluminescence imaging based in vitro cytotoxicity and in vivo homing assays revealed that short-term cultured CIK cells exhibit full cytotoxicity in vitro, but display different tumor homing properties than fully expanded CIK cells in vivo. Our data suggest that short-term cultured CIK cells can be "educated" in vivo, producing fully expanded CIK cells upon IL-12 administration with anti-tumor efficacy in a mouse model. Our findings demonstrate the potential to improve current CIK cell-based immunotherapy by increasing efficacy and shortening ex vivo expansion time. This holds promise for a highly efficacious cancer therapy utilizing synergistic effects of cytokine and cellular immunotherapy.

  View details for DOI 10.1007/s00262-010-0860-y

  View details for Web of Science ID 000279198000003

  View details for PubMedID 20532883

• Indirect imaging of cardiac-specific transgene expression using a bidirectional two-step transcriptional amplification strategy GENE THERAPY Chen, I. Y., Gheysens, O., Ray, S., Wang, Q., Padmanabhan, P., Paulmurugan, R., Loening, A. M., Rodriguez-Porcel, M., Willmann, J. K., Sheikh, A. Y., Nielsen, C. H., Hoyt, G., Contag, C. H., Robbins, R. C., Biswal, S., Wu, J. C., Gambhir, S. S. 2010; 17 (7): 827-838

  Abstract

  Transcriptional targeting for cardiac gene therapy is limited by the relatively weak activity of most cardiac-specific promoters. We have developed a bidirectional plasmid vector, which uses a two-step transcriptional amplification (TSTA) strategy to enhance the expression of two optical reporter genes, firefly luciferase (fluc) and Renilla luciferase (hrluc), driven by the cardiac troponin T (cTnT) promoter. The vector was characterized in vitro and in living mice using luminometry and bioluminescence imaging to assess its ability to mediate strong, correlated reporter gene expression in a cardiac cell line and the myocardium, while minimizing expression in non-cardiac cell lines and the liver. In vitro, the TSTA system significantly enhanced cTnT-mediated reporter gene expression with moderate preservation of cardiac specificity. After intramyocardial and hydrodynamic tail vein delivery of an hrluc-enhanced variant of the vector, long-term fluc expression was observed in the heart, but not in the liver. In both the cardiac cell line and the myocardium, fluc expression correlated well with hrluc expression. These results show the vector's ability to effectively amplify and couple transgene expression in a cardiac-specific manner. Further replacement of either reporter gene with a therapeutic gene should allow non-invasive imaging of targeted gene therapy in living subjects.

  View details for DOI 10.1038/gt.2010.30

  View details for Web of Science ID 000279614600002

  View details for PubMedID 20237511

  View details for PubMedCentralID PMC2900530

• Nanoparticle Formation of Organic Compounds With Retained Biological Activity JOURNAL OF PHARMACEUTICAL SCIENCES Jacobson, G. B., Shinde, R., McCullough, R. L., Cheng, N. J., Creasman, A., Beyene, A., Hickerson, R. P., Quan, C., Turner, C., Kaspar, R. L., Contag, C. H., Zare, R. N. 2010; 99 (6): 2750-2755

  Abstract

  Many pharmaceuticals are formulated as powders to aid drug delivery. A major problem is how to produce powders having high purity, controlled morphology, and retained bioactivity. We demonstrate the use of supercritical carbon dioxide as an antisolvent for meeting this need for two model drug systems, quercetin, a sparingly soluble antioxidant, and short interfering RNA (siRNA), which can silence genes. In both cases we achieve retention of bioactivity as well as a narrow particle size distribution in which the particles are free of impurities.

  View details for DOI 10.1002/jps.22035

  View details for Web of Science ID 000278241800022

  View details for PubMedID 20039390

• Fiber-optic probes enable cancer detection with FTIR spectroscopy TRENDS IN BIOTECHNOLOGY Mackanos, M. A., Contag, C. H. 2010; 28 (6): 317-323

  Abstract

  Fourier transform infrared spectroscopy (FTIR) reveals biochemical 'fingerprints' and has found disease patterns in excised human tissues. Fiber-optic probes have been developed for FTIR in living systems, allowing for cancer detection. There are challenges to making in vivo FTIR a reality, which are being addressed through hardware advances, determining key wavelengths and tissue preparation. Fiber-optic evanescent wave spectroscopy (FEWS)-FTIR with endoscope-compatible fiber-optic silver halide probes is feasible, and could prove useful for distinguishing premalignant and malignant tissues from biopsies or within patients. Developments of smaller silver halide probes as well as in vivo tissue drying methods will move this approach closer to the clinic where it can be used for early cancer detection, disease characterization and guided biopsies.

  View details for DOI 10.1016/j.tibtech.2010.04.001

  View details for Web of Science ID 000278946500005

  View details for PubMedID 20452071

• Assessing delivery and quantifying efficacy of small interfering ribonucleic acid therapeutics in the skin using a dual-axis confocal microscope JOURNAL OF BIOMEDICAL OPTICS Ra, H., Gonzalez-Gonzalez, E., Smith, B. R., Gambhir, S. S., Kino, G. S., Solgaard, O., Kaspar, R. L., Contag, C. H. 2010; 15 (3)

  Abstract

  Transgenic reporter mice and advances in imaging instrumentation are enabling real-time visualization of cellular mechanisms in living subjects and accelerating the development of novel therapies. Innovative confocal microscope designs are improving their utility for microscopic imaging of fluorescent reporters in living animals. We develop dual-axis confocal (DAC) microscopes for such in vivo studies and create mouse models where fluorescent proteins are expressed in the skin for the purpose of advancing skin therapeutics and transdermal delivery tools. Three-dimensional image volumes, through the different skin compartments of the epidermis and dermis, can be acquired in several seconds with the DAC microscope in living mice, and are comparable to histologic analyses of reporter protein expression patterns in skin sections. Intravital imaging with the DAC microscope further enables visualization of green fluorescent protein (GFP) reporter gene expression in the skin over time, and quantification of transdermal delivery of small interfering RNA (siRNA) and therapeutic efficacy. Visualization of transdermal delivery of nucleic acids will play an important role in the development of innovative strategies for treating skin pathologies.

  View details for DOI 10.1117/1.3432627

  View details for Web of Science ID 000280642900042

  View details for PubMedID 20615029

  View details for PubMedCentralID PMC2904026

• Role of nitric oxide in Salmonella typhimurium-mediated cancer cell killing BMC CANCER Barak, Y., Schreiber, F., Thorne, S. H., Contag, C. H., deBeer, D., Matin, A. 2010; 10

  Abstract

  Bacterial targeting of tumours is an important anti-cancer strategy. We previously showed that strain SL7838 of Salmonella typhimurium targets and kills cancer cells. Whether NO generation by the bacteria has a role in SL7838 lethality to cancer cells is explored. This bacterium has the mechanism for generating NO, but also for decomposing it.Mechanism underlying Salmonella typhimurium tumour therapy was investigated through in vitro and in vivo studies. NO measurements were conducted either by chemical assays (in vitro) or using Biosensors (in vivo). Cancer cells cytotoxic assay were done by using MTS. Bacterial cell survival and tumour burden were determined using molecular imaging techniques.SL7838 generated nitric oxide (NO) in anaerobic cell suspensions, inside infected cancer cells in vitro and in implanted 4T1 tumours in live mice, the last, as measured using microsensors. Thus, under these conditions, the NO generating pathway is more active than the decomposition pathway. The latter was eliminated, in strain SL7842, by the deletion of hmp- and norV genes, making SL7842 more proficient at generating NO than SL7838. SL7842 killed cancer cells more effectively than SL7838 in vitro, and this was dependent on nitrate availability. This strain was also ca. 100% more effective in treating implanted 4T1 mouse tumours than SL7838.NO generation capability is important in the killing of cancer cells by Salmonella strains.

  View details for DOI 10.1186/1471-2407-10-146

  View details for Web of Science ID 000277802300001

  View details for PubMedID 20398414

  View details for PubMedCentralID PMC2868810

• Micromirror-scanned dual-axis confocal microscope utilizing a gradient-index relay lens for image guidance during brain surgery JOURNAL OF BIOMEDICAL OPTICS Liu, J. T., Mandella, M. J., Loewke, N. O., Haeberle, H., Ra, H., Piyawattanametha, W., Solgaard, O., Kino, G. S., Contag, C. H. 2010; 15 (2)

  Abstract

  A fluorescence confocal microscope incorporating a 1.8-mm-diam gradient-index relay lens is developed for in vivo histological guidance during resection of brain tumors. The microscope utilizes a dual-axis confocal architecture to efficiently reject out-of-focus light for high-contrast optical sectioning. A biaxial microelectromechanical system (MEMS) scanning mirror is actuated at resonance along each axis to achieve a large field of view with low-voltage waveforms. The unstable Lissajous scan, which results from actuating the orthogonal axes of the MEMS mirror at highly disparate resonance frequencies, is optimized to fully sample 500x500 pixels at two frames per second. Optically sectioned fluorescence images of brain tissues are obtained in living mice to demonstrate the utility of this microscope for image-guided resections.

  View details for DOI 10.1117/1.3386055

  View details for Web of Science ID 000278465300060

  View details for PubMedID 20459274

  View details for PubMedCentralID PMC2869369

• Guided by the light: visualizing biomolecular processes in living animals with bioluminescence CURRENT OPINION IN CHEMICAL BIOLOGY Prescher, J. A., Contag, C. H. 2010; 14 (1): 80-89

  Abstract

  Bioluminescence imaging (BLI) exploits the light-emitting properties of luciferase enzymes for monitoring cells and biomolecular processes in living subjects. Luciferases can be incorporated into a variety of non-luminescent hosts and used to track cells, visualize gene expression, and analyze collections of biomolecules. This article highlights recent applications of BLI to studies of mammalian biology, along with the development of novel bioluminescent probes to 'see' cells and molecules in action. Collectively, these efforts are expanding our understanding of living systems and shedding light on the molecular underpinnings of disease.

  View details for DOI 10.1016/j.cbpa.2009.11.001

  View details for Web of Science ID 000274946700013

  View details for PubMedID 19962933

• Mast Cell-Derived TNF Can Exacerbate Mortality during Severe Bacterial Infections in C57BL/6-KitW-sh/W-sh Mice AMERICAN JOURNAL OF PATHOLOGY Piliponsky, A. M., Chen, C., Grimbaldeston, M. A., Burns-Guydish, S. M., Hardy, J., Kalesnikoff, J., Contag, C. H., Tsai, M., Galli, S. J. 2010; 176 (2): 926-938

  Abstract

  We used mast cell-engrafted genetically mast cell-deficient C57BL/6-Kit(W-sh/W-sh) mice to investigate the roles of mast cells and mast cell-derived tumor necrosis factor in two models of severe bacterial infection. In these mice, we confirmed findings derived from studies of mast cell-deficient WBB6F(1)-Kit(W/W-v) mice indicating that mast cells can promote survival in cecal ligation and puncture (CLP) of moderate severity. However, we found that the beneficial role of mast cells in this setting can occur independently of mast cell-derived tumor necrosis factor. By contrast, using mast cell-engrafted C57BL/6-Kit(W-sh/W-sh) mice, we found that mast cell-derived tumor necrosis factor can increase mortality during severe CLP and can also enhance bacterial growth and hasten death after intraperitoneal inoculation of Salmonella typhimurium. In WBB6F(1)-Kit(W-sh/W-sh) mice, mast cells enhanced survival during moderately severe CLP but did not significantly change the survival observed in severe CLP. Our findings in three types of genetically mast cell-deficient mice thus support the hypothesis that, depending on the circumstances (including mouse strain background, the nature of the mutation resulting in a mast cell deficiency, and type and severity of infection), mast cells can have either no detectable effect or opposite effects on survival during bacterial infections, eg, promoting survival during moderately severe CLP associated with low mortality but, in C57BL/6-Kit(W-sh/W-sh) mice, increasing mortality during severe CLP or infection with S. typhimurium.

  View details for DOI 10.2353/ajpath.2010.090342

  View details for Web of Science ID 000274111400040

  View details for PubMedID 20035049

  View details for PubMedCentralID PMC2808097

• Timing of Bone Marrow Cell Delivery Has Minimal Effects on Cell Viability and Cardiac Recovery After Myocardial Infarction CIRCULATION-CARDIOVASCULAR IMAGING Swijnenburg, R., Govaert, J. A., van der Bogt, K. E., Pearl, J. I., Huang, M., Stein, W., Hoyt, G., Vogel, H., Contag, C. H., Robbins, R. C., Wu, J. C. 2010; 3 (1): 77-U109

  Abstract

  Despite ongoing clinical trials, the optimal time for delivery of bone marrow mononuclear cells (BMCs) after myocardial infarction is unclear. We compared the viability and effects of transplanted BMCs on cardiac function in the acute and subacute inflammatory phases of myocardial infarction.The time course of acute inflammatory cell infiltration was quantified by FACS analysis of enzymatically digested hearts of FVB mice (n=12) after left anterior descending artery ligation. Mac-1(+)Gr-1(high) neutrophil infiltration peaked at day 4. BMCs were harvested from transgenic FVB mice expressing firefly luciferase (Fluc) and green fluorescent protein (GFP). Afterward, 2.5x10(6) BMCs were injected into the left ventricle of wild-type FVB mice either immediately (acute BMC) or 7 days (subacute BMC) after myocardial infarction, or after a sham procedure (n=8 per group). In vivo bioluminescence imaging showed an early signal increase in both BMC groups at day 7, followed by a nonsignificant trend (P=0.203) toward improved BMC survival in the subacute BMC group that persisted until the bioluminescence imaging signal reached<0.01) and 6 weeks (both BMC groups versus saline; P<0.05) but no significant differences between the 2 BMC groups. FACS analysis of BMC-injected hearts at day 7 revealed that GFP(+) BMCs expressed hematopoietic (CD45, Mac-1, Gr-1), minimal progenitor (Sca-1, c-kit), and no endothelial (CD133, Flk-1) or cardiac (Trop-T) cell markers.Timing of BMC delivery has minimal effects on intramyocardial retention and preservation of cardiac function. In general, there is poor long-term engraftment and BMCs tend to adopt inflammatory cell phenotypes.

  View details for DOI 10.1161/CIRCIMAGING.109.872085

  View details for PubMedID 19920031

• From Bench to Bedside with Advanced Dual-Axes Confocal Microendoscope 23rd IEEE International Conference on Micro Electro Mechanical Systems (MEMS 2010) Piyawattanametha, W., Ra, H., Mandella, M. J., Contag, C. H., Solgaard, O. IEEE. 2010: 27–30

  View details for Web of Science ID 000278416400005

• Characterizing deep optical-sectioning microscopy performance with scattering phantoms and numerical simulations Conference on Design and Performance Validation of Phantoms Used in Conjunction with Optical Measurements of Tissue II Liu, J. T., Mandella, M. J., Kino, G. S., Contag, C. H. SPIE-INT SOC OPTICAL ENGINEERING. 2010

  View details for DOI 10.1117/12.844315

  View details for Web of Science ID 000284364900004

• From Bench to Bedside with Advanced Confocal Microendoscope IEEE Photonics Society Winter Topicals Meeting Series Piyawattanametha, W., Mandella, M. J., Ra, H., Liu, J. T., Friedland, S., Qiu, Z., Kino, G. S., Wang, T. D., Contag, C. H., Solgaard, O. IEEE. 2010: 83–84

  View details for Web of Science ID 000283803700044

• Surgical dual-axis confocal microscope for brain tumor resection IEEE Photonics Society Winter Topicals Meeting Series Liu, J. T., Mandella, M. J., Loewke, N. O., Haeberle, H., Ra, H., Piyawattanametha, W., Solgaard, O., Kino, G. S., Contag, C. H. IEEE. 2010: 76–77

  View details for Web of Science ID 000283803700040

• FTIR microspectroscopy for improved prostate cancer diagnosis TRENDS IN BIOTECHNOLOGY Mackanos, M. A., Contag, C. H. 2009; 27 (12): 661-663

  Abstract

  Accurate diagnosis and prognosis is essential for cancer management but is subject to sampling and inter-observer error. In a recent study, Baker et al. compared Fourier transform infrared (FTIR) microspectroscopy with histological pathology to evaluate prostate tissue for disease severity. The authors found that biochemical changes associated with prostate cancer could be discriminated by FTIR to classify confined and locally invasive prostate cancers. These findings could enable the development of improved diagnostic and prognostic methods for the detection and treatment of prostate cancers.

  View details for DOI 10.1016/j.tibtech.2009.09.001

  View details for Web of Science ID 000272277500001

  View details for PubMedID 19853940

• 3-D Near-Infrared Fluorescence Imaging Using an MEMS-Based Miniature Dual-Axis Confocal Microscope IEEE JOURNAL OF SELECTED TOPICS IN QUANTUM ELECTRONICS Piyawattanametha, W., Ra, H., Mandella, M. J., Loewke, K., Wang, T. D., Kino, G. S., Solgaard, O., Contag, C. H. 2009; 15 (5): 1344-1350

  View details for DOI 10.1109/JSTQE.2009.2021533

  View details for Web of Science ID 000270950300008

• siRNA silencing of keratinocyte-specific GFP expression in a transgenic mouse skin model GENE THERAPY Gonzalez-Gonzalez, E., Ra, H., Hickerson, R. P., Wang, Q., Piyawattanametha, W., Mandella, M. J., Kino, G. S., Leake, D., Avilion, A. A., Solgaard, O., Doyle, T. C., Contag, C. H., Kaspar, R. L. 2009; 16 (8): 963-972

  Abstract

  Small interfering RNAs (siRNAs) can be designed to specifically and potently target and silence a mutant allele, with little or no effect on the corresponding wild-type allele expression, presenting an opportunity for therapeutic intervention. Although several siRNAs have entered clinical trials, the development of siRNA therapeutics as a new drug class will require the development of improved delivery technologies. In this study, a reporter mouse model (transgenic click beetle luciferase/humanized monster green fluorescent protein) was developed to enable the study of siRNA delivery to skin; in this transgenic mouse, green fluorescent protein reporter gene expression is confined to the epidermis. Intradermal injection of siRNAs targeting the reporter gene resulted in marked reduction of green fluorescent protein expression in the localized treatment areas as measured by histology, real-time quantitative polymerase chain reaction and intravital imaging using a dual-axes confocal fluorescence microscope. These results indicate that this transgenic mouse skin model, coupled with in vivo imaging, will be useful for development of efficient and 'patient-friendly' siRNA delivery techniques and should facilitate the translation of siRNA-based therapeutics to the clinic for treatment of skin disorders.

  View details for DOI 10.1038/gt.2009.62

  View details for Web of Science ID 000268916800004

  View details for PubMedID 19474811

• Use of an endoscope-compatible probe to detect colonic dysplasia with Fourier transform infrared spectroscopy JOURNAL OF BIOMEDICAL OPTICS Mackanos, M. A., Hargrove, J., Wolters, R., Du, C. B., Friedland, S., Soetikno, R. M., Contag, C. H., Arroyo, M. R., Crawford, J. M., Wang, T. D. 2009; 14 (4)

  Abstract

  Fourier transform infrared (FTIR) spectroscopy is sensitive to the molecular composition of tissue and has the potential to identify premalignant tissue (dysplasia) as an adjunct to endoscopy. We demonstrate collection of mid-infrared absorption spectra with a silver halide (AgCl(0.4)Br(0.6)) optical fiber and use spectral preprocessing to identify optimal subranges that classify colonic mucosa as normal, hyperplasia, or dysplasia. We collected spectra (n=83) in the 950 to 1800 cm(-1) regime on biopsy specimens obtained from human subjects (n=37). Subtle differences in the magnitude of the absorbance peaks at specific wave numbers were observed. The best double binary algorithm for distinguishing normal-versus-dysplasia and hyperplasia-versus-dysplasia was determined from an exhaustive search of spectral intervals and preprocessing techniques. Partial least squares discriminant analysis was used to classify the spectra using a leave-one-subject-out cross-validation strategy. The results were compared with histology reviewed independently by two gastrointestinal pathologists. The optimal thresholds identified resulted in an overall sensitivity, specificity, accuracy, and positive predictive value of 96%, 92%, 93%, and 82%, respectively. These results indicated that mid-infrared absorption spectra collected remotely with an optical fiber can be used to identify colonic dysplasia with high accuracy, suggesting that continued development of this technique for the early detection of cancer is promising.

  View details for DOI 10.1117/1.3174387

  View details for Web of Science ID 000270540100013

  View details for PubMedID 19725718

  View details for PubMedCentralID PMC3232016

• Laser-induced disruption of systemically administered liposomes for targeted drug delivery JOURNAL OF BIOMEDICAL OPTICS Mackanos, M. A., Larabi, M., Shinde, R., Simanovskii, D. M., Guccione, S., Contag, C. H. 2009; 14 (4)

  Abstract

  Liposomal formulations of drugs have been shown to enhance drug efficacy by prolonging circulation time, increasing local concentration and reducing off-target effects. Controlled release from these formulations would increase their utility, and hyperthermia has been explored as a stimulus for targeted delivery of encapsulated drugs. Use of lasers as a thermal source could provide improved control over the release of the drug from the liposomes with minimal collateral tissue damage. Appropriate methods for assessing local release after systemic delivery would aid in testing and development of better formulations. We use in vivo bioluminescence imaging to investigate the spatiotemporal distribution of luciferin, used as a model small molecule, and demonstrate laser-induced release from liposomes in animal models after systemic delivery. These liposomes were tested for luciferin release between 37 and 45 degrees C in PBS and serum using bioluminescence measurements. In vivo studies were performed on transgenic reporter mice that express luciferase constitutively throughout the body, thus providing a noninvasive readout for controlled release following systemic delivery. An Nd:YLF laser was used (527 nm) to heat tissues and induce rupture of the intravenously delivered liposomes in target tissues. These data demonstrate laser-mediated control of small molecule delivery using thermally sensitive liposomal formulations.

  View details for DOI 10.1117/1.3174410

  View details for Web of Science ID 000270540100016

  View details for PubMedID 19725721

• Lymphoid tissue-specific homing of bone marrow-derived dendritic cells BLOOD Creusot, R. J., Yaghoubi, S. S., Chang, P., Chia, J., Contag, C. H., Gambhir, S. S., Fathman, C. G. 2009; 113 (26): 6638-6647

  Abstract

  Because of their potent immunoregulatory capacity, dendritic cells (DCs) have been exploited as therapeutic tools to boost immune responses against tumors or pathogens, or dampen autoimmune or allergic responses. Murine bone marrow-derived DCs (BM-DCs) are the closest known equivalent of the blood monocyte-derived DCs that have been used for human therapy. Current imaging methods have proven unable to properly address the migration of injected DCs to small and deep tissues in mice and humans. This study presents the first extensive analysis of BM-DC homing to lymph nodes (and other selected tissues) after intravenous and intraperitoneal inoculation. After intravenous delivery, DCs accumulated in the spleen, and preferentially in the pancreatic and lung-draining lymph nodes. In contrast, DCs injected intraperitoneally were found predominantly in peritoneal lymph nodes (pancreatic in particular), and in omentum-associated lymphoid tissue. This uneven distribution of BM-DCs, independent of the mouse strain and also observed within pancreatic lymph nodes, resulted in the uneven induction of immune response in different lymphoid tissues. These data have important implications for the design of systemic cellular therapy with DCs, and in particular underlie a previously unsuspected potential for specific treatment of diseases such as autoimmune diabetes and pancreatic cancer.

  View details for DOI 10.1182/blood-2009-02-204321

  View details for Web of Science ID 000267789600024

  View details for PubMedID 19363220

  View details for PubMedCentralID PMC2710920

• The T Cell STAT Signaling Network Is Reprogrammed within Hours of Bacteremia via Secondary Signals JOURNAL OF IMMUNOLOGY Hotson, A. N., Hardy, J. W., Hale, M. B., Contag, C. H., Nolan, G. P. 2009; 182 (12): 7558-7568

  Abstract

  The delicate balance between protective immunity and inflammatory disease is challenged during sepsis, a pathologic state characterized by aspects of both a hyperactive immune response and immunosuppression. The events driven by systemic infection by bacterial pathogens on the T cell signaling network that likely control these responses have not been illustrated in great detail. We characterized how intracellular signaling within the immune compartment is reprogrammed at the single cell level when the host is challenged with a high level of pathogen. To accomplish this, we applied flow cytometry to measure the phosphorylation potential of key signal transduction proteins during acute bacterial challenge. We modeled the onset of sepsis by i.v. administration of avirulent strains of Listeria monocytogenes and Escherichia coli to mice. Within 6 h of bacterial challenge, T cells were globally restricted in their ability to respond to specific cytokine stimulations as determined by assessing the extent of STAT protein phosphorylation. Mechanisms by which this negative feedback response occurred included SOCS1 and SOCS3 gene up-regulation and IL-6-induced endocystosis of the IL-6 receptor. Additionally, macrophages were partially tolerized in their ability to respond to TLR agonists. Thus, in contrast to the view that there is a wholesale immune activation during sepsis, one immediate host response to blood-borne bacteria was induction of a refractory period during which leukocyte activation by specific stimulations was attenuated.

  View details for DOI 10.4049/jimmunol.0803666

  View details for Web of Science ID 000266833900026

  View details for PubMedID 19494279

• Quantifying Cell-Surface Biomarker Expression in Thick Tissues with Ratiometric Three-Dimensional Microscopy BIOPHYSICAL JOURNAL Liu, J. T., Helms, M. W., Mandella, M. J., Crawford, J. M., Kino, G. S., Contag, C. H. 2009; 96 (6): 2405-2414

  Abstract

  The burgeoning fields of in vivo three-dimensional (3D) microscopy and endomicroscopy, as well as ex vivo tissue cytometry have introduced new challenges for tissue preparation and staining with exogenous molecular contrast agents. These challenges include effective delivery of the agents, and once delivered, distinguishing between bound verses unbound molecular probes. If applied topically, there are additional issues with rinsing off unbound probe, which can be nonuniform and inefficient in thick tissues, thus leading to ambiguous contrast and a large nonspecific background that may obscure molecule-specific staining. Therefore, we have developed a ratiometric 3D microscopy scheme that not only reduces the effects of nonspecific sources of contrast, but also enables quantification of the relative binding affinity of imaging probes to their biomarker targets. Here we demonstrate this ratiometric approach by simultaneously imaging a HER2/neu (erbB2)-targeted monoclonal antibody labeled with one fluorophore and an isotype-matched negative control antibody labeled with another fluorophore. By taking a pixel-by-pixel calibrated ratio between the signals from each fluorescent image channel, accurate quantification of specific versus nonspecific binding affinity is achieved with cultured cells, yielding data that are in agreement with analyses via flow cytometry. We also demonstrate quantitative 3D microscopic imaging of biomarker expression in tissue models and in thick human biopsy samples of normal, HER2-negative, and HER2-positive breast tumors. This strategy enables rapid, quantitative, and unambiguous volumetric microscopy of biomarker expression in thick tissues, including whole biopsies, and will enable real-time optical assessment of disease markers in the living body.

  View details for DOI 10.1016/j.bpj.2008.12.3908

  View details for Web of Science ID 000266376700035

  View details for PubMedID 19289065

  View details for PubMedCentralID PMC2907718

• Comparison of Transplantation of Adipose Tissue- and Bone Marrow-Derived Mesenchymal Stem Cells in the Infarcted Heart TRANSPLANTATION van der Bogt, K. E., Schrepfer, S., Yu, J., Sheikh, A. Y., Hoyt, G., Govaert, J. A., Velotta, J. B., Contag, C. H., Robbins, R. C., Wu, J. C. 2009; 87 (5): 642-652

  Abstract

  Mesenchymal stem cells hold promise for cardiovascular regenerative therapy. Derivation of these cells from the adipose tissue might be easier compared with bone marrow. However, the in vivo fate and function of adipose stromal cells (ASC) in the infarcted heart has never been compared directly to bone marrow-derived mesenchymal cells (MSC).ASC and MSC were isolated from transgenic FVB mice with a beta-actin promoter driving firefly luciferase and green fluorescent protein double fusion reporter gene, and they were characterized using flow cytometry, microscopy, bioluminescence imaging and luminometry. FVB mice (n=8 per group) underwent myocardial infarction followed by intramyocardial injection of 5x10(5) ASC, MSC, fibroblasts (Fibro, positive control), or saline (negative control). Cell survival was measured using bioluminescence imaging for 6 weeks and cardiac function was monitored by echocardiography and pressure-volume analysis. Ventricular morphology was assessed using histology.ASC and MSC were CD34(-), CD45(-), c-Kit(-), CD90(+), Sca-1(+), shared similar morphology and had a population doubling time of approximately 2 days. Cells expressed Fluc reporter genes in a number-dependent fashion as confirmed by luminometry. After cardiac transplantation, both cell types showed drastic donor cell death within 4 to 5 weeks. Furthermore, transplantation of either cell type was not capable of preserving ventricular function and dimensions, as confirmed by pressure-volume-loops and histology.This is the first study comparing the in vivo behavior of both cell types in the infarcted heart. ASC and MSC do not tolerate well in the cardiac environment, resulting in acute donor cell death and a subsequent loss of cardiac function similar to control groups.

  View details for DOI 10.1097/TP.0b013e31819609d9

  View details for PubMedID 19295307

• Stem Cell-Mediated Accelerated Bone Healing Observed with in Vivo Molecular and Small Animal Imaging Technologies in a Model of Skeletal Injury JOURNAL OF ORTHOPAEDIC RESEARCH Lee, S., Padmanabhan, P., Ray, P., Gambhir, S. S., Doyle, T., Contag, C., Goodman, S. B., Biswal, S. 2009; 27 (3): 295-302

  Abstract

  Adult stem cells are promising therapeutic reagents for skeletal regeneration. We hope to validate by molecular imaging technologies the in vivo life cycle of adipose-derived multipotent cells (ADMCs) in an animal model of skeletal injury. Primary ADMCs were lentivirally transfected with a fusion reporter gene and injected intravenously into mice with bone injury or sham operation. Bioluminescence imaging (BLI), [(18)F]FHBG (9-(fluoro-hydroxy-methyl-butyl-guanine)-micro-PET, [(18)F]Fluoride ion micro-PET and micro-CT were performed to monitor stem cells and their effect. Bioluminescence microscopy and immunohistochemistry were done for histological confirmation. BLI showed ADMC's traffic from the lungs then to the injury site. BLI microscopy and immunohistochemistry confirmed the ADMCs in the bone defect. Micro-CT measurements showed increased bone healing in the cell-injected group compared to the noninjected group at postoperative day 7 (p < 0.05). Systemically administered ADMC's traffic to the site of skeletal injury and facilitate bone healing, as demonstrated by molecular and small animal imaging. Molecular imaging technologies can validate the usage of adult adipose tissue-derived multipotent cells to promote fracture healing. Imaging can in the future help establish therapeutic strategies including dosage and administration route.

  View details for DOI 10.1002/jor.20736

  View details for PubMedID 18752273

• A general method for conditional regulation of protein stability in living animals. Cold Spring Harbor protocols Sellmyer, M. A., Thorne, S. H., Banaszynski, L. A., Contag, C. H., Wandless, T. J. 2009; 2009 (3): pdb prot5173-?

  View details for DOI 10.1101/pdb.prot5173

  View details for PubMedID 20147108

  View details for PubMedCentralID PMC3215584

• Fibered Confocal Microscopy of Bladder Tumors: An ex Vivo Study JOURNAL OF ENDOUROLOGY Sonn, G. A., Mach, K. E., Jensen, K., Hsiung, P., Jones, S., Contag, C. H., Wang, T. D., Liao, J. C. 2009; 23 (2): 197-201

  Abstract

  The inadequacy of white-light cystoscopy to detect flat bladder tumors is well recognized. Great interest exists in developing other imaging technologies to augment or supplant conventional cystoscopy. Fibered confocal microscopy offers the promise of providing in vivo histopathologic information to help distinguish malignant from benign bladder lesions. We report the initial use of this technology to visualize tumors in the human bladder.We performed ex vivo fibered confocal imaging of fresh radical cystectomy specimens using the Mauna Kea Technologies Cellvizio system. The findings were compared with results from standard histopathology.The bladders of four patients were imaged using the fibered confocal microscope. Normal and neoplastic urothelium manifested differences in cellular and vascular density.This study demonstrates the feasibility of using fibered confocal microscopy to detect histologic differences between normal and neoplastic urothelium, and establishes a foundation for the use of fiber-based confocal microscopy in clinical studies.

  View details for DOI 10.1089/end.2008.0524

  View details for Web of Science ID 000263355500005

  View details for PubMedID 19196063

• CNOB/ChrR6, a new prodrug enzyme cancer chemotherapy MOLECULAR CANCER THERAPEUTICS Thorne, S. H., Barak, Y., Liang, W., Bachmann, M. H., Rao, J., Contag, C. H., Matin, A. 2009; 8 (2): 333-341

  Abstract

  We report the discovery of a new prodrug, 6-chloro-9-nitro-5-oxo-5H-benzo(a)phenoxazine (CNOB). This prodrug is efficiently activated by ChrR6, the highly active prodrug activating bacterial enzyme we have previously developed. The CNOB/ChrR6 therapy was effective in killing several cancer cell lines in vitro. It also efficiently treated tumors in mice with up to 40% complete remission. 9-Amino-6-chloro-5H-benzo(a)phenoxazine-5-one (MCHB) was the only product of CNOB reduction by ChrR6. MCHB binds DNA; at nonlethal concentration, it causes cell accumulation in the S phase, and at lethal dose, it induces cell surface Annexin V and caspase-3 and caspase-9 activities. Further, MCHB colocalizes with mitochondria and disrupts their electrochemical potential. Thus, killing by CNOB involves MCHB, which likely induces apoptosis through the mitochondrial pathway. An attractive feature of the CNOB/ChrR6 regimen is that its toxic product, MCHB, is fluorescent. This feature proved helpful in in vitro studies because simple fluorescence measurements provided information on the kinetics of CNOB activation within the cells, MCHB killing mechanism, its generally efficient bystander effect in cells and cell spheroids, and its biodistribution. The emission wavelength of MCHB also permitted its visualization in live animals, allowing noninvasive qualitative imaging of MCHB in mice and the tumor microenvironment. This feature may simplify exploration of barriers to the penetration of MCHB in tumors and their amelioration.

  View details for DOI 10.1158/1535-7163.MCT-08-0707

  View details for Web of Science ID 000263397300008

  View details for PubMedID 19190118

  View details for PubMedCentralID PMC2670992

• Development of an optimized activatable MMP-14 targeted SPECT imaging probe BIOORGANIC & MEDICINAL CHEMISTRY Watkins, G. A., Jones, E. F., Shell, M. S., VanBrocklin, H. F., Pan, M., Hanrahan, S. M., Feng, J. J., He, J., Sounni, N. E., Dill, K. A., Contag, C. H., Coussens, L. M., Franc, B. L. 2009; 17 (2): 653-659

  Abstract

  Matrix metalloproteinase-14 (MT1-MMP or MMP-14) is a membrane-associated protease implicated in a variety of tissue remodeling processes and a molecular hallmark of select metastatic cancers. The ability to detect MMP-14 in vivo would be useful in studying its role in pathologic processes and may potentially serve as a guide for the development of targeted molecular therapies. Four MMP-14 specific probes containing a positively charged cell penetrating peptide (CPP) d-arginine octamer (r(8)) linked with a MMP-14 peptide substrate and attenuating sequences with glutamate (8e, 4e) or glutamate-glycine (4eg and 4egg) repeating units were modeled using an AMBER force field method. The probe with 4egg attenuating sequence exhibited the highest CPP/attenuator interaction, predicting minimized cellular uptake until cleaved. The in vitro MMP-14-mediated cleavage studies using the human recombinant MMP-14 catalytic domain revealed an enhanced cleavage rate that directly correlated with the linearity of the embedded peptide substrate sequence. Successful cleavage and uptake of a technetium-99m labeled version of the optimal probe was demonstrated in MMP-14 transfected human breast cancer cells. Two-fold reduction of cellular uptake was found in the presence of a broad spectrum MMP inhibitor. The combination of computational chemistry, parallel synthesis and biochemical screening, therefore, shows promise as a set of tools for developing new radiolabeled probes that are sensitive to protease activity.

  View details for DOI 10.1016/j.bmc.2008.11.078

  View details for Web of Science ID 000262708300027

  View details for PubMedID 19109023

  View details for PubMedCentralID PMC2639212

• Trafficking Mesenchymal Stem Cell Engraftment and Differentiation in Tumor-Bearing Mice by Bioluminescence Imaging STEM CELLS Wang, H., Cao, F., De, A., Cao, Y., Contag, C., Gambhir, S. S., Wu, J. C., Chen, X. 2009; 27 (7): 1548-1558

  Abstract

  The objective of the study was to track the distribution and differentiation of mesenchymal stem cells (MSCs) in tumor-bearing mice. The 4T1 murine breast cancer cells were labeled with renilla luciferase-monomeric red fluorescence protein (rLuc-mRFP) reporter gene. The MSCs labeled with firefly luciferase-enhanced green fluorescence protein (fLuc-eGFP) reporter gene (MSCs-R) were isolated from L2G85 transgenic mice that constitutively express fLuc-eGFP reporter gene. To study the tumor tropism of MSCs, we established both subcutaneous and lung metastasis models. In lung metastasis tumor mice, we injected MSCs-R intravenously either on the same day or 4 days after 4T1 tumor cell injection. In subcutaneous tumor mice, we injected MSCs-R intravenously 7 days after subcutaneous 4T1 tumor inoculation. The tumor growth was monitored by rLuc bioluminescence imaging (BLI). The fate of MSCs-R was monitored by fLuc BLI. The localization of MSCs-R in tumors was examined histologically. The osteogenic and adipogenic differentiation of MSCs-R was investigated by alizarin red S and oil red O staining, respectively. The mechanism of the dissimilar differentiation potential of MSCs-R under different tumor microenvironments was investigated. We found that the 4T1 cells were successfully labeled with rLuc-mRFP. The MSCs-R isolated from L2G85 transgenic mice constitutively express fLuc-eGFP reporter gene. When injected intravenously, MSCs-R survived, proliferated, and differentiated in tumor sites but not elsewhere. The localization of GFP(+) MSCs-R in tumor lesions was confirmed ex vivo. In conclusion, the MSCs-R can selectively localize, survive, and proliferate in both subcutaneous tumor and lung metastasis as evidenced by noninvasive bioluminescence imaging and ex vivo validation. The MSCs-R migrated to lung tumor differentiated into osteoblasts, whereas the MSCs-R targeting subcutaneous tumor differentiated into adipocytes.

  View details for DOI 10.1002/stem.81

  View details for PubMedID 19544460

• Sequential in vivo Molecular Imaging with a Dual-Axes Confocal Microscope Conference on Lasers and Electro-Optics/Quantum Electronics and Laser Science Conference (CLEO/QELS 2009) Ra, H., Gonzalez, E., Piyawattanametha, W., Mandella, M. J., Kaspar, R., Contag, C. H., Kino, G. S., Solgaard, O. IEEE. 2009: 1–2

  View details for Web of Science ID 000274751300001

• Fiber Optic FTIR Instrument for In Vivo Detection of Colonic Neoplasia Conference on Endoscopic Microscopy IV Van Nortwick, M., Hargrove, J., Wolters, R., Crawford, J. M., Arroyo, M., Mackanos, M., Contag, C. H., Wang, T. D. SPIE-INT SOC OPTICAL ENGINEERING. 2009

  View details for DOI 10.1117/12.808570

  View details for Web of Science ID 000284823200008

• Foci of Listeria monocytogenes persist in the bone marrow DISEASE MODELS & MECHANISMS Hardy, J., Chu, P., Contag, C. H. 2009; 2 (1-2): 39-46

  Abstract

  Murine listeriosis is one of the most comprehensive and well-studied models of infection, and Listeria monocytogenes has provided seminal information regarding bacterial pathogenesis. However, many aspects of the mouse model remain poorly understood, including carrier states and chronic colonization which represent important features of the spectrum of host-pathogen interaction. Bone marrow has recently been shown to harbor L. monocytogenes, which spreads from this location to the central nervous system. Bone could, therefore, be an important chronic reservoir, but this infection is difficult to study because it involves only a few bacteria and the extent of infection cannot be assessed until after the animal is sacrificed. We employed in vivo bioluminescence imaging to localize L. monocytogenes bone infections over time in live mice, revealing that the bacteria grow in discrete foci. These lesions can persist in many locations in the legs of mice and are not accompanied by a histological indication such as granuloma or a neutrophil infiltratate. We demonstrate that highly attenuated hly mutants, which have defective intracellular replication, are capable of prolonged focal infection of the bone marrow for periods of up to several weeks. These results support the recently proposed hypothesis that the bone marrow is a unique niche for L. monocytogenes.

  View details for DOI 10.1242/dmm.000836

  View details for Web of Science ID 000268254500009

  View details for PubMedID 19132117

  View details for PubMedCentralID PMC2615163

• Heme oxygenase-1 deficiency leads to disrupted response to acute stress in stem cells and progenitors BLOOD Cao, Y., Wagers, A. J., Karsunky, H., Zhao, H., Reeves, R., Wong, R. J., Stevenson, D. K., Weissman, I. L., Contag, C. H. 2008; 112 (12): 4494-4502

  Abstract

  An effective response to extreme hematopoietic stress requires an extreme elevation in hematopoiesis and preservation of hematopoietic stem cells (HSCs). These diametrically opposed processes are likely to be regulated by genes that mediate cellular adaptation to physiologic stress. Herein, we show that heme oxygenase-1 (HO-1), the inducible isozyme of heme degradation, is a key regulator of these processes. Mice lacking one allele of HO-1 (HO-1(+/-)) showed accelerated hematopoietic recovery from myelotoxic injury, and HO-1(+/-) HSCs repopulated lethally irradiated recipients with more rapid kinetics. However, HO-1(+/-) HSCs were ineffective in radioprotection and serial repopulation of myeloablated recipients. Perturbations in key stem cell regulators were observed in HO-1(+/-) HSCs and hematopoietic progenitors (HPCs), which may explain the disrupted response of HO-1(+/-) HPCs and HPCs to acute stress. Control of stem cell stress response by HO-1 presents opportunities for metabolic manipulation of stem cell-based therapies.

  View details for DOI 10.1182/blood-2007-12-127621

  View details for PubMedID 18509090

• Stability Study of Unmodified siRNA and Relevance to Clinical Use OLIGONUCLEOTIDES Hickerson, R. P., Vlassov, A. V., Wang, Q., Leake, D., Ilves, H., Gonzalez-Gonzalez, E., Contag, C. H., Johnston, B. H., Kaspar, R. L. 2008; 18 (4): 345-354

  Abstract

  RNA interference offers enormous potential to develop therapeutic agents for a variety of diseases. To assess the stability of siRNAs under conditions relevant to clinical use with particular emphasis on topical delivery considerations, a study of three different unmodified siRNAs was performed. The results indicate that neither repeated freeze/thaw cycles, extended incubations (over 1 year at 21 degrees C), nor shorter incubations at high temperatures (up to 95 degrees C) have any effect on siRNA integrity as measured by nondenaturing polyacrylamide gel electrophoresis and functional activity assays. Degradation was also not observed following exposure to hair or skin at 37 degrees C. However, incubation in fetal bovine or human sera at 37 degrees C led to degradation and loss of activity. Therefore, siRNA in the bloodstream is likely inactivated, thereby limiting systemic exposure. Interestingly, partial degradation (observed by gel electrophoresis) did not always correlate with loss of activity, suggesting that partially degraded siRNAs retain full functional activity. To demonstrate the functional activity of unmodified siRNA, EGFP-specific inhibitors were injected into footpads and shown to inhibit preexisting EGFP expression in a transgenic reporter mouse model. Taken together, these data indicate that unmodified siRNAs are viable therapeutic candidates.

  View details for DOI 10.1089/oli.2008.0149

  View details for Web of Science ID 000261845200005

  View details for PubMedID 18844576

  View details for PubMedCentralID PMC2829675

• Role of HSP70 in Cellular Thermotolerance LASERS IN SURGERY AND MEDICINE Beckham, J. T., Wilmink, G. J., Mackanos, M. A., Takahashi, K., Contag, C. H., Takahashi, T., Jansen, E. D. 2008; 40 (10): 704-715

  Abstract

  Thermal pretreatment has been shown to condition tissue to a more severe secondary heat stress. In this research we examined the particular contribution of heat shock protein 70 (HSP70) in thermal preconditioning.For optimization of preshock exposures, a bioluminescent Hsp70-luciferase reporter system in NIH3T3 cells tracked the activation of the Hsp70 gene. Cells in 96-well plates were pretreated in a 43 degrees C water bath for 30 minutes, followed 4 hours later with a severe heat shock at 45 degrees C for 50 minutes. Bioluminescence was measured at 2, 4, 6, 8, and 10 hours after preshock only (PS) and at 4 hours after preshock with heatshock (PS+HS). Viability was assessed 48 hours later with a fluorescent viability dye. Preshock induced thermotolerance was then evaluated in hsp70-containing Murine Embryo Fibroblast (+/+) cells and Hsp70-deficient MEF cells (-/-) through an Arrhenius damage model across varying temperatures (44.5-46 degrees C).A time gap of 4 hours between preconditioning and the thermal insult was shown to be the most effective for thermotolerance with statistical confidence of P<0.05. The benefit of preshocking was largely abrogated in Hsp70-deficient cells. The Arrhenius data showed that preshocking leads to increases in the activation energies, E(a), and increases in frequency factors, A. The frequency factor increase was significantly greater in Hsp70-deficient cells.The data shows that HSP70 contributes significantly to cellular thermotolerance but there are other pathways that provide residual thermotolerance in cells deficient in Hsp70.

  View details for DOI 10.1002/lsm.20713

  View details for Web of Science ID 000262030800006

  View details for PubMedID 19065555

• Chemical control of protein stability and function in living mice NATURE MEDICINE Banaszynski, L. A., Sellmyer, M. A., Contag, C. H., Wandless, T. J., Thorne, S. H. 2008; 14 (10): 1123-1127

  Abstract

  Conditional control of protein function in vivo offers great potential for deconvoluting the roles of individual proteins in complicated systems. We recently developed a method in which a small protein domain, termed a destabilizing domain, confers instability to fusion protein partners in cultured cells. Instability is reversed when a cell-permeable small molecule binds this domain. Here we describe the use of this system to regulate protein function in living mammals. We show regulation of secreted proteins and their biological activity with conditional secretion of an immunomodulatory cytokine, resulting in tumor burden reduction in mouse models. Additionally, we use this approach to control the function of a specific protein after systemic delivery of the gene that encodes it to a tumor, suggesting uses for enhancing the specificity and efficacy of targeted gene-based therapies. This method represents a new strategy to regulate protein function in living organisms with a high level of control.

  View details for DOI 10.1038/nm.1754

  View details for Web of Science ID 000259892300044

  View details for PubMedID 18836461

  View details for PubMedCentralID PMC2605277

• Comparison of different adult stem cell types for treatment of myocardial ischemia 80th Annual Scientific Session of the American-Heart-Association (AHA) van der Bogt, K. E., Sheikh, A. Y., Schrepfer, S., Hoyt, G., Cao, F., Ransohoff, K. J., Swijnenburg, R., Pearl, J., Lee, A., Fischbein, M., Contag, C. H., Robbins, R. C., Wu, J. C. LIPPINCOTT WILLIAMS & WILKINS. 2008: S121–U166

  Abstract

  A comparative analysis of the efficacy of different cell candidates for the treatment of heart disease remains to be described. This study is designed to evaluate the therapeutic efficacy of 4 cell types in a murine model of myocardial infarction.Bone marrow mononuclear cells (MN), mesenchymal stem cells (MSC), skeletal myoblasts (SkMb), and fibroblasts (Fibro) expressing firefly luciferase (Fluc) and green fluorescence protein (GFP) were characterized by flow cytometry, bioluminescence imaging (BLI), and luminometry. Female FVB mice (n=70) underwent LAD ligation and intramyocardially received one cell type (5x10(5)) or PBS. Cell survival was measured by BLI and by TaqMan PCR. Cardiac function was assessed by echocardiography and invasive hemodynamic measurements. Fluc expression correlated with cell number in all groups (r(2)>0.93). In vivo BLI revealed acute donor cell death of MSC, SkMb, and Fibro within 3 weeks after transplantation. By contrast, cardiac signals were still present after 6 weeks in the MN group, as confirmed by TaqMan PCR (P<0.01). Echocardiography showed significant preservation of fractional shortening in the MN group compared to controls (P<0.05). Measurements of left ventricular end-systolic/diastolic volumes revealed that the least amount of ventricular dilatation occurred in the MN group (P<0.05). Histology confirmed the presence of MN, although there was no evidence of transdifferentiation by donor MN into cardiomyocytes.This is the first study to show that compared to MSC, SkMB, and Fibro, MN exhibit a more favorable survival pattern, which translates into a more robust preservation of cardiac function.

  View details for DOI 10.1161/CIRCULATIONAHA.107.759480

  View details for Web of Science ID 000259648600018

  View details for PubMedID 18824743

  View details for PubMedCentralID PMC3657517

• In-vivo optical imaging of hsp70 expression to assess collateral tissue damage associated with infrared laser ablation of skin JOURNAL OF BIOMEDICAL OPTICS Wilmink, G. J., Opalenik, S. R., Beckham, J. T., Mackanos, M. A., Nanney, L. B., Contag, C. H., Davidson, J. M., Jansen, E. D. 2008; 13 (5)

  Abstract

  Laser surgical ablation is achieved by selecting laser parameters that remove confined volumes of target tissue and cause minimal collateral damage. Previous studies have measured the effects of wavelength on ablation, but neglected to measure the cellular impact of ablation on cells outside the lethal zone. In this study, we use optical imaging in addition to conventional assessment techniques to evaluate lethal and sublethal collateral damage after ablative surgery with a free-electron laser (FEL). Heat shock protein (HSP) expression is used as a sensitive quantitative marker of sublethal damage in a transgenic mouse strain, with the hsp70 promoter driving luciferase and green fluorescent protein (GFP) expression (hsp70A1-L2G). To examine the wavelength dependence in the mid-IR, laser surgery is conducted on the hsp70A1-L2G mouse using wavelengths targeting water (OH stretch mode, 2.94 microm), protein (amide-II band, 6.45 microm), and both water and protein (amide-I band, 6.10 microm). For all wavelengths tested, the magnitude of hsp70 expression is dose-dependent and maximal 5 to 12 h after surgery. Tissues treated at 6.45 microm have approximately 4x higher hsp70 expression than 6.10 microm. Histology shows that under comparable fluences, tissue injury at the 2.94-microm wavelength was 2x and 3x deeper than 6.45 and 6.10 microm, respectively. The 6.10-microm wavelength generates the least amount of epidermal hyperplasia. Taken together, this data suggests that the 6.10-microm wavelength is a superior wavelength for laser ablation of skin.

  View details for DOI 10.1117/1.2992594

  View details for Web of Science ID 000261764900074

  View details for PubMedID 19021444

• Overcoming multidrug resistance of small-molecule therapeutics through conjugation with releasable octaarginine transporters PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Dubikovskaya, E. A., Thorne, S. H., Pillow, T. H., Contag, C. H., Wender, P. A. 2008; 105 (34): 12128-12133

  Abstract

  Many cancer therapeutic agents elicit resistance that renders them ineffective and often produces cross-resistance to other drugs. One of the most common mechanisms of resistance involves P-glycoprotein (Pgp)-mediated drug efflux. To address this problem, new agents have been sought that are less prone to inducing resistance and less likely to serve as substrates for Pgp efflux. An alternative to this approach is to deliver established agents as molecular transporter conjugates into cells through a mechanism that circumvents Pgp-mediated efflux and allows for release of free drug only after cell entry. Here we report that the widely used chemotherapeutic agent Taxol, ineffective against Taxol-resistant human ovarian cancer cell lines, can be incorporated into a releasable octaarginine conjugate that is effective against the same Taxol-resistant cell lines. It is significant that the ability of the Taxol conjugates to overcome Taxol resistance is observed both in cell culture and in animal models of ovarian cancer. The generality and mechanistic basis for this effect were also explored with coelenterazine, a Pgp substrate. Although coelenterazine itself does not enter cells because of Pgp efflux, its octaarginine conjugate does so readily. This approach shows generality for overcoming the multidrug resistance elicited by small-molecule cancer chemotherapeutics and could improve the prognosis for many patients with cancer and fundamentally alter search strategies for novel therapeutic agents that are effective against resistant disease.

  View details for DOI 10.1073/pnas.0805374105

  View details for Web of Science ID 000258905700008

  View details for PubMedID 18713866

  View details for PubMedCentralID PMC2527877

• Short-duration-focused ultrasound stimulation of Hsp70 expression in vivo PHYSICS IN MEDICINE AND BIOLOGY Kruse, D. E., Mackanos, M. A., O'Connell-Rodwell, C. E., Contag, C. H., Ferrara, K. W. 2008; 53 (13): 3641-3660

  Abstract

  The development of transgenic reporter mice and advances in in vivo optical imaging have created unique opportunities to assess and analyze biological responses to thermal therapy directly in living tissues. Reporter mice incorporating the regulatory regions from the genes encoding the 70 kDa heat-shock proteins (Hsp70) and firefly luciferase (luc) as reporter genes can be used to non-invasively reveal gene activation in living tissues in response to thermal stress. High-intensity-focused ultrasound (HIFU) can deliver measured doses of acoustic energy to highly localized regions of tissue at intensities that are sufficient to stimulate Hsp70 expression. We report activation of Hsp70-luc expression using 1 s duration HIFU heating to stimulate gene expression in the skin of the transgenic reporter mouse. Hsp70 expression was tracked for 96 h following the application of 1.5 MHz continuous-wave ultrasound with spatial peak intensities ranging from 53 W cm(-2) up to 352 W cm(-2). The results indicated that peak Hsp70 expression is observed 6-48 h post-heating, with significant activity remaining at 96 h. Exposure durations were simulated using a finite-element model, and the predicted temperatures were found to be consistent with the observed Hsp70 expression patterns. Histological evaluation revealed that the thermal damage starts at the stratum corneum and extends deeper with increasing intensity. These results indicated that short-duration HIFU may be useful for inducing heat-shock expression, and that the period between treatments needs to be greater than 96 h due to the protective properties of Hsp70.

  View details for DOI 10.1088/0031-9155/53/13/017

  View details for PubMedID 18562783

• Three-dimensional in vivo imaging by a handheld dual-axes confocal microscope OPTICS EXPRESS Ra, H., Piyawattanametha, W., Mandella, M. J., Hsiung, P., Hardy, J., Wang, T. D., Contag, C. H., Kino, G. S., Solgaard, O. 2008; 16 (10): 7224-7232

  Abstract

  We present a handheld dual-axes confocal microscope that is based on a two-dimensional microelectromechanical systems (MEMS) scanner. It performs reflectance and fluorescence imaging at 488 nm wavelength, with three-dimensional imaging capability. The fully packaged microscope has a diameter of 10 mm and acquires images at 4 Hz frame rate with a maximum field of view of 400 microm x 260 microm. The transverse and axial resolutions of the handheld probe are 1.7 microm and 5.8 microm, respectively. Capability to perform real time small animal imaging is demonstrated in vivo in transgenic mice.

  View details for Web of Science ID 000256469800048

  View details for PubMedID 18545427

• Efficient rejection of scattered light enables deep optical sectioning in turbid media with low-numerical-aperture optics in a dual-axis confocal architecture JOURNAL OF BIOMEDICAL OPTICS Liu, J. T., Mandella, M. J., Crawford, J. M., Contag, C. H., Wang, T. D., Kino, G. S. 2008; 13 (3)

  Abstract

  Miniature endoscopic microscopes, with subcellular imaging capabilities, will enable in vivo detection of molecularly-targeted fluorescent probes for early disease detection. To optimize a dual-axis confocal microscope (DACM) design for this purpose, we use a tabletop instrument to determine the ability of this technology to perform optical sectioning deep within tissue. First, we determine how tissue scattering deteriorates the diffraction-limited transverse and vertical responses in reflectance imaging. Specifically, the vertical response of a DACM to a plane reflector is measured at various depths in a scattering phantom and compared with diffraction theory and Monte Carlo scattering simulations. Similarly, transverse line scans across a knife-edge target are performed at various depths in a scattering phantom. Second, as a practical demonstration of deep-tissue fluorescence microscopy that corroborates the findings from our scattering experiments, 3-D fluorescence images are obtained in thick human gastrointestinal mucosal specimens. Our results demonstrate efficient rejection of scattered light in a DACM, which enables deep optical sectioning in tissue with subcellular resolution that can distinguish between normal and premalignant pathologies.

  View details for DOI 10.1117/1.2939428

  View details for Web of Science ID 000257951200048

  View details for PubMedID 18601565

• Integrating the biological characteristics of oncolytic viruses and immune cells can optimize therapeutic benefits of cell-based delivery GENE THERAPY Thorne, S. H., Contag, C. H. 2008; 15 (10): 753-758

  Abstract

  Despite significant advances in the development of tumor-selective agents, strategies for effective delivery of these agents across biological barriers to cells within the tumor microenvironment has been limiting. One tactical approach to overcoming biological barriers is to use cells as delivery vehicles, and a variety of different cell types have been investigated with a range of agents. In addition to transporting agents with targeted delivery, cells can also produce their own tumoricidal effect, conceal a payload from an immune response, amplify a selective agent at the target site and facilitate an antitumor immune response. We have reported a therapeutic combination consisting of cytokine induced killer cells and an oncolytic vaccinia virus with many of these features that led to therapeutic synergy in animal models of human cancer. The synergy was due to the interaction of the two agents to enhance the antitumor benefits of each individual component. As both of these agents display broad tumor-targeting potential and possess unique tumor killing mechanisms, together they were able to recognize and destroy a far greater number of malignant cells within the heterogeneous tumor than either agent alone. Effective cancer therapy will require recognition and elimination of the root of the disease, the cancer stem cell, and the combination of CIK cells and oncolytic vaccinia viruses has this potential. To create effective tumor-selective agents the viruses are modified to take advantage of the unique biology of the cancer cell. Similarly, if we are to develop targeted therapies that are sufficiently multifaceted to eliminate cancer cells at all stages of disease, we should integrate the virus into the unique biology of the cell delivery vehicle.

  View details for DOI 10.1038/gt.2008.42

  View details for Web of Science ID 000255588200007

  View details for PubMedID 18356814

• In vivo analysis of heat-shock-protein-70 induction following pulsed laser irradiation in a transgenic reporter mouse JOURNAL OF BIOMEDICAL OPTICS O'Connell-Rodwell, C. E., Mackanos, M. A., Simanovskii, D., Cao, Y., Bachmann, M. H., Schwettman, H. A., Contag, C. H. 2008; 13 (3)

  Abstract

  Induction of heat shock protein (Hsp) expression appears to correlate with a cytoprotective effect in cultured cells and with improved healing of damaged tissues in animal models and in humans. This family of proteins can also serve as indicators of thermal stress in cases of burn injury or surgical procedures that produce heat. Thus, a rapid in vivo readout for induction of Hsp transcription would facilitate studies of Hsp genes and their encoded proteins as mediators of therapeutic effects and as reporters of thermal damage to tissues. We created a transgenic reporter mouse where expression of luciferase is controlled by the regulatory region of the inducible 70 kDa Hsp, and assessed activation of Hsp70 transcription in live animals in response to rapid, high temperature stresses using in vivo bioluminescence imaging (BLI). This model can be used to noninvasively reveal levels of Hsp70 transcription in living tissues, and has utility in studies of the predictive and protective effects of Hsp70 expression, and of various stress responses in tissues.

  View details for DOI 10.1117/1.2904665

  View details for Web of Science ID 000257951200001

  View details for PubMedID 18601518

• Regulation of maternal and fetal hemodynamics by heme oxygenase in mice BIOLOGY OF REPRODUCTION Zhao, H., Wong, R. J., Doyle, T. C., Nayak, N., Vreman, H. J., Contag, C. H., Stevenson, D. K. 2008; 78 (4): 744-751

  Abstract

  Heme oxygenase (HMOX) regulates vascular tone and blood pressure through the production of carbon monoxide (CO), a vasodilator derived from the heme degradation pathway. During pregnancy, the maternal circulation undergoes significant adaptations to accommodate the hemodynamic demands of the developing fetus. Our objective was to investigate the role of HMOX on maternal and fetal hemodynamics during pregnancy in a mouse model. We measured and compared maternal tissue and placental HMOX activity and endogenous CO production, represented by excreted CO and carboxyhemoglobin levels, during pregnancy (Embryonic Days 12.5-15.5) to nonpregnant controls. Micro-ultrasound was used to monitor maternal abdominal aorta diameters as well as blood flow velocities and diameters of fetal umbilical arteries. Tin mesoporphyrin, a potent HMOX inhibitor, was used to inhibit HMOX activity. Changes in maternal vascular tone were monitored by tail cuff blood pressure measurements. Effects of HMOX inhibition on placental structures were assessed by histology. We showed that maternal tissue and placental HMOX activity and CO production were significantly elevated during pregnancy. When HMOX in the placenta was inhibited, maternal and fetal hemodynamics underwent significant changes, with maternal blood pressures increasing. We concluded that increases in maternal tissue and placental HMOX activity contribute to the regulation of peripheral vascular resistance and therefore are important for the maintenance of normal maternal vascular tone and fetal hemodynamic functions during pregnancy.

  View details for DOI 10.1095/biolreprod.107.064899

  View details for PubMedID 18094356

• Enhancing poxvirus oncolytic effects through increased spread and immune evasion CANCER RESEARCH Kirn, D. H., Wang, Y., Liang, W., Contag, C. H., Thorne, S. H. 2008; 68 (7): 2071-2075

  Abstract

  The antitumoral effects of oncolytic viruses have generally been limited by inefficient spread of the viruses within infected tumors and by inefficient systemic delivery, particularly in preimmunized hosts. Tumor-selective poxviruses have biological characteristics that may overcome these limitations. Nevertheless, physical barriers within the tumor microenvironment, including the extracellular matrix, can still limit intratumoral spread, and neutralizing antibodies can impede systemic delivery. To counter these limitations, we sought to take advantage of a naturally occurring poxvirus form known as extracellular enveloped virus (EEV). The EEV is shrouded by a host cell-derived lipid bilayer containing anticomplement proteins and is typically released from infected cells early during the infection cycle. Therefore, the EEV form evolved for rapid systemic spread within the host and for evasion of immune-mediated clearance. We compared the oncolytic potential of low versus high EEV-producing strains of vaccinia. EEV-enhanced vaccinia strains displayed improved spread within tumors after systemic delivery, resulting in significantly improved antitumor effects. The EEV-enhanced strains also displayed a greater ability to spread between injected and noninjected distant tumors through the blood and, importantly, displayed reduced clearance by neutralizing antibody. Safety was unaffected. The incorporation of EEV-enhancing mutations into next generation oncolytic vaccinia strains may improve the potency of these viruses without sacrificing safety.

  View details for DOI 10.1158/0008-5472.CAN-07-6515

  View details for Web of Science ID 000254738500005

  View details for PubMedID 18381410

• Detection of colonic dysplasia in vivo using a targeted heptapeptide and confocal microendoscopy NATURE MEDICINE Hsiung, P., Hardy, J., Friedland, S., Soetikno, R., Du, C. B., Wu, A. P., Sahbaie, P., Crawford, J. M., Lowe, A. W., Contag, C. H., Wang, T. D. 2008; 14 (4): 454-458

  Abstract

  A combination of targeted probes and new imaging technologies provides a powerful set of tools with the potential to improve the early detection of cancer. To develop a probe for detecting colon cancer, we screened phage display peptide libraries against fresh human colonic adenomas for high-affinity ligands with preferential binding to premalignant tissue. We identified a specific heptapeptide sequence, VRPMPLQ, which we synthesized, conjugated with fluorescein and tested in patients undergoing colonoscopy. We imaged topically administered peptide using a fluorescence confocal microendoscope delivered through the instrument channel of a standard colonoscope. In vivo images were acquired at 12 frames per second with 50-microm working distance and 2.5-microm (transverse) and 20-microm (axial) resolution. The fluorescein-conjugated peptide bound more strongly to dysplastic colonocytes than to adjacent normal cells with 81% sensitivity and 82% specificity. This methodology represents a promising diagnostic imaging approach for the early detection of colorectal cancer and potentially of other epithelial malignancies.

  View details for DOI 10.1038/nm1692

  View details for Web of Science ID 000254674100034

  View details for PubMedID 18345013

  View details for PubMedCentralID PMC3324975

• Prevention of acute graft-versus-host disease by blocking T-cell entry to secondary lymphoid organs BLOOD Beilhack, A., Schulz, S., Baker, J., Beilhack, G. F., Nishimura, R., Baker, E. M., Landan, G., Herman, E. I., Butcher, E. C., Contag, C. H., Negrin, R. S. 2008; 111 (5): 2919-2928

  Abstract

  In acute graft-versus-host disease (aGVHD), donor T cells attack the recipient's gastrointestinal tract, liver, and skin. We hypothesized that blocking access to distinct lymphoid priming sites may alter the specific organ tropism and prevent aGVHD development. In support of this initial hypothesis, we found that different secondary lymphoid organs (SLOs) imprint distinct homing receptor phenotypes on evolving alloreactive effector T cells in vivo. Yet preventing T-cell entry to specific SLOs through blocking monoclonal antibodies, or SLO ablation, did not alter aGVHD pathophysiology. Moreover, transfer of alloreactive effector T cells into conditioned secondary recipients targeted the intestines and liver, irrespective of their initial priming site. Thus, we demonstrate redundancy of SLOs at different anatomical sites in aGVHD initiation. Only prevention of T-cell entry to all SLOs could completely abrogate the onset of aGVHD.

  View details for DOI 10.1182/blood-2007-09-112789

  View details for PubMedID 17989315

• Single-nucleotide-specific siRNA targeting in a dominant-negative skin model JOURNAL OF INVESTIGATIVE DERMATOLOGY Hickerson, R. P., Smith, F. J., Reeves, R. E., Contag, C. H., Leake, D., Leachman, S. A., Milstone, L. M., McLean, W. H., Kaspar, R. L. 2008; 128 (3): 594-605

  Abstract

  RNA interference offers a novel approach for developing therapeutics for dominant-negative genetic disorders. The ability to inhibit expression of the mutant allele without affecting wild-type gene expression could be a powerful new treatment option. Targeting the single-nucleotide keratin 6a (K6a) N171K mutation responsible for the rare monogenic skin disorder pachyonychia congenita (PC), we demonstrate that small interfering RNAs (siRNAs) can potently and selectively block expression of mutant K6a. To test whether lead siRNAs could discriminate mutant mRNA in the presence of both wild-type and mutant forms, a dominant-negative PC cell culture model was developed. As predicted for a dominant-negative disease, simultaneous expression of both wild-type and mutant K6a resulted in defective keratin filament formation. Addition of mutant-specific siRNAs allowed normal filament formation, suggesting selective inhibition of mutant K6a. The effectiveness of our siRNA in skin was tested by co-delivering a firefly luciferase/mutant K6a bicistronic reporter construct and mutant-specific siRNAs to mouse footpads. Potent inhibition of the fluorescent reporter was demonstrated using the Xenogen IVIS200 in vivo imaging system. Additionally, wild type-specific siRNAs knocked down the expression of pre-existing endogenous K6a in human keratinocytes. These results suggest that efficient delivery of these "designer siRNAs" may allow effective treatment of numerous genetic disorders including PC.

  View details for DOI 10.1038/sj.jid.5701060

  View details for Web of Science ID 000253374300012

  View details for PubMedID 17914454

• MEMS based Dual-Axes Confocal Clinical Endoscope for Real Time in vivo Imaging IEEE/LEOS International Conference on Optical MEMS and Nanophotonics Piyawattanametha, W., Mandella, M. J., Ra, H., Liu, J. T., Garai, E., Kino, G. S., Solgaard, O., Contag, C. H. IEEE. 2008: 42–43

  View details for Web of Science ID 000264556700022

• Development of therapeutic siRNAs for pachyonychia congenita JOURNAL OF INVESTIGATIVE DERMATOLOGY Smith, F. J., Hickerson, R. P., Sayers, J. M., Reeves, R. E., Contag, C. H., Leake, D., Kaspar, R. L., McLean, H. I. 2008; 128 (1): 50-58

  Abstract

  Pachyonychia congenita (PC) is an autosomal-dominant keratin disorder where the most painful, debilitating aspect is plantar keratoderma. PC is caused by mutations in one of four keratin genes; however, most patients carry K6a mutations. Knockout mouse studies suggest that ablation of one of the several K6 genes can be tolerated owing to compensatory expression of the others. Here, we have developed potent RNA interference against K6a as a paradigm for treating a localized dominant skin disorder. Four small interfering RNAs (siRNAs) were designed against unique sequences in the K6a 3'-untranslated region. We demonstrated near-complete ablation of endogenous K6a protein expression in two keratinocyte cell lines, HaCaT and NEB-1, by transient transfection of each of the four K6a siRNAs. The siRNAs were effective at very low, picomolar concentrations. One potent lead K6a inhibitor, which was highly specific for K6a, was tested in a mouse model where reporter gene constructs were injected intradermally into mouse paw and luciferase activity was used as an in vivo readout. Imaging in live mice using the Xenogen IVIS system demonstrated that the K6a-specific siRNA strongly inhibited bicistronic K6a-luciferase gene expression in vivo. These data suggest that siRNAs can specifically and very potently target mutated genes in the skin and support development of these inhibitors as potential therapeutics.

  View details for DOI 10.1038/sj.jid.5701040

  View details for Web of Science ID 000251613600009

  View details for PubMedID 17762855

• Sustained release of drugs dispersed in polymer nanoparticles ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Jacobson, G. B., Shinde, R., Contag, C. H., Zare, R. N. 2008; 47 (41): 7880-7882

  View details for DOI 10.1002/anie.200802260

  View details for Web of Science ID 000260062500019

  View details for PubMedID 18773393

  View details for PubMedCentralID PMC2586612

• Design and evaluation of a variable aperture collimator for conformal radiotherapy of small animals using a microCT scanner MEDICAL PHYSICS Graves, E. E., Zhou, H., Chatterjee, R., Keall, P. J., Gambhir, S. S., Contag, C. H., Boyer, A. L. 2007; 34 (11): 4359-4367

  Abstract

  Treatment of small animals with radiation has in general been limited to planar fields shaped with lead blocks, complicating spatial localization of dose and treatment of deep-seated targets. In order to advance laboratory radiotherapy toward what is accomplished in the clinic, we have constructed a variable aperture collimator for use in shaping the beam of microCT scanner. This unit can image small animal subjects at high resolution, and is capable of delivering therapeutic doses in reasonable exposure times. The proposed collimator consists of two stages, each containing six trapezoidal brass blocks that move along a frame in a manner similar to a camera iris producing a hexagonal aperture of variable size. The two stages are offset by 30 degrees and adjusted for the divergence of the x-ray beam so as to produce a dodecagonal profile at isocenter. Slotted rotating driving plates are used to apply force to pins in the collimator blocks and effect collimator motion. This device has been investigated through both simulation and measurement. The collimator aperture size varied from 0 to 8.5 cm as the driving plate angle increased from 0 to 41 degrees. The torque required to adjust the collimator varied from 0.5 to 5 N x m, increasing with increasing driving plate angle. The transmission profiles produced by the scanner at isocenter exhibited a penumbra of approximately 10% of the collimator aperture width. Misalignment between the collimator assembly and the x-ray source could be identified on the transmission images and corrected by adjustment of the collimator location. This variable aperture collimator technology is therefore a feasible and flexible solution for adjustable shaping of radiation beams for use in small animal radiotherapy as well as other applications in which beam shaping is desired.

  View details for DOI 10.1118/1.2789498

  View details for Web of Science ID 000251145900029

  View details for PubMedID 18072501

• Image-guided analyses reveal that non-CD4 splenocytes contribute to CD4+T cell-mediated inflammation leading to islet destruction by altering their local function and not systemic trafficking patterns Lee, M., Lee, W., Van, Y., Contag, C. H., Liu, C. SPRINGER. 2007: S29–S29

  View details for Web of Science ID 000251618000095

• Image-guided analyses reveal that non-CD4 splenocytes contribute to CD4(+) T cell-mediated inflammation leading to islet destruction by altering their local function and not systemic trafficking patterns MOLECULAR IMAGING Lee, M., Lee, W., Van, Y., Contag, C. H., Liu, C. 2007; 6 (6): 369-383

  Abstract

  Recruitment of CD4(+) T cells into islets is a critical component of islet inflammation (insulitis) leading to type 1 diabetes; therefore, determining if conditions used to treat diabetes change their trafficking patterns is relevant to the outcome. Cotransfer of CD4(+)BDC2.5 (BDC) cells with non-CD4 splenocytes obtained from newly diabetic NOD mice, but not when they are transferred alone, induces accelerated diabetes. It is unclear whether these splenocytes affect diabetes development by altering the systemic and/or local trafficking and proliferation patterns of BDC cells in target and nontarget tissues. To address these questions, we developed an animal model to visualize BDC cell trafficking and proliferation using whole-body in vivo bioluminescence imaging and used the images to direct tissue sampling for further analyses of the cell distribution within tissues. The whole-body, or macroscopic, trafficking patterns were not dramatically altered in both groups of recipient mice. However, the local patterns of cell distribution were distinct, which led to invasive insulitis only in cotransferred mice with an increased number of islet-infiltrating CD11b(+) and CD11c(+) cells. Taken together, the non-CD4 splenocytes act locally by promoting invasive insulitis without altering the systemic trafficking patterns or proliferation of BDC cells and thus contributing to diabetes by altering the localization within the tissue.

  View details for DOI 10.2310/7290.2007.00033

  View details for Web of Science ID 000251569400001

  View details for PubMedID 18053408

• Delivery and inhibition of reporter genes by small interfering RNAs in a mouse skin model JOURNAL OF INVESTIGATIVE DERMATOLOGY Wang, Q., Ilves, H., Chu, P., Contag, C. H., Leake, D., Johnston, B. H., Kaspar, R. L. 2007; 127 (11): 2577-2584

  Abstract

  RNA interference offers the potential of a novel therapeutic approach for treating skin disorders. To this end, we investigated delivery of nucleic acids, including a plasmid expressing the reporter gene luciferase, to mouse skin by intradermal injection into footpads using in vivo bioluminescence imaging over multiple time points. In order to evaluate the ability of RNA interference to inhibit skin gene expression, reporter gene constructs were co-injected with specific or non-specific siRNAs and the in vivo effects measured. Our results revealed that specific unmodified and modified siRNAs (but not nonspecific matched controls) strongly inhibit reporter gene expression in mice. These results indicate that small interfering RNA, delivered locally as RNA directly or expressed from viral or non-viral vectors, may be effective agents for treating skin disorders.

  View details for DOI 10.1038/sj.jid.5700891

  View details for Web of Science ID 000250226800014

  View details for PubMedID 17522708

• Functional imaging of colonic mucosa with a fibered Confocal microscope for real-time in vivo pathology CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Wang, T. D., Friedland, S., Sahbaie, P., Soetikno, R., Hsiung, P., Liu, J. T., Crawford, J. M., Contag, C. H. 2007; 5 (11): 1300-1305

  Abstract

  Histologic interpretation of disease currently is performed with static images of excised tissues, and is limited by processing artifact, sampling error, and interpretive variability. The aim of this study was to show the use of functional optical imaging of viable mucosa for quantitative evaluation of colonic neoplasia in real time.Fluorescein (5 mg/mL) was administered topically in 54 human subjects undergoing screening colonoscopy. Fluorescence images were collected with 488-nm excitation at 12 frames/s with the confocal microendoscopy system. Movement of fluorescein in the transient period (<5 s) and the lamina propria:crypt contrast ratio in the steady-state phase (>5 s) were quantified.Normal mucosa showed circular crypts with uniform size, hyperplasia revealed proliferative glands with serrated lumens, and adenomas displayed distorted elongated glands. For t less than 5 seconds, fluorescein passed through normal epithelium with a peak speed of 1.14 +/- 0.09 microm/s at t = 0.5 seconds, and accumulated into lamina propria as points of fluorescence that moved through the interglandular space with an average speed of 41.7 +/- 3.4 microm/s. Passage of fluorescein through adenomatous mucosa was delayed substantially. For t greater than 5 seconds, high sensitivity, specificity, and accuracy was achieved using a discriminant function to evaluate the contrast ratio to distinguish normal from lesional mucosa (91%, 87%, and 89%, respectively; P < .001), hyperplasia from adenoma (97%, 96%, and 96%, respectively; P < .001), and tubular from villous adenoma (100%, 92%, and 93%, respectively; P < .001).Confocal imaging can be performed in vivo to assess the functional behavior of tissue in real time for providing pathologic interpretation, representing a new method for histologic evaluation.

  View details for DOI 10.1016/j.cgh.2007.07.013

  View details for Web of Science ID 000250944900012

  View details for PubMedID 17936692

  View details for PubMedCentralID PMC2104519

• Detection of endogenous biomolecules in Barrett's esophagus by Fourier transform infrared spectroscopy PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Wang, T. D., Triadafilopoulos, G., Crawford, J. M., Dixon, L. R., Bhandari, T., Sahbaie, P., Friediand, S., Soetikno, R., Contag, C. H. 2007; 104 (40): 15864-15869

  Abstract

  Fourier transform infrared (FTIR) spectroscopy provides a unique molecular fingerprint of tissue from endogenous sources of light absorption; however, specific molecular components of the overall FTIR signature of precancer have not been characterized. In attenuated total reflectance mode, infrared light penetrates only a few microns of the tissue surface, and the influence of water on the spectra can be minimized, allowing for the analyses of the molecular composition of tissues. Here, spectra were collected from 98 excised specimens of the distal esophagus, including 38 squamous, 38 intestinal metaplasia (Barrett's), and 22 gastric, obtained endoscopically from 32 patients. We show that DNA, protein, glycogen, and glycoprotein comprise the principal sources of infrared absorption in the 950- to 1,800-cm(-1) regime. The concentrations of these biomolecules can be quantified by using a partial least-squares fit and used to classify disease states with high sensitivity, specificity, and accuracy. Moreover, use of FTIR to detect premalignant (dysplastic) mucosa results in a sensitivity, specificity, positive predictive value, and total accuracy of 92%, 80%, 92%, and 89%, respectively, and leads to a better interobserver agreement between two gastrointestinal pathologists for dysplasia (kappa = 0.72) versus histology alone (kappa = 0.52). Here, we demonstrate that the concentration of specific biomolecules can be determined from the FTIR spectra collected in attenuated total reflectance mode and can be used for predicting the underlying histopathology, which will contribute to the early detection and rapid staging of many diseases.

  View details for DOI 10.1073/pnas.0707567104

  View details for Web of Science ID 000249942700049

  View details for PubMedID 17901200

  View details for PubMedCentralID PMC2000401

• Accelerated bone repair after plasma laser corticotomies ANNALS OF SURGERY Leucht, P., Lam, K., Kim, J., Mackanos, M. A., Simanovskii, D. M., Longaker, M. T., Contag, C. H., Schwettman, H. A., Helms, J. A. 2007; 246 (1): 140-150

  Abstract

  To reveal, on a cellular and molecular level, how skeletal regeneration of a corticotomy is enhanced when using laser-plasma mediated ablation compared with conventional mechanical tissue removal.Osteotomies are well-known for their most detrimental side effect: thermal damage. This thermal and mechanical trauma to adjacent bone tissue can result in the untoward consequences of cell death and eventually in a delay in healing.Murine tibial corticotomies were performed using a conventional saw and a Ti:Sapphire plasma-generated laser that removes tissue with minimal thermal damage. Our analyses began 24 hours after injury and proceeded to postsurgical day 6. We investigated aspects of wound repair ranging from vascularization, inflammation, cell proliferation, differentiation, and bone remodeling.Histology of mouse corticotomy sites uncovered a significant difference in the onset of bone healing; whereas laser corticotomies showed abundant bone matrix deposition at postsurgical day 6, saw corticotomies only exhibited undifferentiated tissue. Our analyses uncovered that cutting bone with a saw caused denaturation of the collagen matrix due to thermal effects. This denatured collagen represented an unfavorable scaffold for subsequent osteoblast attachment, which in turn impeded deposition of a new bony matrix. The matrix degradation induced a prolonged inflammatory reaction at the cut edge to create a surface favorable for osteochondroprogenitor cell attachment. Laser corticotomies were absent of collagen denaturation, therefore osteochondroprogenitor cell attachment was enabled shortly after surgery.In summary, these data demonstrate that corticotomies performed with Ti:Sapphire lasers are associated with a reduced initial inflammatory response at the injury site leading to accelerated osteochondroprogenitor cell migration, attachment, differentiation, and eventually matrix deposition.

  View details for DOI 10.1097/01.sla.0000258559.07435.b3

  View details for Web of Science ID 000247672300022

  View details for PubMedID 17592303

  View details for PubMedCentralID PMC1899222

• Real-time analysis of uptake and bioactivatable cleavage of luciferin-transporter conjugates in transgenic reporter mice PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Wender, P. A., Goun, E. A., Jones, L. R., Pillow, T. H., Rothbard, J. B., Shinde, R., Contag, C. H. 2007; 104 (25): 10340-10345

  Abstract

  Many therapeutic leads fail to advance clinically because of bioavailability, selectivity, and formulation problems. Molecular transporters can be used to address these problems. Molecular transporter conjugates of otherwise poorly soluble or poorly bioavailable drugs or probes exhibit excellent solubility in water and biological fluids and at the same time an enhanced ability to enter tissues and cells and with modification to do so selectively. For many conjugates, however, it is necessary to release the drug/probe cargo from the transporter after uptake to achieve activity. Here, we describe an imaging method that provides quantification of transporter conjugate uptake and cargo release in real-time in animal models. This method uses transgenic (luciferase) reporter mice and whole-body imaging, allowing noninvasive quantification of transporter conjugate uptake and probe (luciferin) release in real time. This process effectively emulates drug-conjugate delivery, drug release, and drug turnover by an intracellular target, providing a facile method to evaluate comparative uptake of new transporters and efficacy and selectivity of linker release as required for fundamental studies and therapeutic applications.

  View details for DOI 10.1073/pnas.0703919104

  View details for Web of Science ID 000247500000010

  View details for PubMedID 17563383

  View details for PubMedCentralID PMC1965515

• Combining immune cell and viral therapy for the treatment of cancer CELLULAR AND MOLECULAR LIFE SCIENCES Thorne, S. H., Contag, C. H. 2007; 64 (12): 1449-1451

  Abstract

  A variety of viral-based and immune cell therapies have been proposed for use in the treatment of cancer. One possible approach to improve the effectiveness of these biological agents may be to combine them such that we can take advantage of natural immune cell-pathogen relationships. Here we discuss these potential approaches with particular emphasis on the use of immune cells as carrier vehicles to deliver viral therapies to the tumor.

  View details for DOI 10.1007/s00018-007-6550-z

  View details for Web of Science ID 000247272100001

  View details for PubMedID 17404689

• Therapeutic si RNAs for treatment of pachyonychia congenita 68th Annual Meeting of the Society-for-Investigative-Dermatology Smith, F. J., Hickerson, R. P., Sayers, J., Reeves, R. E., Contag, C. H., Leake, D., Kaspar, R. L., McLean, W. NATURE PUBLISHING GROUP. 2007: S94–S94

  View details for Web of Science ID 000245387800557

• In vivo dynamics of regulatory T-cell trafficking and survival predict effective strategies to control graft-versus-host disease following allogeneic transplantation BLOOD Nguyen, V. H., Zeiser, R., daSilva, D. L., Chang, D. S., Beilhack, A., Contag, C. H., Negrin, R. S. 2007; 109 (6): 2649-2656

  Abstract

  CD4(+)CD25(+) regulatory T cells (Tregs) suppress immune responses to alloantigens. The in vivo circulation and tissue localization of Tregs during an adaptive immune response remain unclear. We noninvasively tracked luciferase-expressing Tregs over time in an allogeneic bone marrow transplant model and demonstrated colocalization with effector T cells and initial expansion in secondary lymphoid organs before migration into inflamed tissues. Inflammation induced by irradiation and the allogeneic setting provided crucial stimuli for early Treg expansion and migration, leading to parallel reduction of effector T-cell proliferation in lymphoid organs and peripheral tissues. Treg transplants conferred long-term protection from systemic inflammatory challenge consistent with Treg in vivo survival. Suppression occurred during multiple phases of inflammation, but is optimal in the initial phase, providing protection from graft-versus-host disease while maintaining the graft-versus-tumor effect even at physiologic doses of Tregs due to their in vivo expansion, hence overcoming a major barrier to potential clinical applications of Tregs given their rarity.

  View details for DOI 10.1182/blood-2006-08-044529

  View details for PubMedID 17095616

• Early CD30 signaling is critical for adoptively transferred CD4(+)CD25(+) regulatory T cells in prevention of acute graft-versus-host disease BLOOD Zeiser, R., Nguyen, V. H., Hou, J., Beilhack, A., Zambricki, E., Buess, M., Contag, C. H., Negrin, R. S. 2007; 109 (5): 2225-2233

  Abstract

  Murine CD4+CD25+ regulatory T cells (Treg cells) reduce acute graft-versus-host disease (aGvHD). However, surface molecules critical for suppression are unclear. Deficiency of CD30 (CD30-/-) leads to impaired thymic negative selection and augmented T-cell autoreactivity. Therefore, we investigated the role of CD30 signaling in Treg-cell function during aGvHD. Treg cells derived from CD30-/- animals were significantly less effective in preventing aGvHD lethality. Early blockade of the CD30/CD153 pathway with a neutralizing anti-CD153 mAb reduced Treg-mediated protection from proinflammatory cytokine accumulation and donor-type T-cell apoptosis. In vivo bioluminescence imaging demonstrated intact homing but reduced expansion of luciferase-expressing Treg cells when CD153 was blocked during the early phase after adoptive transfer. CD30 surface expression on Treg cells increased with alloantigen exposure, and CD153 expression on recipient-type dendritic cells increased in the presence of a proinflammatory environment. These data demonstrate that early CD30 signaling is critical for Treg-mediated aGvHD protection after major MHC-mismatch bone marrow transplantation.

  View details for DOI 10.1182/blood-2006-07-038455

  View details for PubMedID 17068147

• Prevention of acute graft-versus-host disease by blocking T cell entry to secondary lymphoid organs 96th Annual Meeting of the United-States-and-Canadian-Academy-of-Pathology Schulz, S., Beilhack, A., Baker, J., Nishimura, R., Landan, G., Baker, E., Herman, E., BUTCHER, E. C., Contag, C. H., Negrin, R. S. NATURE PUBLISHING GROUP. 2007: 259A–259A

  View details for Web of Science ID 000244922402023

• Prevention of acute-versus-host disease by blocking t cell entry to secondary lymphoid organs 96th Annual Meeting of the United-States-and-Canadian-Academy-of-Pathology Schultz, S., Beilhack, A., Baker, J., Nishimura, R., Landan, G., Baker, E., Herman, E. I., BUTCHER, E. C., Contag, C. H., Negrin, R. S. NATURE PUBLISHING GROUP. 2007: 259A–259A

  View details for Web of Science ID 000244935302023

• Miniature near-infrared dual-axes confocal microscope utilizing a two-dimensional microelectromechanical systems scanner OPTICS LETTERS Liu, J. T., Mandella, M. J., Ra, H., Wong, L. K., Solgaard, O., Kino, G. S., Piyawattanametha, W., Contag, C. H., Wang, T. D. 2007; 32 (3): 256-258

  Abstract

  The first, to our knowledge, miniature dual-axes confocal microscope has been developed, with an outer diameter of 10 mm, for subsurface imaging of biological tissues with 5-7 microm resolution. Depth-resolved en face images are obtained at 30 frames per second, with a field of view of 800 x 100 microm, by employing a two-dimensional scanning microelectromechanical systems mirror. Reflectance and fluorescence images are obtained with a laser source at 785 nm, demonstrating the ability to perform real-time optical biopsy.

  View details for Web of Science ID 000244278900018

  View details for PubMedID 17215937

• CD4+CD25+regulatory T cells enhance immune reconstitution following allogeneic hematopoietic cell transplantation by protecting thymic and lymphoid compartments from graft-versus-host disease damage without impacting T cell repertoire development Tandem BMT Meeting 2007 Nguyen, V. H., Chang, D., Shashidhar, S., Bachmann, M., Contag, C. H., Brown, J. M., Negrin, R. S. ELSEVIER SCIENCE INC. 2007: 20–20

  View details for Web of Science ID 000244028300048

• Donor CD8(+) T cells mediate graft-versus-leukemia activity without clinical signs of graft-versus-host disease in recipients conditioned with anti-CD3 monoclonal antibody JOURNAL OF IMMUNOLOGY Zhang, C., Lou, J., Li, N., Todorov, I., Lin, C., Cao, Y., Contag, C. H., Kandeel, F., Forman, S., Zeng, D. 2007; 178 (2): 838-850

  Abstract

  Donor CD8(+) T cells play a critical role in mediating graft-vs-leukemia (GVL) activity, but also induce graft-vs-host disease (GVHD) in recipients conditioned with total body irradiation (TBI). In this study, we report that injections of donor C57BL/6 (H-2(b)) or FVB/N (H-2(q)) CD8(+) T with bone marrow cells induced chimerism and eliminated BCL1 leukemia/lymphoma cells without clinical signs of GVHD in anti-CD3-conditioned BALB/c (H-2(d)) recipients, but induced lethal GVHD in TBI-conditioned recipients. Using in vivo and ex vivo bioluminescent imaging, we observed that donor CD8(+) T cells expanded rapidly and infiltrated GVHD target tissues in TBI-conditioned recipients, but donor CD8(+) T cell expansion in anti-CD3-conditioned recipients was confined to lymphohematological tissues. This confinement was associated with lack of up-regulated expression of alpha(4)beta(7) integrin and chemokine receptors (i.e., CXCR3) on donor CD8(+) T cells. In addition, donor CD8(+) T cells in anti-CD3-conditioned recipients were rendered unresponsive, anergic, Foxp3(+), or type II cytotoxic T phenotype. Those donor CD8(+) T cells showed strong suppressive activity in vitro and mediated GVL activity without clinical signs of GVHD in TBI-conditioned secondary recipients. These results indicate that anti-CD3 conditioning separates GVL activity from GVHD via confining donor CD8(+) T cell expansion to host lymphohemological tissues as well as tolerizing them in the host.

  View details for Web of Science ID 000243320400024

  View details for PubMedID 17202345

• Methods for imaging cell fates in hematopoiesis. Methods in molecular medicine Duda, J., Karimi, M., Negrin, R. S., Contag, C. H. 2007; 134: 17-34

  Abstract

  Modern imaging technologies that allow for in vivo monitoring of cells in intact research subjects have opened up broad new areas of investigation. In the field of hematopoiesis and stem cell research, studies of cell trafficking involved in injury repair and hematopoietic engraftment have made great progress using these new tools. Multiple imaging modalities are available, each with its own advantages and disadvantages, depending on the specific application. For mouse models, clinically validated technologies such as magnetic resonance imaging (MRI) and positron emission tomography (PET) have been joined by optical imaging techniques such as in vivo bioluminescence imaging (BLI) and fluorescence imaging, and all have been used to monitor bone marrow and stem cells after transplantation into mice. Each modality requires that the cells of interest be marked with a distinct label that makes them uniquely visible using that technology. For each modality, there are several labels to choose from. Finally, multiple methods for applying these different labels are available. This chapter provides an overview of the imaging technologies and commonly used labels for each, as well as detailed protocols for gene delivery into hematopoietic cells for the purposes of applying these labels. The goal of this chapter is to provide adequate background information to allow the design and implementation of an experimental system for in vivo imaging in mice.

  View details for PubMedID 17666740

• Compact optical design for dual-axes confocal endoscopic microscopes Conference on Three-Dimensional and Multidimensional Microscopy - Image Acquisition and Processing XIV Mandella, M. J., Liua, J. T., Piyawattanametha, W., Ra, H., Hsiung, P., Wong, L. K., Solgaard, O., Wang, T. D., Contag, C. H., Kino, G. S. SPIE-INT SOC OPTICAL ENGINEERING. 2007

  View details for DOI 10.1117/12.699484

  View details for Web of Science ID 000245855700007

• Three-dimensional In vivo real time imaging by a miniature dual-axes confocal microscope based on a two-dimensional mems scanner 14th International Conference on Solid-State Sensors, Actuators and Microsystems/21st European Conference on Solid-State Transducers Piyawattanametha, W., Ra, H., Mandella, M. J., Liu, J. T., Wong, L. K., Du, C. B., Wang, T. D., Contag, C. H., Kino, G. S., Solgaard, O. IEEE. 2007

  View details for Web of Science ID 000249603700105

• Wavelength-dependent dynamics of heat shock protein 70 expression in free electron laser wounds Conference on Thermal Treatment of Tissue - Energy Delivery and Assessment IV Wilmink, G. J., Beckham, J. T., Mackanos, M., Contag, C. H., Davidson, J. M., Jansen, E. D. SPIE-INT SOC OPTICAL ENGINEERING. 2007

  View details for DOI 10.1117/12.705085

  View details for Web of Science ID 000246494100002

• Three-Dimensional in vivo Reflectance and Fluorescence Imaging by a Handheld Dual-Axes Confocal Microscope Conference on Lasers and Electro-Optics/Quantum Electronics and Laser Science Conference Ra, H., Piyawattanametha, W., Mandella, M. J., Liu, J. T., Wong, L. K., Wang, T. D., Contag, C. H., Kino, G. S., Solgaard, O. IEEE. 2007: 795–796

  View details for Web of Science ID 000268751000400

• Molecular Imaging of bone marrow mononuclear cell homing and engraftment in ischemic myocardium STEM CELLS Sheikh, A. Y., Lin, S., Cao, F., Cao, Y., van der Bogt, K. E., Chu, P., Chang, C., Contag, C. H., Robbins, R. C., Wu, J. C. 2007; 25 (10): 2677-2684

  Abstract

  Bone marrow mononuclear cell (BMMC) therapy shows promise as a treatment for ischemic heart disease. However, the ability to monitor long-term cell fate remains limited. We hypothesized that molecular imaging could be used to track stem cell homing and survival after myocardial ischemia-reperfusion (I/R) injury. We first harvested donor BMMCs from adult male L2G85 transgenic mice constitutively expressing both firefly luciferase (Fluc) and enhanced green fluorescence protein reporter gene. Fluorescence-activated cell sorting analysis revealed approximately 0.07% of the population to consist of classic hematopoietic stem cells (lin-, thy-int, c-kit+, Sca-1+). Afterward, adult female FVB recipients (n = 38) were randomized to sham surgery or acute I/R injury. Animals in the sham (n = 16) and I/R (n = 22) groups received 5 x 10(6) of the L2G85-derived BMMCs via tail vein injection. Bioluminescence imaging (BLI) was used to track cell migration and survival in vivo for 4 weeks. BLI showed preferential homing of BMMCs to hearts with I/R injury compared with sham hearts within the first week following cell injection. Ex vivo analysis of explanted hearts by histology confirmed BLI imaging results, and quantitative real-time polymerase chain reaction (for the male Sry gene) further demonstrated a greater number of BMMCs in hearts with I/R injury compared with the sham group. Functional evaluation by echocardiography demonstrated a trend toward improved left ventricular fractional shortening in animals receiving BMMCs. Taken together, these data demonstrate that molecular imaging can be used to successfully track BMMC therapy in murine models of heart disease. Specifically, we have demonstrated that systemically delivered BMMCs preferentially home to and are retained by injured myocardium. Disclosure of potential conflicts of interest is found at the end of this article.

  View details for DOI 10.1634/stemcells.2007-0041

  View details for Web of Science ID 000249929900031

  View details for PubMedID 17628019

  View details for PubMedCentralID PMC3657503

• In vivo pathology: Seeing with molecular specificity and cellular resolution in the living body ANNUAL REVIEW OF PATHOLOGY-MECHANISMS OF DISEASE Contag, C. H. 2007; 2: 277-305

  Abstract

  The emerging tools of in vivo molecular imaging are enabling dynamic cellular and molecular analyses of disease mechanisms in living animal models and humans. These advances have the potential to dramatically change a number of fields of study, including pathology, and to contribute to the development of regenerative medicine and stem cell therapies. The new tools of molecular imaging, which have already had a tremendous impact on preclinical studies, hold great promise for bringing important and novel information to the clinician and the patient. These approaches are likely to enable early diagnosis, rapid typing of molecular markers, immediate assessment of therapeutic outcome, and ready measures of the extent of tissue regeneration after damage. However, the full impact of these new techniques will be determined by our ability to translate them to the clinic and to develop a general strategy that integrates them with other advances in molecular diagnostics and molecular medicine.

  View details for DOI 10.1146/annurev.pathol.2.010506.091930

  View details for Web of Science ID 000245663900012

  View details for PubMedID 18039101

• Comparison of heme oxygenase activity in gut-associated lymphoid tissues and the intestine. 8th Conference of the Western Student Medical Research Forum/Western Section of the American-Federation-for-Medical-Research/Western Association-of-Physicians/Western-Society-for-Pediatric-Research/Western-Society-for-Clinical-Investigation Burns-Guydisli, S. M., Wong, R. J., Kafsh, F., Contag, C. H., Stevenson, D. K. LIPPINCOTT WILLIAMS & WILKINS. 2007: S117–S118

  View details for Web of Science ID 000247692400262

• Alloreactive T-cell trafficking after hematopoietic stem cell transplantation. Journal of stem cells & regenerative medicine Beilhack, A., Schulz, S., Baker, J., Beilhack, G., Herman, E., Baker, E., Landan, G., Nishimura, R., Butcher, E., Contag, C., Negrin, R. 2007; 2 (1): 107-?

  View details for PubMedID 24692934

• Alloreactive T-cell trafficking after hematopoietic stem cell transplantation. Journal of stem cells & regenerative medicine Beilhack, A., Schulz, S., Baker, J., Beilhack, G., Herman, E., Baker, E., Landan, G., Nishimura, R., Butcher, E., Contag, C., Negrin, R. 2007; 2 (1): 17-?

  View details for PubMedID 24692881

• The potential Salmonella aroA(-) vaccine strain is safe and effective in young BALB/c mice NEONATOLOGY Burns-Guydish, S. M., Zhao, H., Stevenson, D. K., Contag, C. H. 2007; 91 (2): 114-120

  Abstract

  Due to the increased susceptibility of neonates to pathogens including those with mutations, the use of live vaccine strategies in the human population may present a potential risk to the young.The specific aim of this study was to assess the risk that prospective Salmonella enterica serovar Typhimurium vaccine strains pose for the neonate and determine whether the strains are an effective vaccine by assessing the adaptive immune response.To evaluate the susceptibility of young mice to potential vaccine strains, S. typhimuriumaroA(-) and Delta phoP mutant strains were labeled by chromosomal insertion of the lux operon--this serves as a readily traceable marker of infection using noninvasive imaging methods. BALB/c mice ages 1, 2, 4, and 6 weeks of age were fed the bioluminescent aroA(-) or Delta phoP strains and the course of infection was monitored by in vivobioluminescence imaging. In addition, blood samples were collected post-inoculation to assess the IgG response of mice to S. typhimurium LPS.Young BALB/c mice were not susceptible to the aroA(-) strain in contrast to their susceptibility to the Delta phoP strain at a dose of 10(9) colony forming units. Delivery by oral feeding of the aroA(-) and Delta phoP strains in young mice also produced a robust IgG anti-LPS response.Here, we report that young 2-week-old mice orally fed the bioluminescent aroA(-) S. typhimurium strain were not susceptible to infection and elicited a protective immune response.

  View details for DOI 10.1159/000097128

  View details for PubMedID 17344661

• A signal hierarchy model of alloreactive T cell trafficking for the organ manifestation in acute graft-versus-host disease. 48th Annual Meeting of the American-Society-of-Hematology Beilhack, A., Schulz, S., Baker, J., Beilhack, G. F., Nishimura, R., Landan, G., Baker, E. M., Herman, E. I., Contag, C. H., Negrin, R. S. AMER SOC HEMATOLOGY. 2006: 26A–26A

  View details for Web of Science ID 000242440000073

• Heme oxygenase 1 deficiency compromises stress responses of hematopoietic stem cells. 48th Annual Meeting of the American-Society-of-Hematology Cao, Y., Wagers, A. J., Karsunky, H., Zhao, H., Reeves, R., Wang, R. J., Stevenson, D. K., Weissman, I. L., Contag, C. H. AMER SOC HEMATOLOGY. 2006: 395A–395A

  View details for Web of Science ID 000242440001612

• Early CD30 signaling is critical for adoptively transferred CD4(+)CD25(+) regulatory T cells in prevention of acute graft versus host disease. 48th Annual Meeting of the American-Society-of-Hematology Zeiser, R., Nguyen, V. H., Hou, J., Beilhack, A., Zambricki, E. A., Buess, M., Contag, C. H., Negrin, R. AMER SOC HEMATOLOGY. 2006: 906A–907A

  View details for Web of Science ID 000242440004224

• CD4+CD25+ Regulatory T cells enhance immune reconstitution following allogeneic hematopoietic cell transplantation by protecting thymic and lymphoid compartments from graft-versus-host disease damage without impacting T cell repertoire development. 48th Annual Meeting of the American-Society-of-Hematology Nguyen, V. H., Chang, D., Shashidhar, S., Bachmann, M., Contag, C. H., Brown, J. M., Negrin, R. S. AMER SOC HEMATOLOGY. 2006: 25A–25A

  View details for Web of Science ID 000242440000071

• Expression and regulation of heme oxygenase isozymes in the developing mouse cortex PEDIATRIC RESEARCH Zhao, H., Wong, R. J., Nguyen, X., Kalish, F., Mizobuchi, M., Vreman, H. J., Stevenson, D. K., Contag, C. H. 2006; 60 (5): 518-523

  Abstract

  Heme oxygenase (HO), the rate-limiting enzyme in heme degradation, plays a role in neonatal jaundice. Understanding the regulation of the developmental expression patterns of the two HO isozymes, HO-1 and HO-2, is essential for targeting HO to control pathologic jaundice, and uncovering the fundamental role that they play in mammalian development. Here we characterized the ontogeny of HO-1 and HO-2 expression in the developing mouse cortex by in vivo bioluminescence imaging, quantitative RT-PCR, and Western blot. HO-2, the predominant isoform in the adult cortex, was relatively stable throughout all ages. HO-1 was observed to be progressively down-regulated in an age-related manner. HO-1 expression in the adult cortex was also the lowest among the eight adult tissues analyzed. Because there is a 283-bp CpG island region in the HO-1 promoter, we hypothesized that methylation of the island is responsible for the age-related HO-1 down-regulation in the cortex. Methylation status was assessed using regular and quantitative methylation-specific PCR and the CpG island was found to be hypomethylated at all ages. Therefore, we conclude that HO-1 gene expression in the cortex is developmentally-regulated and that methylation of the HO-1 CpG island is not associated with the down-regulation of the gene.

  View details for DOI 10.1203/01.PDR.0000242374.21415.f5

  View details for PubMedID 16966352

• Molecular imaging using visible light to reveal biological changes in the brain NEUROIMAGING CLINICS OF NORTH AMERICA Contag, C. H. 2006; 16 (4): 633-?

  Abstract

  Advances in imaging have enabled the study of cellular and molecular processes in the context of the living body that include cell migration patterns, location and extent of gene expression, degree of protein-protein interaction, and levels of enzyme activity. These tools, which operate over a range of scales, resolutions, and sensitivities, have opened up broad new areas of investigation where the influence of organ systems and functional circulation is intact. There are a myriad of imaging modalities available, each with its own advantages and disadvantages, depending on the specific application. Among these modalities, optical imaging techniques, including in vivo bioluminescence imaging and fluorescence imaging, use visible light to interrogate biology in the living body. Optimal imaging with these modalities require that the appropriate marker be used to tag the process of interest to make it uniquely visible using a particular imaging technology. For each optical modality, there are various labels to choose from that range from dyes that permit tissue contrast and dyes that can be activated by enzymatic activity, to gene-encoding proteins with optical signatures that can be engineered into specific biological processes. This article provides and overview of optical imaging technologies and commonly used labels, focusing on bioluminescence and fluorescence, and describes several examples of how these tools are applied to biological questions relating to the central nervous system.

  View details for DOI 10.1016/j.nic.2006.08.002

  View details for Web of Science ID 000243152800009

  View details for PubMedID 17148024

• Luciferin derivatives for enhanced in vitro and in vivo bioluminescence assays BIOCHEMISTRY Shinde, R., Perkins, J., Contag, C. H. 2006; 45 (37): 11103-11112

  Abstract

  In vivo bioluminescence imaging has become a cornerstone technology for preclinical molecular imaging. This imaging method is based on light-emitting enzymes, luciferases, which require specific substrates for light production. When linked to a specific biological process in an animal model of human biology or disease, the enzyme-substrate interactions become biological indicators that can be studied noninvasively in living animals. Signal intensity in these animal models depends on the availability of the substrate for the reaction within living cells in intact organs. The biodistribution and clearance rates of the substrates are therefore directly related to optimal imaging times and signal intensities and ultimately determine the sensitivity of detection and predictability of the model. Modifications of d-luciferin, the substrate for the luciferases obtained from beetle, including fireflies, result in novel properties and offer opportunities for improved bioassays. For this purpose, we have synthesized a conjugate, glycine-d-aminoluciferin, and investigated its properties relative to those of d-aminoluciferin and d-luciferin. The three substrates exhibited different kinetic properties and different intracellular accumulation profiles due to differences in their molecular structure, which in turn influenced their biodistribution in animals. Glycine-d-aminoluciferin had a longer in vivo circulation time than the other two substrates. The ability to assay luciferase in vitro and in vivo using these substrates, which exhibit different pharmacokinetic and pharmacodynamic properties, will provide flexibility and improve current imaging capabilities.

  View details for DOI 10.1021/bi060475o

  View details for Web of Science ID 000240436800007

  View details for PubMedID 16964971

• Dual-axes confocal reflectance microscope for distinguishing colonic neoplasia JOURNAL OF BIOMEDICAL OPTICS Liu, J. T., Mandella, M. J., Friedland, S., Soetikno, R., Crawford, J. M., Contag, C. H., Kino, G. S., Wang, T. D. 2006; 11 (5)

  Abstract

  A dual-axes confocal reflectance microscope has been developed that utilizes a narrowband laser at 1310 nm to achieve high axial resolution, image contrast, field of view, and tissue penetration for distinguishing among normal, hyperplastic, and dysplastic colonic mucosa ex vivo. Light is collected off-axis using a low numerical aperture objective to obtain vertical image sections, with 4- to 5-microm resolution, at tissue depths up to 610 microm. Post-objective scanning enables a large field of view (610 x 640 microm), and balanced-heterodyne detection provides sensitivity to collect vertical sections at one frame per second. System optics are optimized to effectively reject out-of-focus scattered light without use of a low-coherence gate. This design is scalable to millimeter dimensions, and the results demonstrate the potential for a miniature instrument to detect precancerous tissues, and hence to perform in vivo histopathology.

  View details for DOI 10.1117/1.2363363

  View details for Web of Science ID 000242576900023

  View details for PubMedID 17092168

  View details for PubMedCentralID PMC2104521

• Cellular tolerance to pulsed hyperthermia PHYSICAL REVIEW E Simanovskii, D. M., Mackanos, M. A., Irani, A. R., O'Connell-Rodwell, C. E., Contag, C. H., Schwettman, H. A., Palanker, D. V. 2006; 74 (1)

  Abstract

  Transient heating of tissues leading to cellular stress or death is very common in medicine and biology. In procedures involving a mild (below 70 degrees C) and prolonged (minutes) heating, such as hyperthermal tumor therapy, the cellular response to thermal stress is relatively well studied. However, there is practically no data on cell viability at higher temperatures and shorter exposures, while the demand for this knowledge is growing. Two main reasons motivate this research: (i) a growing number of laser therapies and surgical procedures involving pulsed heating, and (ii) cellular viability data at short exposures to high temperatures provide a unique insight into the understanding of processes leading to thermally induced cellular death. We designed a technique and performed a study of cell viability under pulses of heat from 0.3 to 100 ms in duration with peak temperatures as high as 130 degrees C. We found that the threshold of cellular death in this range can be accurately approximated by the Arrhenius law with the activation energy of 1 eV, a significantly lower value than was reported in studies based on multisecond exposures.

  View details for DOI 10.1103/PhysRevE.74.011915

  View details for Web of Science ID 000239425600098

  View details for PubMedID 16907135

• Inhibition of CD4(+)CD25(+) regulatory T-cell function by calcineurin-dependent interleukin-2 production BLOOD Zeiser, R., Nguyen, V. H., Beilhack, A., Buess, M., Schulz, S., Baker, J., Contag, C. H., Negrin, R. S. 2006; 108 (1): 390-399

  Abstract

  CD4+CD25+ regulatory T (Treg) cells control immunologic tolerance and antitumor immune responses. Therefore, in vivo modification of Treg function by immunosuppressant drugs has broad implications for transplantation biology, autoimmunity, and vaccination strategies. In vivo bioluminescence imaging demonstrated reduced early proliferation of donor-derived luciferase-labeled conventional T cells in animals treated with Treg cells after major histocompatibility complex mismatch bone marrow transplantation. Combining Treg cells with cyclosporine A (CSA), but not rapamycin (RAPA) or mycophenolate mofetil (MMF), suppressed Treg function assessed by increased T-cell proliferation, graft-versus-host disease (GVHD) severity, and reduced survival. Expansion of Treg and FoxP3 expression within this population was lowest in conjunction with CSA, suggesting that calcineurin-dependent interleukin 2 (IL-2) production is critically required for Treg cells in vivo. The functional defect of Treg cells after CSA exposure could be reversed by exogenous IL-2. Further, the Treg plus RAPA combination preserved graft-versus-tumor (GVT) effector function against leukemia cells. Our data indicate that RAPA and MMF rather than CSA preserve function of Treg cells in pathologic immune responses such as GVHD without weakening the GVT effect.

  View details for DOI 10.1182/blood-2006-01-0329

  View details for PubMedID 16522809

• Cellular immunotherapy redirected by bispecific antibodies in primary ovarian cancer cells: A preclinical study. 42nd Annual Meeting of the American-Society-of-Clinical-Oncology Chan, J. K., Hamilton, C. A., Cheung, M. K., Schulz, S., Thorne, S. H., Anderson, E. M., Baker, J., Husain, A., Teng, N. N., Contag, C. H., Negrin, R. S. AMER SOC CLINICAL ONCOLOGY. 2006: 104S–104S

  View details for Web of Science ID 000239009400406

• In vivo imaging using bioluminescence: a tool for probing graft-versus-host disease. Nature reviews. Immunology Negrin, R. S., Contag, C. H. 2006; 6 (6): 484-490

  Abstract

  Immunological reactions have a key role in health and disease and are complex events characterized by coordinated cell trafficking to specific locations throughout the body. Clarification of these cell-trafficking events is crucial for improving our understanding of how immune reactions are initiated, controlled and recalled. As we discuss here, an emerging modality for revealing cell trafficking is bioluminescence imaging, which harnesses the light-emitting properties of enzymes such as luciferase for quantification of cells and uses low-light imaging systems. This strategy could be useful for the study of a wide range of biological processes, such as the pathophysiology of graft-versus-host and graft-versus-leukaemia reactions.

  View details for PubMedID 16724101

• Innovation - In vivo imaging using bioluminescence: a tool for probing graft-versus-host disease NATURE REVIEWS IMMUNOLOGY Negrin, R. S., Contag, C. H. 2006; 6 (6): 484-U2

  Abstract

  Immunological reactions have a key role in health and disease and are complex events characterized by coordinated cell trafficking to specific locations throughout the body. Clarification of these cell-trafficking events is crucial for improving our understanding of how immune reactions are initiated, controlled and recalled. As we discuss here, an emerging modality for revealing cell trafficking is bioluminescence imaging, which harnesses the light-emitting properties of enzymes such as luciferase for quantification of cells and uses low-light imaging systems. This strategy could be useful for the study of a wide range of biological processes, such as the pathophysiology of graft-versus-host and graft-versus-leukaemia reactions.

  View details for DOI 10.1038/nri1879

  View details for Web of Science ID 000238529700017

• Releasable luciferin-transporter conjugates: Tools for the real-time analysis of cellular uptake and release JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Jones, L. R., Goun, E. A., Shinde, R., Rothbard, J. B., Contag, C. H., Wender, P. A. 2006; 128 (20): 6526-6527

  Abstract

  The design, synthesis, and evaluation of conjugates of arginine-rich transporters and luciferin are described that release luciferin only after entry into cells that are stably transfected with luciferase. Each molecule of free luciferin that is released after entry generates a photon that can be measured allowing for real-time quantification of uptake and release in cells. The process provides a method to assay uptake and release of free luciferin as a function of variations in the releasable linker and in the transporter.

  View details for DOI 10.1021/ja0586283

  View details for Web of Science ID 000237590500002

  View details for PubMedID 16704230

• Intracellular cargo delivery by an octaarginine transporter adapted to target prostate cancer cells through cell surface protease activation BIOCONJUGATE CHEMISTRY Goun, E. A., Shinde, R., Dehnert, K. W., Adams-Bond, A., Wender, P. A., Contag, C. H., Franc, B. L. 2006; 17 (3): 787-796

  Abstract

  Delivery of therapeutics and imaging agents to target tissues requires localization and activation strategies with molecular specificity. Cell-associated proteases can be used for these purposes in a number of pathologic conditions, and their enzymatic activities can be exploited for activation strategies. Here, molecules based on the d-arginine octamer (r8) protein-transduction domain (PTD, also referred to as molecular transporters) have been adapted for selective uptake into cells only after proteolytic cleavage of a PTD-attenuating sequence by the prostate-specific antigen (PSA), an extracellular protease associated with the surface and microenvironment of certain prostate cancer cells. Convergent syntheses of these activatable PTDs (APTDs) are described, and the most effective r8 PTD-attenuating sequence is identified. The conjugates are shown to be stable in serum, cleaved by PSA, and taken up into Jurkat (human T cells) and PC3M prostate cancer cell lines only after cleavage by PSA. These APTD peptide-based molecules may facilitate targeted delivery of therapeutics or imaging agents to PSA-expressing prostate cancers.

  View details for DOI 10.1021/bc0503216

  View details for PubMedID 16704219

• Systemic effects of orally-administered zinc and tin (IV) metalloporphyrins on heme oxygenase expression in mice PEDIATRIC RESEARCH Morioka, I., Wong, R. J., Abate, A., Vreman, H. J., Contag, C. H., Stevenson, D. K. 2006; 59 (5): 667-672

  Abstract

  Some metalloporphyrins (Mps) inhibit heme oxygenase (HO), the rate-limiting enzyme in the production of bilirubin, and are potential compounds for the treatment of neonatal jaundice. We studied the safety and efficacy of Mps following oral administration. Adult HO-1-luc reporter mice were administered 30 micromol/kg body weight of tin mesoporphyrin (SnMP), zinc bis glycol deuteroporphyrin (ZnBG), or zinc protoporphyrin (ZnPP), or vehicle by oral gavage. Bilirubin production was measured as total body carbon monoxide (CO) excretion (VeCO). HO activity was quantitated via CO measurements by gas chromatography. HO-1 protein was determined by Western blot. HO-1 transcription levels were assessed by in vivo bioluminescence imaging. A significant 28% decrease in bilirubin production occurred within 3 h of SnMP treatment and persisted beyond 48 h. Bilirubin production decreased 15% and 9% by 3 h after administration of ZnBG and ZnPP, respectively, but returned to baseline within 48 h. Maximal inhibition of liver, spleen, and intestine HO activity was seen at 3 h with inhibitory effects decreasing in the order: SnMP > or = ZnBG > or = ZnPP. After SnMP treatment, HO-1 transcription increased 5.7-fold after 24 h. Furthermore, liver and spleen HO-1 protein significantly increased 3.7- and 2.0-fold, respectively, after 24 h. HO-1 transcription and protein were not affected in ZnBG- or ZnPP-treated mice. We conclude that the three Mps are absorbed at different rates in the mouse and affect bilirubin production and HO-1 expression in a tissue- and time-dependent manner.

  View details for DOI 10.1203/01.pdr.0000215088.71481.a6

  View details for PubMedID 16627879

• Selective intratumoral amplification of an antiangiogenic vector by an oncolytic virus produces enhanced antivascular and anti-tumor efficacy MOLECULAR THERAPY Thorne, S. H., Tam, B. Y., Kirn, D. H., Contag, C. H., Kuo, C. J. 2006; 13 (5): 938-946

  Abstract

  The development of effective cancer therapy will require the simultaneous targeting of multiple steps in tumor development. We have previously described an antiangiogenic gene therapy vector, Ad Flk1-Fc, which expresses a soluble VEGF receptor capable of inhibiting tumor angiogenesis and growth. We have also described an oncolytic virus, dl922/947, whose replication and subsequent cytotoxicity are restricted to cancer cells with a loss of the G1-S cell cycle checkpoint. Here we have optimized methods for combining these therapies, yielding significantly greater anti-tumor effects than the respective monotherapies. In cultured tumor lines, co-infection with both Ad Flk1-Fc and dl922/947 allowed replication and repackaging of the replication-deficient Ad Flk1-Fc and enhanced soluble VEGF receptor expression. Similar repackaging and increased gene expression were demonstrated in vivo using bioluminescence imaging studies. Finally, coadministration of these therapeutic viral therapies in vivo produced significantly enhanced anti-tumor effects in colon HCT 116 and prostate PC-3 xenografts in mice. This increased therapeutic benefit correlated with replication of Ad Flk1-Fc viral genomes, increased intratumoral levels of Flk1-Fc protein, and decreased microvessel density, consistent with enhanced antiangiogenic activity.

  View details for DOI 10.1016/j.ymthe.2005.12.010

  View details for Web of Science ID 000237523000016

  View details for PubMedID 16469543

• Enhanced killing of primary ovarian cancer by retargeting autologous cytokine-induced killer cells with bispecific antibodies: A preclinical study CLINICAL CANCER RESEARCH Chan, J. K., Hamilton, C. A., Cheung, M. K., Karimi, M., Baker, J., Gall, J. M., Schulz, S., Thorne, S. H., Teng, N. N., Contag, C. H., Lum, L. G., Negrin, R. S. 2006; 12 (6): 1859-1867

  Abstract

  Cytokine-induced killer (CIK) cells are ex vivo activated and expanded CD8+ natural killer T cells that have been shown to have antitumor activity. This is the first study exploring cell killing of primary ovarian carcinoma cells with and without bispecific antibodies. Primary cancer cells and autologous CIK cells were collected from women with epithelial ovarian cancer. Bispecific antibodies against cancer antigen-125 (BSAbxCA125) and Her2 (BSAbxHer2) were developed using chemical heteroconjugation. On fluorescence-activated cell sorting analysis, the expansion of CIK cells resulted in a significant increase of CD3+CD8+ and CD3+CD56+ T cells. With enhancement by bispecific antibodies, the mean percent lysis in a 51Cr release assay of fresh ovarian cancer cells exposed to autologous CIK cells increased from 21.7 +/- 0.3% to 89.4 +/- 2.1% at an E:T ratio of 100:1 (P < 0.001). Anti-NKG2D antibodies attenuated the CIK activity by 56.8% on primary cells (P < 0.001). In a xenograft severe combined immunodeficient mouse model, real-time tumor regression and progression was visualized using a noninvasive in vivo bioluminescence imaging system. Four hours after CIK cell injection, we were able to visualize CD8+NKG2D+ CIK cells infiltrating Her2-expressing cancer cells on fluorescence microscopy. Mice that underwent adoptive transfer of CIK cells redirected with BSAbxCA125 and BSAbxHer2 had significant reduction in tumor burden (P < 0.001 and P < 0.001) and improvement in survival (P = 0.05 and P = 0.006) versus those treated with CIK cells alone. Bispecific antibodies significantly enhanced the cytotoxicity of CIK cells in primary ovarian cancer cells and in our in vivo mouse model. The mechanism of cytolysis seems to be mediated in part by the NKG2D receptor.

  View details for DOI 10.1158/1078-0432.CCR-05-2019

  View details for PubMedID 16551871

• Induced biliary excretion of Listeria monocytogenes INFECTION AND IMMUNITY Hardy, J., Margolis, J. J., Contag, C. H. 2006; 74 (3): 1819-1827

  Abstract

  Listeria monocytogenes is a ubiquitous gram-positive bacterium that can cause systemic and often life-threatening disease in immunocompromised hosts. This organism is largely an intracellular pathogen; however, we have determined that it can also grow extracellularly in animals, in the lumen of the gallbladder. The significance of growth in the gallbladder with respect to the pathogenesis and spread of listeriosis depends on the ability of the bacterium to leave this organ and be disseminated to other tissues and into the environment. Should this process be highly inefficient, growth in the gallbladder would have no impact on pathogenesis or spread, but if it occurs efficiently, bacterial growth in this organ may contribute to listeriosis and dissemination of this organism. Here, we use whole-body imaging to determine the efficacy and kinetics of food- and hormone-induced biliary excretion of L. monocytogenes from the murine gallbladder, demonstrating that transit through the bile duct into the intestine can occur within 5 min of induction of gallbladder contraction by food or cholecystokinin and that movement of bacteria through the intestinal lumen can occur very rapidly in the absence of fecal material. These studies demonstrate that L. monocytogenes bacteria replicating in the gallbladder can be expelled from the organ efficiently and that the released bacteria move into the intestinal tract, where they pass into the environment and may possibly reinfect the animal.

  View details for DOI 10.1128/IAI.74.3.1819-1827.2006

  View details for Web of Science ID 000235817500043

  View details for PubMedID 16495556

  View details for PubMedCentralID PMC1418634

• Differential effects of immunosuppressants on adoptively transferred CD4+CD25(high) regulatory T cells in prevention of experimental acute graft-versus-host disease 32nd Annual Meeting of the American-Society-for-Blood-and-Marrow-Transplantation Zeiser, R., Nguyen, V., Beilhack, A., Schulz, S., Baker, J., Contag, C. H., Negrin, R. S. ELSEVIER SCIENCE INC. 2006: 69–69

  View details for Web of Science ID 000235344100197

• In vivo kinetics of acute graft-versus-host disease in conditioned vs. unconditioned hosts 32nd Annual Meeting of the American-Society-for-Blood-and-Marrow-Transplantation Beilhack, A., Wai, L. E., Schulz, S., Belhack, G. F., Nishimura, R., Baker, E. M., Herman, E. I., Landan, G., Olson, J. A., Baker, J., Contag, C. H., Negrin, R. S. ELSEVIER SCIENCE INC. 2006: 51–52

  View details for Web of Science ID 000235344100145

• Prevention of acute graft-versus-host disease despite compensatory function of lymphoid organs in vivo 32nd Annual Meeting of the American-Society-for-Blood-and-Marrow-Transplantation Beilback, A., Schulz, S., Baker, J., Beilback, G. F., Nishimura, R., Landan, G., Baker, E. M., Herman, E. I., BUTCHER, E. C., Contag, C. H., Negrin, R. S. ELSEVIER SCIENCE INC. 2006: 11–11

  View details for Web of Science ID 000235344100024

• The effectiveness of oral tin mesoporphyrin prophylaxis in reducing bilirubin production after an oral heme load in a transgenic mouse model BIOLOGY OF THE NEONATE DeSandre, G. H., Wong, R. J., Morioka, I., Contag, C. H., Stevenson, D. K. 2006; 89 (3): 139-146

  Abstract

  Neonatal jaundice is commonly encountered and rarely associated with morbidity and mortality. Nonetheless, infants with glucose-6-phosphate dehydrogenase deficiency often have hemolysis (a heme load) caused by an environmental oxidant trigger, thus increasing their risk for serious morbidity. The use of tin mesoporphyrin (SnMP) has been proposed for interdicting the development of severe hyperbilirubinemia in a variety of conditions.We studied the in vivo effects of prophylactic oral SnMP on heme oxygenase (HO) activity and bilirubin production, as indexed by the excretion rate of carbon monoxide (VeCO), following a subsequent oral heme load.Adult mice were exposed serially to heme and assessed for in vivo bilirubin production rates, HO-1 transcription and protein, and HO activity. The effect of prophylaxis with a single oral dose of SnMP prior to an oral heme load was assessed by measuring VeCOand tissue HO activities.After serial heme exposures, VeCO, HO-1 transcription and protein, and liver and spleen HO activities increased incrementally. After pretreatment with oral SnMP, bilirubin production decreased in response to an oral heme load. Also, heme-mediated increases in liver, spleen, and intestine HO activities were significantly dampened.A single oral dose of SnMP results in durable inhibition of bilirubin production and HO activity for at least 24 h in a mouse model of oral heme loading. Further studies are needed to fully elucidate the duration of this protection against hyperbilirubinemia due to a delayed heme load and any long-term consequences of prophylaxis with SnMP on HO-1 transcription and HO-1 protein.

  View details for DOI 10.1159/000088717

  View details for PubMedID 16205054

• A narrowband dual-axes confocal reflectance microscope for distinguishing colonic neoplasia Conference on Three-Dimensional and Multidimensional Microscopy - Image Acquisition and Processing XIII Liu, J. T., Mandella, M. J., Contag, C. H., Kino, G. S., Wang, T. D. SPIE-INT SOC OPTICAL ENGINEERING. 2006

  View details for DOI 10.1117/12.649019

  View details for Web of Science ID 000237132200001

• Inhibition of hepatitis CIRFS-mediated gene expression by small hairpin RNAs in human hepatocytes and mice 1st Annual Meeting of the Oligonucleotide-Therapeutics-Society Ilves, H., Kaspar, R. L., Wang, Q., Seyhan, A. A., Vlassov, A. V., Contag, C. H., Leake, D., Johnston, B. H. BLACKWELL PUBLISHING. 2006: 52–55

  Abstract

  The ability of small hairpin RNAs (shRNAs) to inhibit hepatitis C virus internal ribosome entry site (HCV IRES)-dependent gene expression was investigated in cultured cells and a mouse model. The results indicate that shRNAs, delivered as naked RNA or expressed from vectors, may be effective agents for the control of HCV and related viruses.

  View details for DOI 10.1196/annals.1348.060

  View details for Web of Science ID 000243449900008

  View details for PubMedID 17145925

• New enzyme for reductive cancer chemotherapy, YieF, and its improvement by directed evolution MOLECULAR CANCER THERAPEUTICS Barak, Y., Thorne, S. H., Ackerley, D. F., Lynch, S. V., Contag, C. H., Matin, A. 2006; 5 (1): 97-103

  Abstract

  Reductive prodrugs, mitomycin C and 5-aziridinyl-2,4-dinitrobenzamide (CB 1954), are nontoxic in their native form but become highly toxic upon reduction. Their effectiveness in cancer chemotherapy can be enhanced by delivering to tumors enzymes with improved prodrug reduction kinetics. We report the discovery of a new prodrug-reducing enzyme, YieF, from Escherichia coli, and the improvement of its kinetics for reducing mitomycin C and CB 1954. A YieF-derived enzyme, Y6, killed HeLa spinner cells with >or=5-fold efficiency than the wild-type enzymes, YieF and NfsA, at a variety of drug and enzyme concentrations and incubation times. With adhered HeLa cells and Salmonella typhimurium SL 7838 bacteria as enzyme delivery vehicle, at least an order of magnitude less of Y6-producing bacteria were required to kill >90% of tumor cells compared with bacteria expressing the wild-type enzymes, which at a comparable level killed < 5% of the cells. Thus, Y6 is a promising enzyme for use in cancer chemotherapy, and Salmonella strain SL 7838, which specifically targets tumors, may be used to deliver the prodrug-activating enzymes to tumors.

  View details for DOI 10.1158/1535-7163.MCT-05-0365

  View details for Web of Science ID 000234772900011

  View details for PubMedID 16432167

• Immunotherapy of primary ovarian cancer using autologous cytokine induced killer cells retargeted with bispecific antibodies: A preclinical study. 47th Annual Meeting of the American-Society-of-Hematology Chan, J. K., Hamilton, C. A., Cheung, M. K., Karimi, M., Baker, J., Gall, J. M., Lum, L. G., Schulz, S., Thorne, S. H., Teng, N. N., Contag, C. H., Negrin, R. S. AMER SOC HEMATOLOGY. 2005: 669A–670A

  View details for Web of Science ID 000233426004226

• Early treatment with CD4+ CD25+ regulatory T cells provides prolonged suppressive effects which control evolving but not established graft-versus-host-disease. 47th Annual Meeting of the American-Society-of-Hematology Nguyen, V. H., Zeiser, R., Dasilva, D., Contag, C. N., Negrin, R. S. AMER SOC HEMATOLOGY. 2005: 379A–379A

  View details for Web of Science ID 000233426002238

• Prevention of acute graft-versus-host disease despite compensatory function of lymphoid organs in vivo. 47th Annual Meeting of the American-Society-of-Hematology Beilhack, A., Schulz, S., Baker, J., Beilhack, G. F., Nishimura, R., Landan, G., Baker, E. M., Herman, E. I., BUTCHER, E. C., Contag, C. H., Negrin, R. S. AMER SOC HEMATOLOGY. 2005: 173A–173A

  View details for Web of Science ID 000233426001051

• In vivo effects of cyclosporine a, mycophenolate mofetile and rapamycin on adoptively transferred CD4(+)CD25(+) regulatory T cells in prevention of experimental acute graft-versus-host disease. 47th Annual Meeting of the American-Society-of-Hematology Zeiser, R., Nguyen, V., Beilhack, A., Baker, J., Schulz, S., Contag, C. H., Negrin, R. S. AMER SOC HEMATOLOGY. 2005: 136A–137A

  View details for Web of Science ID 000233426000453

• Ex vivo expanded dendritic cells home to T-cell zones of lymphoid organs and survive in vivo after allogeneic bone marrow transplantation AMERICAN JOURNAL OF PATHOLOGY Schimmelpfennig, C. H., Schulz, S., Arber, C., Baker, J., Tarner, I., McBride, J., Contag, C. H., Negrin, R. S. 2005; 167 (5): 1321-1331

  Abstract

  Little is known about adoptive transfer of allogeneic ex vivo expanded dendritic cells (eDCs). We investigated the trafficking pattern of eDCs in mice after allogeneic bone marrow transplantation by using bioluminescence imaging. eDCs were expanded from bone marrow precursors in the presence of GM-CSF, interleukin-4, and Flt3L and retrovirally transduced to express luciferase (luc) and green fluorescence protein (gfp). Flow cytometry showed polyclonal DC populations after expansion that consisted of CD11c+CD11b+ and CD11c-CD11b+ cells that co-expressed CD40, CD80, CD86, and MHCII. eDCs were functional in mixed lymphocyte reactions and produced tumor necrosis factor-alpha on phytohemagglutinin stimulation. The eDCs were then injected intravenously into BALB/c recipient mice that had received allogeneic bone marrow transplantation 6 weeks previously. On day 1 after transfer, eDCs were detected by bioluminescence imaging throughout the lungs and spleen. In the later course, signals were observed throughout thymus, lower abdomen, and spleen throughout a period of more than 42 days. Immunofluorescence microscopy confirmed CD11c positivity on the gfp+ donor cells, which localized in T-cell zones of mesenteric lymph nodes, Peyer's patches, spleen, and thymus. These findings are important for adoptive immunotherapies because they indicate that eDCs migrate efficiently in vivo and are capable of surviving long term.

  View details for PubMedID 16251416

• Hemin-activated macrophages home to the pancreas and protect from acute pancreatitis via heme oxygenase-1 induction JOURNAL OF CLINICAL INVESTIGATION Nakamichi, I., Habtezion, A., Zhong, B. H., Contag, C. H., BUTCHER, E. C., Omary, M. B. 2005; 115 (11): 3007-3014

  Abstract

  Hemin upregulates heme oxygenase-1 (HO-1), a stress-induced enzyme implicated in protection from a variety of injuries while its related isoform HO-2 is constitutively expressed. The role of hemin or HO-1 in the pancreas and their potential modulation of pancreatic injury are unknown. We show that HO-1 is induced in pancreatitis caused by caerulein and more prominently in severe pancreatitis caused by feeding a choline-deficient diet (CDD). Intraperitoneal hemin administration dramatically increases peritoneal and pancreas macrophages that overexpress HO-1 in association with pancreatic induction of the chemoattractants monocyte chemotactic protein-1 and macrophage inflammatory protein-1alpha but not RANTES or macrophage inflammatory protein-2. Hemin administration before CDD feeding protected 8 of 8 mice from lethality while 7 of 16 controls died. Protection is mediated by HO-1-overexpressing macrophages since hemin-primed macrophages home to the pancreas after transfer to naive mice and protect from CDD-induced pancreatitis. Suppression of hemin-primed peritoneal cell HO-1 using HO-1-specific small interfering RNA prior to cell transfer abolishes protection from CDD-induced pancreatitis. Similarly, hemin pretreatment in caerulein-induced pancreatitis reduces serum amylase and lipase, decreases pancreatic trypsin generation, and protects from lung injury. Therefore, hemin-like compounds or hemin-activated macrophages may offer novel therapeutic approaches for preventing acute pancreatitis and its pulmonary complication via upregulation of HO-1.

  View details for DOI 10.1172/JCI24912

  View details for Web of Science ID 000233022100011

  View details for PubMedID 16239966

  View details for PubMedCentralID PMC1257535

• Breast-milk shedding of drug-resistant HIV-1 subtype C in women exposed to single-dose nevirapine 11th Conference on Retroviruses and Opportunistic Infections Lee, E. J., Kantor, R., Zijenah, L., Sheldon, W., Emel, L., Mateta, P., Johnston, E., Wells, J., Shetty, A. K., Coovadia, H., Maldonado, Y., Jones, S. A., Mofenson, L. M., Contag, C. H., Bassett, M., Katzenstein, D. A. UNIV CHICAGO PRESS. 2005: 1260–64

  Abstract

  Single-dose nevirapine reduces intrapartum human immunodeficiency virus 1 type (HIV-1) transmission but may also select for nonnucleoside reverse-transcriptase inhibitor (NNRTI) resistance in breast milk (BM) and plasma. Among 32 Zimbabwean women, median 8-week postpartum plasma and BM HIV-1 RNA levels were 4.57 and 2.13 log(10) copies/mL, respectively. BM samples from women with laboratory-diagnosed mastitis (defined as elevated BM Na(+) levels) were 5.4-fold more likely to have HIV-1 RNA levels above the median. BM RT sequences were not obtained for 12 women with BM HIV-1 RNA levels below the lower limit of detection of the assay used. In 20 paired BM and plasma samples, 65% of BM and 50% of plasma RT sequences had NNRTI-resistance mutations, with divergent mutation patterns.

  View details for PubMedID 16136470

• Small hairpin RNAs efficiently inhibit hepatitis CIRES-mediated gene expression in human tissue culture cells and a mouse model MOLECULAR THERAPY Wang, Q., Contag, C. H., Ilves, H., Johnston, B. H., Kaspar, R. L. 2005; 12 (3): 562-568

  Abstract

  Treatment and prevention of hepatitis C virus (HCV) infections remain a major challenge for controlling this worldwide health problem; existing therapies are only partially effective and no vaccine is currently available. RNA interference offers the potential of a novel therapeutic approach for treating HCV infections. Toward this end, we evaluated small hairpin interfering RNAs (shRNAs) targeting the conserved internal ribosome entry site (IRES) element of the HCV genome for their ability to control gene expression in human cells and animals. We used a reporter gene plasmid in which firefly luciferase (fLuc) expression is dependent on the HCV IRES. Direct delivery of HCV IRES shRNAs efficiently blocked HCV IRES-mediated fLuc expression in transfected human 293FT cells as well as in a mouse model in which nucleic acids were delivered to liver cells by hydrodynamic transfection via the tail vein. These results indicate that shRNAs, delivered as RNA or expressed from viral or nonviral vectors, may be effective agents for the control of HCV and related viruses.

  View details for Web of Science ID 000231592400025

  View details for PubMedID 15953767

• In vivo visualization of cardiac allograft rejection and trafficking passenger leukocytes using bioluminescence imaging CIRCULATION Tanaka, M., Swijnenburg, R. J., Gunawan, F., Cao, Y. A., Yang, Y., Caffarelli, A. D., de Bruin, J. L., Contag, C. H., Robbins, R. C. 2005; 112 (9): I105-I110

  Abstract

  We investigated the feasibility of bioluminescence imaging (BLI) for the in vivo assessment of cardiac allograft viability and visualization of passenger leukocytes during the course of acute rejection.Hearts of FVB (H-2q) luciferase-green fluorescent protein transgenic mice (beta-actin promoter) or FVB luciferase transgenic mice (CD5 promoter) were heterotopically transplanted into either BALB/c (H-2d) or FVB recipients. Light intensity emitting from the recipient animals was measured daily by in vivo BLI until 12 days after transplantation. Graft beating score (0 to 4) was assessed by daily abdominal palpation until 12 days after transplantation. Inflammatory cell infiltration (CD45 stain) and structural changes of green fluorescent protein-positive cardiomyocytes were followed by immunohistochemistry. All cardiac allografts were acutely rejected by 12 days after transplantation. The intensity of light emitting from cardiac allografts declined 4 days after transplantation and correlated with graft beating scores (R2=0.91, P=0.02). Immunohistochemistry confirmed these results by showing an increase of CD45+ inflammatory cell infiltration and destruction of green fluorescent protein-positive cardiomyocytes in the cardiac allografts during acute rejection. In vivo BLI visualized migration and proliferation of CD5+ passenger leukocytes in both syngeneic and allogeneic recipients. In the allograft recipients, light signal from CD5+ passenger leukocytes peaked at 6 hours and diminished by 12 hours, whereas in the syngeneic recipients, the signal remained high until 10 days after transplantation.BLI is a useful modality for the quantitative assessment of in vivo cardiac graft viability and tracking of passenger leukocytes in vivo during the course of acute rejection.

  View details for DOI 10.1161/CIRCULATIONAHA.104.524777

  View details for Web of Science ID 000231741600016

  View details for PubMedID 16159800

• In vivo analyses of early events in acute graft-versus-host disease reveal sequential infiltration of T-cell subsets BLOOD Beilhack, A., Schulz, S., Baker, J., Beilhack, G. F., Wieland, C. B., Herman, E. I., Baker, E. M., Cao, Y. A., Contag, C. H., Negrin, R. S. 2005; 106 (3): 1113-1122

  Abstract

  Graft-versus-host disease (GVHD) is a major obstacle in allogeneic hematopoietic cell transplantation. Given the dynamic changes in immune cell subsets and tissue organization, which occur in GVHD, localization and timing of critical immunological events in vivo may reveal basic pathogenic mechanisms. To this end, we transplanted luciferase-labeled allogeneic splenocytes and monitored tissue distribution by in vivo bioluminescence imaging. High-resolution analyses showed initial proliferation of donor CD4+ T cells followed by CD8+ T cells in secondary lymphoid organs with subsequent homing to the intestines, liver, and skin. Transplantation of purified naive T cells caused GVHD that was initiated in secondary lymphoid organs followed by target organ manifestation in gut, liver, and skin. In contrast, transplanted CD4+ effector memory T (T(EM)) cells did not proliferate in secondary lymphoid organs in vivo and despite their in vitro alloreactivity in mixed leukocyte reaction (MLR) assays did not cause acute GVHD. These findings underline the potential of T-cell subsets with defined trafficking patterns for immune reconstitution without the risk of GVHD.

  View details for DOI 10.1182/blood-2005-02-0509

  View details for PubMedID 15855275

• Bioluminescence regenerative cycle (BRC) system: Theoretical considerations for nucleic acid quantification assays BIOPHYSICAL CHEMISTRY Hassibi, A., Contag, C., Vlad, M. O., Hafezi, M., Lee, T. H., Davis, R. W., Pourmand, N. 2005; 116 (3): 175-185

  Abstract

  A novel application of bioluminescence for nucleic acid quantification, the bioluminescence regenerative cycle (BRC), is described in theoretical terms and supported by preliminary experimental data. In the BRC system, pyrophosphate (PPi) molecules are released during biopolymerization and are counted and correlated to DNA copy number. The enzymes ATP-sulfurylase and firefly luciferase are employed to generate photons quantitatively from PPi. Enzymatic unity-gain positive feedback is implemented to amplify photon generation and to compensate for decay in light intensity by self-regulation. The cumulative total of photons can be orders of magnitude higher than in typical chemiluminescent processes. A system level theoretical model is developed, taking into account the kinetics of the regenerative cycle, contamination, and detector noise. Data and simulations show that the photon generation process achieves steady state for the time range of experimental measurements. Based on chain reaction theory, computations show that BRC is very sensitive to variations in the efficiencies of the chemical reactions involved and less sensitive to variations in the quantum yield of the process. We show that BRC can detect attomolar quantities of DNA (10(-18) mol), and that the useful dynamic range is five orders of magnitude. Sensitivity is not constrained by detector performance but rather by background bioluminescence caused by contamination by either PPi or ATP (adenosine triphosphate).

  View details for DOI 10.1016/j.bpc.2005.04.002

  View details for Web of Science ID 000230717400001

  View details for PubMedID 15882922

  View details for PubMedCentralID PMC2096776

• Molecular imaging using labeled donor tissues reveals patterns of engraftment, rejection, and survival in transplantation TRANSPLANTATION Cao, Y. A., Bachmann, M. H., Beilhack, A., Yang, Y., Tanaka, M., Swijnenburg, R. J., Reeves, R., Taylor-Edwards, C., Schulz, S., Doyle, T. C., Fathman, C. G., Robbins, R. C., Herzenberg, L. A., Negrin, R. S., Contag, C. H. 2005; 80 (1): 134-139

  Abstract

  Tissue regeneration and transplantation of solid organs involve complex processes that can only be studied in the context of the living organism, and methods of analyzing these processes in vivo are essential for development of effective transplantation and regeneration procedures. We utilized in vivo bioluminescence imaging (BLI) to noninvasively visualize engraftment, survival, and rejection of transplanted tissues from a transgenic donor mouse that constitutively expresses luciferase. Dynamic early events of hematopoietic reconstitution were accessible and engraftment from as few as 200 transplanted whole bone marrow (BM) cells resulted in bioluminescent foci in lethally irradiated, syngeneic recipients. The transplantation of autologous pancreatic Langerhans islets and of allogeneic heart revealed the tempo of transplant degeneration or immune rejection over time. This imaging approach is sensitive and reproducible, permits study of the dynamic range of the entire process of transplantation, and will greatly enhance studies across various disciplines involving transplantation.

  View details for DOI 10.1097/01.TP.0000164347.50559.A3

  View details for Web of Science ID 000230473800023

  View details for PubMedID 16003245

• Monitoring age-related susceptibility of young mice to oral Salmonella enterica serovar typhimurium infection using an in vivo murine model PEDIATRIC RESEARCH Burns-Guydish, S. M., Olomu, I. N., Zhao, H., Wong, R. J., Stevenson, D. K., Contag, C. H. 2005; 58 (1): 153-158

  Abstract

  Neonates and young children are acutely susceptible to infections by gastrointestinal bacterial pathogens, such as Salmonella enterica serovar Typhimurium (S. typhimurium). To reveal age-related differences in susceptibility to this pathogen, we used in vivo bioluminescence imaging (BLI) to monitor the progression of infection in neonatal (1-wk-old), suckling (2-wk-old), juvenile (4-wk-old), and adult (6-wk-old) BALB/c mice. Mice were orally infected with various doses of a bioluminescent-labeled wild-type or mutant S. typhimurium strain, and progression of infection was monitored by BLI for 2 wks. We found that neonatal and suckling mice were more susceptible to the wild-type strain at inoculum sizes 4 and 2 log(10)'s lower for neonatal and suckling mice, respectively, than those for adult mice. At the lower inocula, newborn mice showed disseminated systemic infection as indicated by the pattern of photon emission assessed by BLI, whereas no bioluminescent signals were detectable in adult mice. In addition, an orgA(-) mutant strain of S. typhimurium with reduced virulence in adult mice produced systemic infection in newborn, suckling, and juvenile mice. Furthermore, as low as 3 log(10) CFU could be detected by BLI in tissue. The present study demonstrates that susceptibility to S. typhimurium infection decreases with age. Also, we established that BLI can be used to monitor the progression of infection in mice. Thus, this model of age-related susceptibility to S. typhimurium using BLI can be used to advance our understanding of the mechanisms involved in newborn susceptibility to infection.

  View details for DOI 10.1203/01.PDR.0000157725.44213.C4

  View details for PubMedID 15774831

• Real-time in vivo imaging of stem cells following transgenesis by transposition. Molecular therapy : the journal of the American Society of Gene Therapy Tolar, J., Osborn, M., Bell, S., McElmurry, R., Xia, L., Riddle, M., Panoskaltsis-Mortari, A., Jiang, Y., McIvor, R. S., Contag, C. H., Yant, S. R., Kay, M. A., Verfaillie, C. M., Blazar, B. R. 2005; 12 (1): 42-48

  Abstract

  Previous studies have identified Sleeping Beauty transposons as efficient vectors for nonviral gene delivery in mammalian cells. However, studies demonstrating the usefulness of transposons as gene delivery vehicles into adult stem cells are lacking. Multipotent adult progenitor cells (MAPC) are nonhematopoietic stem cells with the capacity to form most, if not all, cell types of the body and as such hold great therapeutic potential. The whole-body biodistribution and persistence of MAPC are unknown, and such data would help direct clinical applications. We have nucleofected murine MAPC with two plasmid-based Sleeping Beauty transposons encoding the red fluorescent protein (DsRed2) and firefly luciferase. Transgenic euploid MAPC clones maintained their characteristic multilineage differentiation potential in vitro. DsRed2 and luciferase expression allowed for MAPC detection in vivo and in tissue sections. To confirm that transgenesis occurred by transposition into the genome of MAPC, we mapped Sleeping Beauty transposon integration sites in two MAPC clones using splinkerette PCR. This novel dual-reporter imaging approach based on the transgenesis of MAPC with Sleeping Beauty transposons sheds light on the homing patterns of MAPC and paves the way for quantification of MAPC engraftment in real time in vivo.

  View details for PubMedID 15963919

• Real-time in vivo imaging transgenesis by of stem cells following transposition MOLECULAR THERAPY Tolar, J., Osborn, M., Bell, S., McElmurry, R., Xia, L., Riddle, M., Panoskaltsis-Mortari, A., Jiang, Y. H., McIvor, R. S., Contag, C. H., Yant, S. R., Kay, M. A., Verfaillie, C. M., Blazar, B. R. 2005; 12 (1): 42-48

  Abstract

  Previous studies have identified Sleeping Beauty transposons as efficient vectors for nonviral gene delivery in mammalian cells. However, studies demonstrating the usefulness of transposons as gene delivery vehicles into adult stem cells are lacking. Multipotent adult progenitor cells (MAPC) are nonhematopoietic stem cells with the capacity to form most, if not all, cell types of the body and as such hold great therapeutic potential. The whole-body biodistribution and persistence of MAPC are unknown, and such data would help direct clinical applications. We have nucleofected murine MAPC with two plasmid-based Sleeping Beauty transposons encoding the red fluorescent protein (DsRed2) and firefly luciferase. Transgenic euploid MAPC clones maintained their characteristic multilineage differentiation potential in vitro. DsRed2 and luciferase expression allowed for MAPC detection in vivo and in tissue sections. To confirm that transgenesis occurred by transposition into the genome of MAPC, we mapped Sleeping Beauty transposon integration sites in two MAPC clones using splinkerette PCR. This novel dual-reporter imaging approach based on the transgenesis of MAPC with Sleeping Beauty transposons sheds light on the homing patterns of MAPC and paves the way for quantification of MAPC engraftment in real time in vivo.

  View details for DOI 10.1016/j.ymthe.2005.02.023

  View details for Web of Science ID 000230282200011

• Emission spectra of bioluminescent reporters and interaction with mammalian tissue determine the sensitivity of detection in vivo JOURNAL OF BIOMEDICAL OPTICS Zhao, H., Doyle, T. C., Coquoz, O., Kalish, F., Rice, B. W., Contag, C. H. 2005; 10 (4)

  Abstract

  In vivo bioluminescence imaging depends on light emitted by luciferases in the body overcoming the effect of tissue attenuation. Understanding this relationship is essential for detection and quantification of signal. We have studied four codon optimized luciferases with different emission spectra, including enzymes from firefly (FLuc), click beetle (CBGr68, CBRed) and Renilla reniformins (hRLuc). At 25 degrees C, the in vitro lambda(max) of these reporters are 578, 543, 615, and 480 nm, respectively; at body temperature, 37 degrees C, the brightness increases and the firefly enzyme demonstrates a 34-nm spectral red shift. Spectral shifts and attenuation due to tissue effects were evaluated using a series of 20-nm bandpass filters and a cooled charge-coupled device (CCD) camera. Attenuation increased and the spectra of emitted light was red shifted for signals originating from deeper within the body relative to superficial origins. The tissue attenuation of signals from CBGr68 and hRLuc was greater than from those of Fluc and CBRed. To further probe tissue effects, broad spectral emitters were created through gene fusions between CBGr68 and CBRed. These resulted in enzymes with broader emission spectra, featuring two peaks whose intensities are differentially affected by temperature and tissue depth. These spectral measurement data allow for improved understanding of how these reporters can be used in vivo and what they can reveal about biological processes in living subjects.

  View details for DOI 10.1117/1.2032388

  View details for Web of Science ID 000232799200010

  View details for PubMedID 16178634

• Global analysis of Smad2/3-dependent TGF-beta signaling in living mice reveals prominent tissue-specific responses to injury JOURNAL OF IMMUNOLOGY Lin, A. H., Luo, J., Mondshein, L. H., ten Dijke, P., Vivien, D., Contag, C. H., Wyss-Coray, T. 2005; 175 (1): 547-554

  Abstract

  Smad2 and Smad3 (Smad2/3) proteins are key signaling molecules for TGF-beta and some related family members regulating the transcription of several hundred genes. TGF-beta have key roles in development, tissue homeostasis, and the pathogenesis of many human diseases, including cancer, fibrotic disorders, developmental defects, and neurodegeneration. To study the temporal and spatial patterns of Smad2/3-dependent signaling in normal and pathological conditions in the living organism, we engineered transgenic mice with a Smad-responsive luciferase reporter construct (SBE-luc mice). Using bioluminescent imaging, we assessed Smad2/3 signaling activity noninvasively in living mice. At baseline, this activity was highest in brain, intestine, heart, and skin, and correlated with biochemical measurements of reporter activity. Primary astrocytes cultured from SBE-luc mice showed specific activation of the reporter in response to Smad2/3-activating TGF-beta family members. Treatment of mice with the endotoxin LPS resulted in a fast and vigorous, but transient activation of the reporter in the intestine. Although the response was similarly rapid in brain, it remained increased, indicating important but different cellular responses to endotoxin challenge in these organs. Traumatic brain injury with a needle stab resulted in local activation of Smad2/3-dependent genes and a severalfold increase in bioluminescence in living mice. SBE-luc mice can therefore be used to study temporal, tissue-specific activation of Smad2/3-dependent signaling in living mice in normal or pathological conditions as well as for the identification of endogenous or synthetic modulators of this pathway.

  View details for Web of Science ID 000230050900069

  View details for PubMedID 15972691

• Low levels of Her2/neu expressed by Ewing's family tumor cell lines can redirect cytokine-induced killer cells CLINICAL CANCER RESEARCH Verneris, M. R., Arshi, A., Edinger, M., Kornacker, M., Natkunam, Y., Karami, M., Cao, Y. A., Marina, N., Contag, C. H., Negrin, R. S. 2005; 11 (12): 4561-4570

  Abstract

  To identify novel treatments for pediatric solid tumors and/or for malignancies with low-level Her2/neu expression.Using fluorescence-activated cell sorting and immunohistochemistry, Her2/neu expression was determined on cell lines derived vfrom Ewing's family tumors (EFT) and neuroblastoma. Sensitivity to trastuzumab treatment was investigated using an in vitro proliferation assay. Cytotoxicity against EFT cell lines was done with either freshly isolated or ex vivo activated and expanded T cells (cytokine-induced killer cells, CIK cells), with or without addition of a CD3xHer2/neu bispecific antibody. The effects of either trastuzumab, CIK cells alone, or CD3xHer2/neu bispecific antibody redirected CIK cells was determined using a SCID/hu model of EFTs and serial, noninvasive bioluminescent imaging.EFT cell lines express 5- to 10-fold lower levels of her2/neu than either breast (BT-474) or ovarian (SK-OV-3) cell lines. Treatment of EFT cell lines with trastuzumab did not induce growth inhibition either in vitro or in vivo. In contrast, Her2/neu could be used to redirect CIK cell to mediate cytotoxicity against EFTs both in vitro and in vivo (using two different treatment schemas).CD3xHer2/neu bispecific antibody and CIK cells may be a suitable approach to treat malignancies with low-level Her2/neu expression not responsive to trastuzumab.

  View details for Web of Science ID 000229725900037

  View details for PubMedID 15958642

• Adoptive transfer of mast cells does not enhance the impaired survival of Kit(W)/Kit(W-v) mice in a model of low dose intraperitoneal infection with bioluminescent Salmonella typhimurium IMMUNOLOGY LETTERS Chatterjea, D., Burns-Guydish, S. M., Sciuto, T. E., Dvorak, A., Contag, C. H., Galli, S. J. 2005; 99 (1): 122-129

  Abstract

  Mast cells are important effector cells in IgE-associated immune responses, but also can contribute to host defense in certain examples of bacterial infection. We found that genetically mast cell-deficient WBB6F1-Kit(W)/Kit(W-v) mice exhibited more bacterial CFUs per spleen by 6 days after intraperitoneal injection of bioluminescent Salmonella typhimurium, and died more rapidly after infection, than did the congenic WBB6F1-Kit(+/+) wild type mice. Adoptive transfer of bone marrow-derived cultured mast cells of Kit(+/+) origin to the peritoneal cavity of Kit(W)/Kit(W-v) mice resulted in engraftment of mast cells in the peritoneal cavity and mesentery of the recipient mice, and the development of large numbers of mast cells in the spleen. However, such mast cell-engrafted Kit(W)/Kit(W-v) mice appeared sicker after intraperitoneal injection with S. typhimurium than did mast cell-deficient Kit(W)/Kit(W-v) mice, and exhibited numbers of CFUs of bacteria per spleen, and a survival curve, that were not significantly different than those of Kit(W)/Kit(W-v) mice. These results, when taken together with prior studies investigating the roles of mast cells in innate immunity, strongly suggest that whether mast cells can be shown to have a significant role in enhancing survival during bacterial infections may depend critically on the details of the particular experimental systems examined.

  View details for DOI 10.1016/j.imlet.2005.02.015

  View details for Web of Science ID 000229659300019

  View details for PubMedID 15894120

• Using in vivo bioluminescence imaging to shed light on cancer biology PROCEEDINGS OF THE IEEE Thorne, S. H., Contag, C. H. 2005; 93 (4): 750-762

  View details for DOI 10.1109/JPROC.2005.844261

  View details for Web of Science ID 000227956600004

• Bone morphogenetic protein 2 and retinoic acid accelerate in vivo bone formation, osteoclast recruitment, and bone turnover TISSUE ENGINEERING Cowan, C. M., Aalami, O. O., Shi, Y. Y., Chou, Y. F., Mari, C., Thomas, R., Quarto, N., Nacamuli, R. P., Contag, C. H., Wu, B., Longaker, M. T. 2005; 11 (3-4): 645-658

  Abstract

  Reconstruction of craniofacial defects presents a substantial biomedical burden, and requires complex surgery. Interestingly, children after age 2 years and adults are unable to heal large skull defects. This nonhealing paradigm provides an excellent model system for craniofacial skeletal tissueengineering strategies. Previous studies have documented the in vivo osteogenic potential of adipose-derived stromal (ADS) cells and bone marrow-derived stromal (BMS) cells. This study investigates the ability to accelerate in vivo osteogenesis on ex vivo recombinant human bone morphogenetic protein 2 (BMP-2) and retinoic acid stimulation. Mouse osteoblasts, ADS cells, and BMS cells were seeded onto apatite-coated PLGA scaffolds, stimulated with rhBMP-2 and retinoic acid ex vivo for 4 weeks, and subsequently implanted into critically sized (4 mm) calvarial defects. Samples were harvested after 2, 4, 8, and 12 weeks. Areas of complete bony bridging were noted as early as 2 weeks in vivo; however, osteoclasts were attracted to the scaffold as identified by calcitonin receptor staining and tartrate-resistant acid phosphatase activity staining. Although the optimal method of in vitro osteogenic priming for mesenchymal cells remains unknown, these results provide evidence that BMP-2 and retinoic acid stimulation of multipotent cells ex vivo can subsequently induce significant quantities of bone formation within a short time period in vivo.

  View details for PubMedID 15869441

• In vivo spatial and temporal analyses in mice reveal redundancy of lymphoid tissues in inducing acute GVHD after allogeneic hematopoietic cell transplantation Joint Meeting of the American-Society-for-Blood-and-Marrow-Transplantation/Center-for-International-Blood-and-Marrow-Transplant-Research Beilhack, A., Schulz, S., Baker, J., Beilhack, G. F., Wieland, C. B., Baker, E. M., Herman, E. I., Landan, G., Olson, J. A., Cao, Y. A., Contag, C. H., Negrin, R. S. ELSEVIER SCIENCE INC. 2005: 35–35

  View details for Web of Science ID 000227329000100

• In vivo trafficking of CD4+CD25+ regulatory T-cells in allogeneic recipients using bioluminescence imaging Joint Meeting of the American-Society-for-Blood-and-Marrow-Transplantation/Center-for-International-Blood-and-Marrow-Transplant-Research Nguyen, V. H., Wieland, C., Contag, C., Negrin, R. ELSEVIER SCIENCE INC. 2005: 13–13

  View details for Web of Science ID 000227329000036

• Cellular tolerance to pulsed heating Conference on Optical Interactions with Tissue and Cells XVI Simanovskii, D., Sarkar, M., Irani, A., O'Connell-Rodwell, C., Contag, C., SCHWETTMAN, A., Palanker, D. SPIE-INT SOC OPTICAL ENGINEERING. 2005: 254–259

  View details for DOI 10.1117/12.601774

  View details for Web of Science ID 000229361500030

• Comparison between PET and bioluminescence imaging for quantitative assessment of tumor burden 47th Annual Meeting of the American-Society-for-Therapeutic-Radiology-and-Oncology Maxim, P. G., Thorndyke, B. R., Dandekar, M., Mayer, D., Contag, C. H., Gambhir, S. S., Xing, L. ELSEVIER SCIENCE INC. 2005: S490–S490

  View details for Web of Science ID 000232083301348

• T cell trafficking in acute GVHD: In vivo bioluminescence evaluation to elucidate the role of different lymphatic organs. 46th Annual Meeting of the American-Society-of-Hematology Beilhack, A., Schulz, S., Baker, J., Beilhack, G. F., Wieland, C. B., Baker, E. M., Herman, E. I., Landan, G., Kime, J. A., Cao, Y. A., Contag, C. H., Negrin, R. S. AMER SOC HEMATOLOGY. 2004: 171A–171A

  View details for Web of Science ID 000225127500596

• Real-time in vivo biodistribution of multipotent adult progenitor cells (MAPC): Role of the immune system in MAPC resistance in non-transplanted and bone marrow transplanted mice. 46th Annual Meeting of the American-Society-of-Hematology Tolar, J., Bell, S., McElmurry, R., Xia, L. L., McIvor, R. S., Yam, S. R., Kay, M. A., Contag, C. H., Verfaillie, C. M., Blazar, B. R. AMER SOC HEMATOLOGY. 2004: 147A–148A

  View details for Web of Science ID 000225127500510

• Transgenesis of multipotent adult progenitor cells (MAPC) with sleeping beauty transposons to determine MAPC homing and persistence in real-time in vivo. 46th Annual Meeting of the American-Society-of-Hematology Tolar, J., Osborn, M., Bell, S., Xia, L., Riddle, M., Panoskaltsis-Mortari, A., McIvor, S., Stephen, Y. R., Kay, M. A., Contag, C. H., Verfaillie, C. M., Blazar, B. R. AMER SOC HEMATOLOGY. 2004: 577A–578A

  View details for Web of Science ID 000225127502100

• In vivo visualization of cardiac allograft rejection and trafficking passenger leukocytes using bioluminescence imaging 77th Scientific Meeting of the American-Heart-Association Tanaka, M., Swijnenburg, R. J., Gunawan, F., Cao, Y. A., Kofidis, T., Contag, C. H., Robbins, R. C. LIPPINCOTT WILLIAMS & WILKINS. 2004: 585–85

  View details for Web of Science ID 000224783503173

• Tumor imaging using a standardized radiolabeled adapter protein docked to vascular endothelial growth factor JOURNAL OF NUCLEAR MEDICINE Blankenberg, F. G., Mandl, S., Cao, Y. A., O'Connell-Rodwell, C., Contag, C., Mari, C., Gaynutdinov, T. I., Vanderheyden, J. L., Backer, M. V., Backer, J. M. 2004; 45 (8): 1373-1380

  Abstract

  Direct radiolabeling of proteins can result in the loss of targeting activity, requires highly customized procedures, and yields heterogeneous products. Here we describe a novel imaging complex comprised of a standardized (99m)Tc-radiolabeled adapter protein noncovalently bound to a "Docking tag" fused to a "Targeting protein". The assembly of this complex is based on interactions between human 109-amino acid (HuS) and 15-amino acid (Hu-tag) fragments of ribonuclease I, which serve as an "Adapter protein" and a Docking tag, respectively.HuS modified with hydrazinonicotinamide (HYNIC) was radiolabeled using (99m)Tc-tricine to a specific activity of 3.4-7.4 MBq/microg. Protein complexes were then formed by mixing (99m)Tc-HuS with equimolar amounts of either Hu-tagged VEGF(121) (Hu-VEGF [vascular endothelial growth factor]) or Hu-tagged anti-VEGFR-2 single-chain antibody (Hu-P4G7) and incubating on ice for 15 min. 4T1 luc/gfp luciferase-expressing murine mammary adenocarcinoma cells (1 x 10(4)) were implanted subcutaneously or injected intravenously into BALB/c mice. Bioluminescent imaging (BLI) was performed 10 d later. Immediately after BLI visualization of tumor, 18.5-37 MBq of tracer (5-10 microg of protein) were injected via tail vein. One hour later planar or SPECT images were obtained, followed by killing the mice.There was significantly (P = 0.0128) increased uptake of (99m)Tc-HuS/Hu-VEGF (n = 10) within subcutaneous tumor as compared with (99m)Tc-HuS/Hu-P4G7 (n = 5) at biodistribution assay (2.68 +/- 0.75 vs. 1.8 +/- 0.21; tumor-to-subcutaneous tissue [ratio of specific activities], respectively), despite similar molecular weights. The focal (99m)Tc-HuS/Hu-VEGF uptake seen on planar images (3.44 +/- 1.16 [tumor to soft-tissue background]) corresponded directly to the locations of tumor observed by BLI. Region of interest analyses of SPECT images revealed a significant increase of (99m)Tc-HuS/Hu-VEGF (n = 5) within the lungs with BLI-detectable pulmonary tumor nodules as compared with controls (n = 4) (right: 4.47 +/- 2.07 vs. 1.79 +/- 0.56; left: 3.66 +/- 1.65 vs. 1.62 +/- 0.45, tumor lung [counts/pixel]/normal lung [counts/pixel], respectively).(99m)Tc-HuS/Hu-VEGF complex is stable for at least 1 h in vivo and can be effectively used to image mouse tumor neovasculature in lesions as small as several millimeters in soft tissue. We expect that a similar approach can be adapted for in vivo delivery of other targeting proteins of interest without affecting their bioactivity.

  View details for PubMedID 15299064

• Confocal fluorescence microscope with dual-axis architecture and biaxial postobjective scanning JOURNAL OF BIOMEDICAL OPTICS Wang, T. D., Contag, C. H., Mandella, M. J., Chan, N. Y., Kino, G. S. 2004; 9 (4): 735-742

  Abstract

  We present a novel confocal microscope that has dual-axis architecture and biaxial postobjective scanning for the collection of fluorescence images from biological specimens. This design uses two low-numerical-aperture lenses to achieve high axial resolution and long working distance, and the scanning mirror located distal to the lenses rotates along the orthogonal axes to produce arc-surface images over a large field of view (FOV). With fiber optic coupling, this microscope can potentially be scaled down to millimeter dimensions via microelectromechanical systems (MEMS) technology. We demonstrate a benchtop prototype with a spatial resolution < or =4.4 microm that collects fluorescence images with a high SNR and a good contrast ratio from specimens expressing GFP. Furthermore, the scanning mechanism produces only small differences in aberrations over the image FOV. These results demonstrate proof of concept of the dual-axis confocal architecture for in vivo molecular and cellular imaging.

  View details for Web of Science ID 000222865800008

  View details for PubMedID 15250760

• Apoptosis in a rodent model of cranial suture fusion: In situ imaging and gene expression analysis PLASTIC AND RECONSTRUCTIVE SURGERY Fong, K. D., Song, H. M., Nacamuli, R. P., Franc, B. L., Mari, C., Fang, T. D., Warren, S. M., Contag, C. H., Blankenberg, F. G., Longaker, M. T. 2004; 113 (7): 2037-2047

  Abstract

  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.

  View details for DOI 10.1097/01.prs.000012118201199.c1

  View details for PubMedID 15253194

• Adipose-derived adult stromal cells heal critical-size mouse calvarial defects NATURE BIOTECHNOLOGY Cowan, C. M., Shi, Y. Y., Aalami, O. O., Chou, Y. F., Mari, C., Thomas, R., Quarto, N., Contag, C. H., Wu, B., Longaker, M. T. 2004; 22 (5): 560-567

  Abstract

  In adults and children over two years of age, large cranial defects do not reossify successfully, posing a substantial biomedical burden. The osteogenic potential of bone marrow stromal (BMS) cells has been documented. This study investigates the in vivo osteogenic capability of adipose-derived adult stromal (ADAS) cells, BMS cells, calvarial-derived osteoblasts and dura mater cells to heal critical-size mouse calvarial defects. Implanted, apatite-coated, PLGA scaffolds seeded with ADAS or BMS cells produced significant intramembranous bone formation by 2 weeks and areas of complete bony bridging by 12 weeks as shown by X-ray analysis, histology and live micromolecular imaging. The contribution of implanted cells to new bone formation was 84-99% by chromosomal detection. These data show that ADAS cells heal critical-size skeletal defects without genetic manipulation or the addition of exogenous growth factors.

  View details for DOI 10.1038/nbt958

  View details for PubMedID 15077117

• Site-specific integration enhances expression of DNA introduced into skeletal muscle 7th Annual Meeting of the American-Society-of-Gene-Therapy Wheeler, T. M., Jarrahian, S., Bertoni, C., Olivares, E. C., Doyle, T., Contag, C. H., Calos, M. P., Rando, T. A. NATURE PUBLISHING GROUP. 2004: S307–S307

  View details for Web of Science ID 000222316600811

• Dynamics of heme oxygenase-1 expression in infection of young mice Annual Meeting of the Pediatric-Academic-Societies Burns, S. M., Wong, R. J., Contag, C. H., Stevenson, D. K. NATURE PUBLISHING GROUP. 2004: 472A–472A

  View details for Web of Science ID 000220591102744

• In vivo bioluminescence imaging for integrated studies of infection CELLULAR MICROBIOLOGY Doyle, T. C., Burns, S. M., Contag, C. H. 2004; 6 (4): 303-317

  Abstract

  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.

  View details for DOI 10.1111/j.1462-5822.2004.00378.x

  View details for Web of Science ID 000220006600001

  View details for PubMedID 15009023

• Role of CpG island methylation in the developmental regulation of heme oxygenase-1 expression in the developing mouse brain Annual Meeting of the Pediatric-Academic-Societies Zhao, H., Wong, R. J., Mizobuchi, M., Contag, C. H., Stevenson, D. K. NATURE PUBLISHING GROUP. 2004: 422A–422A

  View details for Web of Science ID 000220591102464

• Expression of heme oxygenase-1 in response to infection in young mice Experimental Biology 2004 Annual Meeting Burns, S. M., Wong, R. J., Stevenson, D. K., Contag, C. H. FEDERATION AMER SOC EXP BIOL. 2004: A1312–A1312

  View details for Web of Science ID 000220470702642

• Tissue-specific effects of dexamethasone on heme oxygenase-1 expression Experimental Biology 2004 Annual Meeting Wong, R. J., Mizobuchi, M., Zhao, H., Abate, A., Vreman, H. J., Contag, C. H., Stevenson, D. K. FEDERATION AMER SOC EXP BIOL. 2004: A1311–A1311

  View details for Web of Science ID 000220470702638

• The E47 transcription factor negatively regulates CD5 expression during thymocyte development PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Yang, Y., Contag, C. H., Felsher, D., Shachaf, C. M., Cao, Y., Herzenberg, L. A., Herzenberg, L. A., Tung, J. W. 2004; 101 (11): 3898-3902

  Abstract

  The expression of CD5 increases progressively as thymocytes mature. We have shown that CD5 expression is controlled by a tissue-specific regulatory promoter located upstream of the CD5 translation start sites. Deletion of this regulatory promoter, which contains three potential transcription factor binding sites (CCAAT, kappa E2, and ets) reduces the promoter activity to basal level. Of these sites, only ets proved essential for CD5 expression in T cell lines. Here, we introduce a role for the E47 transcription factor and the CD5 promoter kappa E2 site in regulating CD5 expression during thymocyte development. Using T cell lines, we show that (i) mutation of the kappa E2 site in the CD5 regulatory promoter results in a significant elevation of CD5 promoter activity; (ii) the E47 transcription factor binds to the kappa E2 site; and (iii) overexpression of E47 inhibits CD5 expression. We then show, in high-dimensional fluorescence-activated cell sorting studies with primary thymocytes at successive developmental stages, that (i) intracellular E47 levels decrease as surface CD5 expression increases; (ii) E47 expression is down-regulated and CD5 expression is correspondingly up-regulated in DN3 thymocytes in RAG-2-deficient mice injected with anti-CD3 to mimic pre-T cell receptor stimulation; and (iii) E47 expression is down-regulated and CD5 expression is up-regulated when double positive thymocytes are stimulated in vitro with anti-CD3. Based on these data, we propose that E47 negatively regulates CD5 expression by interacting with the kappa E2 site in the CD5 regulatory promoter and that decreases in E47 in response to developmental signals are critical to the progressive increase in CD5 expression as thymocytes mature.

  View details for DOI 10.1073/pnas.0308764101

  View details for Web of Science ID 000220314500034

  View details for PubMedID 15001710

  View details for PubMedCentralID PMC374341

• A genetic reporter of thermal stress defines physiologic zones over a defined temperature range FASEB JOURNAL O'Connell-Rodwell, C. E., Shriver, D., Simanovskii, D. M., Mcclure, C., Cao, Y. A., Zhang, W. S., Bachmann, M. H., Beckham, J. T., Jansen, E. D., Palanker, D., Schwettman, H. A., Contag, C. H. 2004; 18 (2): 264-271

  Abstract

  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.

  View details for DOI 10.1096/fj.03-0585com

  View details for Web of Science ID 000220425000006

  View details for PubMedID 14769820

• p47(phox) deficiency impairs NF-kappa B activation and host defense in Pseudomonas pneumonia JOURNAL OF IMMUNOLOGY Sadikot, R. T., Zeng, H., Yull, F. E., Li, B., Cheng, D. S., Kernodle, D. S., Jansen, E. D., Contag, C. H., Segal, B. H., Holland, S. M., Blackwell, T. S., Christman, J. W. 2004; 172 (3): 1801-1808

  Abstract

  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.

  View details for Web of Science ID 000188378700057

  View details for PubMedID 14734763

• The role of Peyer's patches and mesenteric lymph nodes in acute GVHD: A study guided by bioluminescence imaging in vivo Tandem BMT 2004 Meeting Beilback, A., Schulz, S., Wieland, C. B., Beilback, G. F., Baker, J., Cao, Y. A., Herman, E. I., Baker, E. M., Contag, C. H., Negrin, R. S. ELSEVIER SCIENCE INC. 2004: 5–5

  View details for Web of Science ID 000188877500003

• Characterization of coelenterazine analogs for measurements of Renilla luciferase activity in live cells and living animals. Molecular imaging Zhao, H., Doyle, T. C., Wong, R. J., Cao, Y., Stevenson, D. K., Piwnica-Worms, D., Contag, C. H. 2004; 3 (1): 43-54

  Abstract

  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.

  View details for PubMedID 15142411

• Assessment of cellular response to thermal laser injury through bioluminescence imaging of heat shock protein 70 Photonics West Conference 2003 Beckham, J. T., Mackanos, M. A., Crooke, C., Takahashi, T., O'Connell-Rodwell, C., Contag, C. H., Jansen, E. D. AMER SOC PHOTOBIOLOGY. 2004: 76–85

  Abstract

  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.

  View details for Web of Science ID 000188259900011

  View details for PubMedID 14974719

• Performance of dual axes confocal microscope for in vivo molecular and cellular imaging Conference on 3-Dimensional and Multidimensional Microscopy - Image Acquisition and Processing XI Kino, G., Wang, T., Contag, C., Mandella, M., Chan, N. SPIE-INT SOC OPTICAL ENGINEERING. 2004: 35–46

  View details for DOI 10.1117/12.530016

  View details for Web of Science ID 000223297800005

• Role of CpG island methylation in the transcriptional regulation of heme oxygenase-1 in the developing brain. Western Regional Meeting of the American-Federation-for-Medical-Research Zhao, H., Wong, R. J., Mizobuchi, M., Stevenson, D. K., Contag, C. H. LIPPINCOTT WILLIAMS & WILKINS. 2004: S171–S172

  View details for Web of Science ID 000188254600550

• Response of heme oxygenase-1 to infection in young mice. Western Regional Meeting of the American-Federation-for-Medical-Research Burns, S. M., Wong, R. J., Stevenson, D. K., Contag, C. H. LIPPINCOTT WILLIAMS & WILKINS. 2004: S123–S123

  View details for Web of Science ID 000188254600271

• Multi-modality imaging identifies key times for annexin V imaging as an early predictor of therapeutic outcome. Molecular imaging Mandl, S. J., Mari, C., Edinger, M., Negrin, R. S., Tait, J. F., Contag, C. H., Blankenberg, F. G. 2004; 3 (1): 1-8

  Abstract

  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.

  View details for PubMedID 15142407

• In vivo visualization of the spatial and temporal events in bone marrow engraftment and graft vs. host disease induction by bioluminescence. 45th Annual Meeting and Exhibition of the American-Society-of-Hematology Beilhack, A., Schulz, S., Wieland, C. B., Beilhack, G. F., Cao, Y. A., Baker, J., Herman, E. I., Baker, E. M., Contag, C. H., Negrin, R. S. AMER SOC HEMATOLOGY. 2003: 944A–944A

  View details for Web of Science ID 000186536703518

• Survival and homing of ex vivo expanded donor derived dendritic cells after allogeneic BMT. 45th Annual Meeting and Exhibition of the American-Society-of-Hematology Schimmelpfennig, C. H., Schulz, S., Arber, C., Baker, J., Tarner, I. H., Mcbride, J. M., Fathman, C. G., Contag, C. H., Negrin, R. S. AMER SOC HEMATOLOGY. 2003: 695A–695A

  View details for Web of Science ID 000186536702572

• In vivo trafficking of activated T cells in allogeneic recipients using bioluminescent imaging. 45th Annual Meeting and Exhibition of the American-Society-of-Hematology Wieland, C. B., Beilhack, A., Baker, J., Cao, Y. A., Herman, E., Baker, E., Contag, C. H., Negrin, R. S. AMER SOC HEMATOLOGY. 2003: 948A–948A

  View details for Web of Science ID 000186536703532

• In vivo dynamics of hematopoietic reconstitution from purified stem and progenitor cells. 45th Annual Meeting and Exhibition of the American-Society-of-Hematology Wagers, A. J., Cao, Y. A., Beilhack, A., Dusich, J., Bachmann, M. H., Negrin, R. S., Weissman, I. L., Contag, C. H. AMER SOC HEMATOLOGY. 2003: 332A–332A

  View details for Web of Science ID 000186536701196

• Dual-axes confocal microscopy with post-objective scanning and low-coherence heterodyne detection OPTICS LETTERS Wang, T. D., Contag, C. H., Mandella, M. J., Chan, N. Y., Kino, G. S. 2003; 28 (20): 1915-1917

  Abstract

  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.

  View details for Web of Science ID 000185746300019

  View details for PubMedID 14587774

• Evaluation of effector cell fate and function by in vivo bioluminescence imaging METHODS Edinger, M., Hoffmann, P., Contag, C. H., Negrin, R. S. 2003; 31 (2): 172-179

  Abstract

  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.

  View details for DOI 10.1016/S1046-2023(03)00127-0

  View details for Web of Science ID 000185379400009

  View details for PubMedID 12957575

• Breaching biological barriers: protein translocation domains as tools for molecular imaging and therapy. Molecular imaging Franc, B. L., Mandl, S. J., Siprashvili, Z., Wender, P., Contag, C. H. 2003; 2 (4): 313-323

  Abstract

  The lipid bilayer of a cell presents a significant barrier for the delivery of many molecular imaging reagents into cells at target sites in the body. Protein translocation domains (PTDs) are peptides that breach this barrier. Conjugation of PTDs to imaging agents can be utilized to facilitate the delivery of these agents through the cell wall, and in some cases, into the cell nucleus, and have potential for in vitro and in vivo applications. PTD imaging conjugates have included small molecules, peptides, proteins, DNA, metal chelates, and magnetic nanoparticles. The full potential of the use of PTDs in novel in vivo molecular probes is currently under investigation. Cells have been labeled in culture using magnetic nanoparticles derivatized with a PTD and monitored in vivo to assess trafficking patterns relative to cells expressing a target antigen. In vivo imaging of PTD-mediated gene transfer to cells of the skin has been demonstrated in living animals. Here we review several natural and synthetic PTDs that have evolved in the quest for easier translocation across biological barriers and the application of these peptide domains to in vivo delivery of imaging agents.

  View details for PubMedID 14717330

• Gene transfer via reversible plasmid condensation with cysteine-flanked, internally spaced arginine-rich peptides HUMAN GENE THERAPY Siprashvili, Z., Scholl, F. A., Oliver, S. F., Adams, A., Contag, C. H., Wender, P. A., Khavari, P. A. 2003; 14 (13): 1225-1233

  Abstract

  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.

  View details for Web of Science ID 000184794500002

  View details for PubMedID 12952594

• A potent and specific morpholino antisense inhibitor of hepatitis C translation in mice HEPATOLOGY McCaffrey, A. P., Meuse, L., Karimi, M., Contag, C. H., Kay, M. A. 2003; 38 (2): 503-508

  Abstract

  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.

  View details for DOI 10.1053/jhep.2003.50330

  View details for Web of Science ID 000184531300026

  View details for PubMedID 12883495

• Effects of metalloporphyrins on heme oxygenase-1 transcription: correlative cell culture assays guide in vivo imaging. Molecular imaging Hajdena-Dawson, M., Zhang, W., Contag, P. R., Wong, R. J., Vreman, H. J., Stevenson, D. K., Contag, C. H. 2003; 2 (3): 138-149

  Abstract

  Heme oxygenase (HO) is the rate-limiting step in the heme degradation pathway and is a potential target for the control, or prevention, of pathologic jaundice in neonates. Metalloporphyrins (Mps), a diverse set of synthetic derivatives of heme, can competitively inhibit the HO enzymes. However, certain Mps are phototoxic and some increase transcription of HO-1, the inducible HO isozyme. Therefore, effective development of this class of compounds as therapeutics for treating pathologic jaundice will require rapid and integrated biological screens to identify the most efficacious and safe Mps. To study the safety of these compounds, we assessed their cytotoxic effects and measured luciferase activity by bioluminescent imaging (BLI) as an index of HO-1 transcription, first in live cell cultures and then in living transgenic reporter mice. A total of 12 Mps were first evaluated in the correlative cell culture assay. Based on results from this study, 2 Mps, zinc protoporphyrin (ZnPP) and zinc bis glycol porphyrin (ZnBG), were selected for further studies in the live animal model. In vitro BLI showed ZnPP to be a strong inducer of HO-1 transcription in comparison to ZnBG, which showed minimal induction. Cytotoxicity studies revealed that ZnPP was phototoxic, whereas ZnBG had no effect on cell viability. In vivo BLI showed that both ZnPP and ZnBG had minimal effects on the levels of HO-1 transcription in the animals. Furthermore, serum enzyme assays indicated that neither caused detectable liver toxicity. These findings, and especially those with ZnBG, support the use of selected Mps as therapies for pathologic jaundice. Coupling the high throughput advantage of cell culture with the capability of imaging for whole-body temporal analyses could accelerate and refine the preclinical phases of drug development. Thus, this study serves as a model for understanding the effects of specific compounds in relation to defined targets using an integrated approach.

  View details for PubMedID 14649057

• Comparison of gene expression after intraperitoneal delivery of AAV2 or AAV5 in utero MOLECULAR THERAPY Lipshutz, G. S., Titre, D., Brindle, M., Bisconte, A. R., Contag, C. H., Gaensler, K. M. 2003; 8 (1): 90-98

  Abstract

  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.

  View details for DOI 10.1016/S1525-0016(03)00132-1

  View details for Web of Science ID 000184066900015

  View details for PubMedID 12842432

• In vivo activation of the human CYP3A4 promoter in mouse liver and regulation by pregnane X receptors BIOCHEMICAL PHARMACOLOGY Zhang, W. S., Purchio, A., Chen, K., Burns, S. M., Contag, C. H., Contag, P. R. 2003; 65 (11): 1889-1896

  Abstract

  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.

  View details for DOI 10.1016/S0006-2952(03)00188-6

  View details for Web of Science ID 000183429900015

  View details for PubMedID 12781341

• Optical monitoring and treatment of potentially lethal wound infections in vivo 8th International-Photodynamic-Association Congress Hamblin, M. R., Zahra, T., Contag, C. H., McManus, A. T., Hasan, T. OXFORD UNIV PRESS. 2003: 1717–25

  Abstract

  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.

  View details for Web of Science ID 000183279200006

  View details for PubMedID 12751029

• Advancing molecular therapies through in vivo bioluminescent imaging. Molecular imaging McCaffrey, A., Kay, M. A., Contag, C. H. 2003; 2 (2): 75-86

  Abstract

  Effective development of therapeutics that target the molecular basis of disease is dependent on testing new therapeutic moieties and delivery strategies in animal models of human disease. Accelerating the analyses of these models and improving their predictive value through whole animal imaging methods, which provide data in real time and are sensitive to the subtle changes, are crucial for rapid advancement of these approaches. Modalities based on optics are rapid, sensitive, and accessible methods for in vivo analyses with relatively low instrumentation costs. In vivo bioluminescent imaging (BLI) is one of these optically based imaging methods that enable rapid in vivo analyses of a variety of cellular and molecular events with extreme sensitivity. BLi is based on the use of light-emitting enzymes as internal biological light sources that can be detected externally as biological indicators. BLI has been used to test spatio-temporal expression patterns of both target and therapeutic genes in living laboratory animals where the contextual influences of whole biological systems are preserved. BLI has also been used to analyze gene delivery, immune cell therapies, and the in vivo efficacy of inhibitory RNAs. New tools for BLI are being developed that will offer greater flexibility in detection and analyses. BLI can be used to accelerate the evaluation of experimental therapeutic strategies and whole body imaging offers the opportunity of revealing the effects of novel approaches on key steps in disease processes.

  View details for PubMedID 12964305

• Dual-axis confocal microscope for high-resolution in vivo imaging OPTICS LETTERS Wang, T. D., Mandella, M. J., Contag, C. H., Kino, G. S. 2003; 28 (6): 414-416

  Abstract

  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.

  View details for Web of Science ID 000181411300012

  View details for PubMedID 12659264

• Animal models of bone metastasis 3rd North American Symposium on Skeletal Complications of Malignancy Rosol, T. J., Tannehill-Gregg, S. H., LeRoy, B. E., Mandl, S., Contag, C. H. JOHN WILEY & SONS INC. 2003: 748–57

  Abstract

  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.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.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.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.

  View details for DOI 10.1002/cncr.11150

  View details for Web of Science ID 000180666300006

  View details for PubMedID 12548572

• Direct intestinal administration of metalloporphyrins and heme oxygenase expression. Western Regional Meeting of the American-Federation-for-Medical-Research Wong, R. J., Abate, A., Dennery, P. A., Vreman, H. J., Contag, C. H., Stevenson, D. K. LIPPINCOTT WILLIAMS & WILKINS. 2003: S140–S141

  View details for Web of Science ID 000180569600291

• Revealing lymphoma growth and the efficacy of immune cell therapies using in vivo bioluminescence imaging BLOOD Edinger, M., Cao, Y. A., Verneris, M. R., Bachmann, M. H., Contag, C. H., Negrin, R. S. 2003; 101 (2): 640-648

  Abstract

  Cancer therapeutics have achieved success in the treatment of a variety of malignancies, however, relapse of disease from small numbers of persistent tumor cells remains a major obstacle. Advancement of treatment regimens that effectively control minimal residual disease and prevent relapse would be greatly accelerated if sensitive and noninvasive assays were used to quantitatively assess tumor burden in animal models of minimal residual disease that are predictive of the human response. In vivo bioluminescence imaging (BLI) is an assay for the detection of small numbers of cells noninvasively and enables the quantification of tumor growth within internal organs. Fusion genes that encode bioluminescent and fluorescent reporter proteins effectively couple the powerful in vivo capabilities of BLI with the subset-discriminating capabilities of fluorescence-activated cell sorting. We labeled 2 murine lymphoma cell lines with dual function reporter genes and monitored radiation and chemotherapy as well as immune-based strategies that employ the tumorcidal activity of ex vivo-expanded CD8(+) natural killer (NK)-T cells. Using BLI we were able to visualize the entire course of malignant disease including engraftment, expansion, metastasis, response to therapy, and unique patterns of relapse. We also labeled the effector NK-T cells and monitored their homing to the sites of tumor growth followed by tumor eradication. These studies reveal the efficacy of immune cell therapies and the tempo of NK-T cell trafficking in vivo. The complex cellular processes in bone marrow transplantation and antitumor immunotherapy, previously inaccessible to investigation, can now be revealed in real time in living animals.

  View details for DOI 10.1182/blood-2002-06-1751

  View details for PubMedID 12393519

• Treatment of autoimmune disease by adoptive cellular gene therapy 10th International Conference on Myasthenia Gravis and Related Disorders Tarner, I. H., Slavin, A. J., McBride, J., Levicnik, A., Smith, R., Nolan, G. P., Contag, C. H., Fathman, C. G. NEW YORK ACAD SCIENCES. 2003: 512–519

  Abstract

  Autoimmune disorders represent inappropriate immune responses directed at self-tissue. Antigen-specific CD4+ T cells and antigen-presenting dendritic cells (DCs) are important mediators in the pathogenesis of auto-immune disease and thus are ideal candidates for adoptive cellular gene therapy, an ex vivo approach to therapeutic gene transfer. Using retrovirally transduced cells and luciferase bioluminescence, we have demonstrated that primary T cells, T cell hybridomas, and DCs rapidly and preferentially home to the sites of inflammation in animal models of multiple sclerosis, arthritis, and diabetes. These cells, transduced with retroviral vectors to drive expression of various "regulatory proteins" such as IL-4, IL-10, IL-12p40, and anti-TNF scFv, deliver these immunoregulatory proteins to the inflamed lesions, providing therapy for experimental autoimmune encephalitis (EAE), collagen-induced arthritis (CIA), and nonobese diabetic mice (NOD).

  View details for Web of Science ID 000186107400066

  View details for PubMedID 14592922

• Apoptosis in a rat model of cranial suture fusion 10th International Congress of the International-Society-of-Craniofacial-Surgery Fong, K. D., Song, H. M., Nacamuli, R. P., Franc, B., Fang, T. D., Salim, A., Contag, C., Blankenburg, F., Longaker, M. T. MEDIMOND PUBLISHING CO. 2003: 405–407

  View details for Web of Science ID 000227470500077

• Bioluminescence imaging as a marker for cellular Hsp70 response to thermal laser injury Conference on Laser-Tissue Interaction XIV Beckham, J. T., Baran, J. A., Mackanos, M., Crooke, C., Takahashi, T., O' Connell-Rodwell, C., Contag, C. H., Jansen, E. D. SPIE-INT SOC OPTICAL ENGINEERING. 2003: 153–164

  View details for Web of Science ID 000184961100021

• Myeloid progenitors protect against invasive aspergillosis and Pseudomonas aeruginosa infection following hematopoietic stem cell transplantation BLOOD Bitmansour, A., Burns, S. M., Traver, D., Akashi, K., Contag, C. H., Weissman, I. L., Brown, J. M. 2002; 100 (13): 4660-4667

  Abstract

  Myelotoxic treatments for oncologic diseases are often complicated by neutropenia, which renders patients susceptible to potentially lethal infections. In these studies of murine hematopoietic stem cell transplantation (HSCT), cotransplantation of lineage-restricted progenitors known as common myeloid progenitors (CMP) and granulocyte-monocyte progenitors (GMP) protects against death following otherwise lethal challenge with either of 2 pathogens associated with neutropenia: Aspergillus fumigatus and Pseudomonas aeruginosa. Cotransplantation of CMP/GMP resulted in a significant and rapid increase in the absolute number of myeloid cells in the spleen, most of which were derived from the donor CMP/GMP. Despite persistent peripheral neutropenia, improved survival correlated with the measurable appearance of progenitor-derived myeloid cells in the spleen. A marked reduction or elimination of tissue pathogen load was confirmed by culture and correlated with survival. Localization of infection by P aeruginosa and extent of disease was also assessed by in vivo bioluminescent imaging using a strain of P aeruginosa engineered to constitutively express a bacterial luciferase. Imaging confirmed that transplantation with a graft containing hematopoietic stem cells and CMP/GMP reduced the bacterial load as early as 18 hours after infection. These results demonstrate that enhanced reconstitution of a tissue myeloid pool offers protection against lethal challenge with serious fungal and bacterial pathogens.

  View details for DOI 10.1182/blood-2002-05-1552

  View details for Web of Science ID 000179759800058

  View details for PubMedID 12393415

• Retroviral gene therapy of collagen-induced arthritis by local delivery of IL-4 CLINICAL IMMUNOLOGY Tarner, I. H., Nakajima, A., Seroogy, C. M., Ermann, J., Levicnik, A., Contag, C. H., Fathman, C. G. 2002; 105 (3): 304-314

  Abstract

  Rheumatoid arthritis (RA) is an autoimmune arthritis, for which treatment options remain limited. This study investigated the potential role of adoptive cellular gene therapy as a novel means for treating the RA animal model collagen-induced arthritis (CIA). Adoptive transfer of antigen-specific T-cell hybridomas retrovirally transduced to express IL-4 1 day before booster immunization significantly reduced the number of inflamed joints. Cell transfer after clinical onset of disease had no therapeutic effect. Bioluminescence imaging showed that the hybridomas migrated to the inflamed joints, thus delivering the regulatory protein locally at the site of inflammation. The homing was, at least in part, due to chemotaxis in response to proinflammatory chemokines that are expressed in inflamed joints. There were no significant changes in the cytokine milieu of the draining lymph nodes, nor in the systemic levels of anti-collagen antibodies in treated mice. We conclude that the beneficial clinical effects observed in our model were most likely based on the local action(s) of IL-4 in the inflamed joints and that the local delivery (and effects) of regulatory cytokines, like IL-4, constitutes a novel and effective method of preventing organ-specific autoimmune disease and of minimizing systemic adverse effects.

  View details for DOI 10.1006/clim.2002.5299

  View details for Web of Science ID 000180187600008

  View details for PubMedID 12498812

• Trafficking of ex vivo expanded donor dendritic cells after allogeneic BMT. 44th Annual Meeting of the American-Society-of-Hematology Schimmelpfennig, C. H., Mcbride, J. M., Tarner, I. H., Baker, J., McGuire, J., Fathman, C. G., Contag, C. H., Negrin, R. S. AMER SOC HEMATOLOGY. 2002: 408A–408A

  View details for Web of Science ID 000179184701586

• Impact of three non-myeloablative conditioning regimens on allogeneic bone marrow transplantation in mice with B-cell lymphoma. 44th Annual Meeting of the American-Society-of-Hematology Schimmelpfennig, C. H., Baker, J., Mont-Reynaud, M. J., Edinger, M., Contag, C. H., Blazar, B. R., Negrin, R. S. AMER SOC HEMATOLOGY. 2002: 402B–402B

  View details for Web of Science ID 000179184801716

• Advancing animal models of neoplasia through in vivo bioluminescence imaging EUROPEAN JOURNAL OF CANCER Edinger, M., Cao, Y. A., Hornig, Y. S., Jenkins, D. E., Verneris, M. R., Bachmann, M. H., Negrin, R. S., Contag, C. H. 2002; 38 (16): 2128-2136

  Abstract

  Malignant disease is the final manifestation of complex molecular and cellular events leading to uncontrolled cellular proliferation and eventually tissue destruction and metastases. While the in vitro examination of cultured tumour cells permits the molecular dissection of early pathways in tumorigenesis on cellular and subcellular levels, only interrogation of these processes within the complexity of organ systems of the living animal can reveal the full range of pathophysiological changes that occur in neoplastic disease. Such analyses require technologies that facilitate the study of biological processes in vivo, and several approaches have been developed over the last few years. These strategies, in the nascent field of in vivo molecular and cellular imaging, combine molecular biology with imaging modalities as a means to real-time acquisition of functional information about disease processes in living systems. In this review, we will summarise recent developments in in vivo bioluminescence imaging (BLI) and discuss the potential of this imaging strategy for the future of cancer research.

  View details for Web of Science ID 000179242500008

  View details for PubMedID 12387838

• Visualization of effective tumor targeting by CD8+natural killer T cells redirected with bispecific antibody F(ab ')(2)HER2xCD3 CANCER RESEARCH Scheffold, C., Kornacker, M., Scheffold, Y. C., Contag, C. H., Negrin, R. S. 2002; 62 (20): 5785-5791

  Abstract

  HER2 is an attractive immunotherapeutic target for neoplastic disease because this cell surface molecule is overexpressed on a large fraction of malignant tumor cells. To directly assess therapeutic responses to targeted therapy by noninvasive in vivo imaging in small animals, human HER2-expressing ovarian carcinoma cells were genetically modified with a firefly luciferase gene, and light emission was used for visualization of tumor growth and response to therapy. This imaging approach was able to demonstrate in real-time tumor regression in a HER2 xenograft mouse model by adoptive transfer of in vitro induced and expanded cytotoxic CD8+ natural killer T (NKT) cells retargeted with a humanized bispecific antibody F(ab')(2)HER2xCD3. Immunotherapy with effector cells alone or a humanized monoclonal antibody anti-p185(HER2) (4D5-8) resulted in significant but slower reduction in tumor burden. Long-term survival of tumor xenografts correlated inversely with visible residual tumor burden. In vitro, F(ab')(2)HER2xCD3 substantially augmented cytotoxic activity of CD8+ NKT cells. By flow-sorting, CD8+ NKT cells coexpressing CD56 were found to have the highest redirected killing ability. Treatment with concanamycin A or EGTA abrogated CD8+ NKT cytotoxicity indicating that perforin is a major pathway of tumor cell lysis. In contrast, when CD8+ NKT cell were cross-linked with F(ab')(2)HER2xCD3 neither the immunosuppressants cyclosporine A and FK506, nor the increase of intracellular cyclic AMP by dibutyryl cyclic AMP were able to inhibit cytotoxicity demonstrating that signaling via the CD3 antigen changes the biological activity of non-MHC-restricted effector cells. These studies have demonstrated that CD8+ NKT cells can be successfully redirected to tumor cells using bispecific antibodies and offer a promising strategy for adoptive immunotherapy of neoplastic diseases.

  View details for PubMedID 12384539

• Selection of potential therapeutics based on in vivo spatiotemporal transcription patterns of heme oxygenase-1 JOURNAL OF MOLECULAR MEDICINE-JMM Zhang, W. S., Contag, P. R., Hardy, J., Zhao, H., Vreman, H. J., Hajdena-Dawson, M., Wong, R. J., Stevenson, D. K., Contag, C. H. 2002; 80 (10): 655-664

  Abstract

  Heme oxygenase (HO), a key catabolic enzyme in the conversion of heme to bilirubin, is an ideal target for reducing bilirubin production and preventing pathological jaundice in newborn infants. Metalloporphyrins (Mps) have been well characterized as competitive inhibitors of HO and have been evaluated as potential chemopreventive agents for neonatal jaundice. However, in addition to reducing HO activity, many Mps have been shown to increase HO-1 transcription, which would likely reduce their potential therapeutic utility. The differential effects of Mps on the transcription of HO-1 were therefore evaluated in living transgenic (Tg) reporter mice. Of the compounds evaluated, we observed that zinc bis-glycol porphyrin (ZnBG), a potent inhibitor of HO enzyme activity, did not alter HO-1 transcription patterns in Tg mice. Whole body images of HO-1 transcription patterns did, however; reveal increases in HO-1 transcription in Tg mice after treatment with other Mps, heme and cadmium chloride (CdCl(2)). Intravenous injections of CdCl(2) resulted in expression patterns that differed in tempo and location from those observed in Tg mice treated with intraperitoneal injections. Spatiotemporal analyses of transcriptional regulation in living animals accelerated the assessment of an adverse effect of Mps by revealing different patterns of HO-1 transcription. Among the known inhibitors of HO enzyme activity that were evaluated in this study, ZnBG did not significantly affect HO-1 transcription and therefore may be well suited for the prevention of neonatal jaundice.

  View details for DOI 10.1007/s00109-002-0375-x

  View details for PubMedID 12395150

• It's not just about anatomy: In vivo bioluminescence imaging as an eyepiece into biology JOURNAL OF MAGNETIC RESONANCE IMAGING Contag, C. H., Ross, B. D. 2002; 16 (4): 378-387

  Abstract

  Among the newly described tools that enable analyses of cellular and molecular events in living animals, in vivo bioluminescence imaging (BLI) offers important opportunities for investigating a wide variety of disease processes. BLI utilizes luciferase as an internal biological light source that can be genetically programmed to noninvasively "report" the presence or activation of specific biological events. Applications of BLI have included the use of luciferase to demonstrate expression of cell- and tissue-specific promoters, label cell populations, guide detection by other imaging modalities, and detect protein-protein interaction. These applications of BLI technology have allowed quantitative measurements of tumor burden and treatment response, immune cell trafficking, and detection of gene transfer. Spatiotemporal information can be rapidly obtained in the context of whole biological systems in vivo, which can accelerate the development of experimental therapeutic strategies. This paper provides a review of the biological applications in which in vivo BLI has been utilized to nondestructively monitor biological processes in intact small animal models, and highlights some of the advancements that will increase the versatility of BLI as a molecular imaging tool.

  View details for DOI 10.1002/jmri.10178

  View details for Web of Science ID 000178445700006

  View details for PubMedID 12353253

• Adoptive cellular gene therapy of autoimmune disease. Autoimmunity reviews Slavin, A. J., Tarner, I. H., Nakajima, A., Urbanek-Ruiz, I., McBride, J., Contag, C. H., Fathman, C. G. 2002; 1 (4): 213-219

  Abstract

  Autoimmune disorders represent inappropriate immune responses directed at self-tissue. Because CD4+ T cells are important mediators in the pathogenesis of autoimmune disease, they are ideal candidates for cell-based gene therapy. Using retrovirally-transduced cells and luciferase bioluminescence, we have demonstrated that primary T cells and hybridomas, rapidly and preferentially home to the sites of inflammation in organ-specific autoimmune disease. These cells, transduced with retroviral vectors to drive expression of various 'regulatory proteins', such as IL-4, IL-10 and IL-12p40, deliver these immunoregulatory proteins to the inflamed lesions, providing therapy for experimental models of autoimmune disease such as EAE, CIA and NOD mice. This technique was originally developed in our lab in the murine model of multiple sclerosis, EAE, where T cell hybridomas reactive with myelin basic protein (MBP) were transduced to express and used to deliver the modulatory cytokine, IL-4. Recently we have observed that the cytokine receptor antagonist, IL-12p40 transduced anti-myelin basic protein (MBP) TCR-transgenic T cells (but not CII-reactive T cells) were effective in preventing EAE whereas the CII-reactive, but not MBP-reactive T cells, transduced to express IL-12p40, would treat CIA.

  View details for PubMedID 12848998

• Bioluminescent indicators for in vivo measurements of gene expression TRENDS IN BIOTECHNOLOGY O'Connell-Rodwell, C. E., Burns, S. M., Bachmann, M. H., Contag, C. H. 2002; 20 (8): S19-S23

  View details for Web of Science ID 000208536200004

• Visualization of tumor and effector cell trafficking in hematologic malignancies Negrin, R., Edinger, M., Verneris, M., Cao, Y. A., Bachmann, M., Contag, C. ELSEVIER SCIENCE INC. 2002: 105–

  View details for Web of Science ID 000176187000270

• Dissection of promoter control modules that direct Bmp4 expression in the epithelium-derived components of hair follicles BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Zhang, J. H., Tan, X. Y., Contag, C. H., Lu, Y. B., Guo, D. Y., Harris, S. E., Feng, J. Q. 2002; 293 (5): 1412-1419

  Abstract

  Bone morphogenetic protein 4 (Bmp4) plays a significant role in development. Using transgenic approaches, we studied the mechanisms controlling Bmp4 expression during primordial and mature tissue development, as well as in epithelium- and mesenchyme-derived components with hair follicles as a model. In this report, we demonstrated that the promoter region between the -0.26 and the -1.14 kb, highly conserved between human and mouse, controls Bmp4 expression in the epithelium-derived tissues but not in mesenchyme-derived tissues of hair follicles, suggesting that control modules for Bmp4 expression in epithelium-derived tissues and mesenchyme originated tissues are in separate regions. Using live embryos and mice, we demonstrated the temporal and spatial activities of these modules. We also demonstrated that control regions for Bmp4 expression in primordial and differentiated hair follicle tissues are separated. Therefore we present a model to explain a mechanism controlling expression of the Bmp4 gene in different tissue types, as well as different development stages as related to hair development.

  View details for Web of Science ID 000176107900018

  View details for PubMedID 12054672

• Regulation of intestine-specific spatiotemporal expression by the rat lactase promoter JOURNAL OF BIOLOGICAL CHEMISTRY Lee, S. Y., Wang, Z., Lin, C. K., Contag, C. H., Olds, L. C., Cooper, A. D., Sibley, E. 2002; 277 (15): 13099-13105

  Abstract

  Lactase gene transcription is spatially restricted to the proximal and middle small intestine of the developing mouse. To identify regions of the lactase gene involved in mediating the spatiotemporal expression pattern, transgenic mice harboring 0.8-, 1.3-, and 2.0-kb fragments of the 5'-flanking region cloned upstream of a firefly-luciferase reporter were generated. Transgene expression was assessed noninvasively in living mice using a sensitive low light imaging system. Two independent, 1.3- and 2.0-kb, lactase promoter-reporter transgenic lines expressed appropriate high levels of luciferase activity in the small intestine (300-3,000 relative light units/microg) with maximal expression in the middle segments. Post-weaned 30-day transgenic offspring also demonstrated an appropriate 4-fold maturational decline in luciferase expression in the small intestine. The pattern of the 2.0-kb promoter transgene mRNA abundance most closely mimicked that of the endogenous lactase gene with respect to spatiotemporal restriction. In contrast, a 0.8-kb promoter-reporter construct expressed low level luciferase activity (<25 relative light units/microg) in multiple organs and throughout the gastrointestinal tract in transgenic mice. Thus, a distinct 5'-region of the lactase promoter directs intestine-specific expression in the small intestine of transgenic mice, and regulatory sequences have been localized to a 1.2-kb region upstream of the lactase transcription start site. In addition, we have demonstrated that in vivo bioluminescence imaging can be utilized for assessment of intestinal expression patterns of a luciferase reporter gene driven by lactase promoter regions in transgenic mice.

  View details for DOI 10.1074/jbc.M112152200

  View details for PubMedID 11812796

• Developmentally regulated pattern of heme oxygenase-1 expression in the mouse brain Wong, R. J., Nguyen, X. N., Zhao, H., Vreman, H. J., Contag, C. H., Stevenson, D. K. NATURE PUBLISHING GROUP. 2002: 328A–328A

  View details for Web of Science ID 000174714601906

• Rapid in vivo assessment of heme oxygenase-1 gene transcription: Involvement of regulatory regions in metalloporphyrin-mediated activation Kazerounizadeh, H. R., Gruber, C. A., Burns, S. M., Hardy, J. W., Wong, R. J., Vreman, H. J., Stevenson, D. K., Contag, C. H. NATURE PUBLISHING GROUP. 2002: 327A–328A

  View details for Web of Science ID 000174714601904

• Understanding immune cell trafficking patterns via in vivo bioluminescence imaging JOURNAL OF CELLULAR BIOCHEMISTRY Mandl, S., Schimmelpfennig, C., Edinger, M., Negrin, R. S., Contag, C. H. 2002: 239-248

  Abstract

  Cell migration is a key aspect of the development of the immune system and mediating an immune response. There is extensive and continual redistribution of cells to different anatomic sites throughout the body. These trafficking patterns control immune function, tissue regeneration, and host responses to insult. The ability to monitor the fate and function of cells, therefore, is imperative to both understanding the role of specific cells in disease processes and to devising rational therapeutic strategies. Determining the fate of immune cells and understanding the functional changes associated with migration and proliferation require effective means of obtaining in vivo measurements in the context of intact organ systems. A variety of imaging methods are available to provide structural information, such as X-ray CT and MRI, but only recently new tools have been developed that reveal cellular and molecular changes as they occur within living animals. We have pioneered one of these techniques that is based on the observations that light passes through mammalian tissues, and that luciferases can serve as internal biological sources of light in the living body. This method, called in vivo bioluminescence imaging, is a rapid and noninvasive functional imaging method that employs light-emitting reporters and external photon detection to follow biological processes in living animals in real time. This imaging strategy enables the studies of trafficking patterns for a variety of cell types in live animal models of human biology and disease. Using this approach we have elucidated the spatiotemporal trafficking patterns of lymphocytes within the body. In models of autoimmune disease we have used the migration of "pathogenic" immune cells to diseased tissues as a means to locally deliver and express therapeutic proteins. Similarly, we have determined the tempo of NK-T cell migration to neoplastic lesions and measured their life span in vivo. Using bioluminescence imaging individual groups of animals can be followed over time significantly reducing the number of animals per experiment, and improving the statistical significance of a study since changes in a given population can be studied over time. Such rapid assays that reveal cell fates in vivo will increase our basic understanding of the molecular signals that control these migratory pathways and will substantially speed up the development and evaluation of therapies.

  View details for DOI 10.1002/jcb.10454

  View details for PubMedID 12552623

• Rapid control of wound infections by targeted photodynamic therapy monitored by in vivo bioluminescence imaging PHOTOCHEMISTRY AND PHOTOBIOLOGY Hamblin, M. R., O'Donnell, D. A., Murthy, N., Contag, C. H., Hasan, T. 2002; 75 (1): 51-57

  Abstract

  The worldwide rise in antibiotic resistance necessitates the development of novel antimicrobial strategies. In this study we report on the first use of a photochemical approach to destroy bacteria infecting a wound in an animal model. Following topical application, a targeted polycationic photosensitizer conjugate between poly-L-lysine and chlorin(e6) penetrated the gram (-) outer bacterial membrane, and subsequent activation with 660 nm laser light rapidly killed Escherichia coli infecting excisional wounds in mice. To facilitate real-time monitoring of infection, we used bacteria that expressed the lux operon from Photorhabdus luminescens; these cells emitted a bioluminescent signal that allowed the infection to be rapidly quantified, using a low-light imaging system. There was a light-dose dependent loss of luminescence in the wound treated with conjugate and light, not seen in untreated wounds. Treated wounds healed as well as control wounds, showing that the photodynamic treatment did not damage the host tissue. Our study points to the possible use of this methodology in the rapid control of wounds and other localized infections.

  View details for Web of Science ID 000173525200008

  View details for PubMedID 11837327

• Advances in vivo bioluminescence imaging of gene expression ANNUAL REVIEW OF BIOMEDICAL ENGINEERING Contag, C. H., Bachmann, M. H. 2002; 4: 235-260

  Abstract

  To advance our understanding of biological processes as they occur in living animals, imaging strategies have been developed and refined that reveal cellular and molecular features of biology and disease in real time. One rapid and accessible technology for in vivo analysis employs internal biological sources of light emitted from luminescent enzymes, luciferases, to label genes and cells. Combining this reporter system with the new generation of charge coupled device (CCD) cameras that detect the light transmitted through the animal's tissues has opened the door to sensitive in vivo measurements of mammalian gene expression in living animals. Here, we review the development and application of this imaging strategy, in vivo bioluminescence imaging (BLI), together with in vivo fluorescence imaging methods, which has enabled the real-time study of immune cell trafficking, of various genetic regulatory elements in transgenic mice, and of in vivo gene transfer. BLI has been combined with fluorescence methods that together offer access to in vivo measurements that were not previously available. Such studies will greatly facilitate the functional analysis of a wide range of genes for their roles in health and disease.

  View details for DOI 10.1146/annurev.bioeng.4.111901.093336

  View details for Web of Science ID 000177827800011

  View details for PubMedID 12117758

• Targeted photodynamic therapy for infected wounds in mice. Conference on Optical Methods for Tumor Treatment and Detection - Mechanisms and Techniques in Photodynamic Therapy XI Hamblin, M. R., O'Donnell, D. A., Zahra, T., Contag, C. H., McManus, A., Hasan, T. SPIE-INT SOC OPTICAL ENGINEERING. 2002: 48–58

  View details for Web of Science ID 000177417600006

• Visualization of tumor growth and response to NK-T cell based immunotherapy using bioluminescence 3rd International Symposium on Transplantation in Hematology and Oncology Negrin, R. S., Edinger, M., Verneris, M., Cao, Y. A., Bachmann, M., Contag, C. H. SPRINGER. 2002: S44–S45

  View details for Web of Science ID 000178591900015

  View details for PubMedID 12611073

• In vivo patterns of heme oxygenase-1 transcription. Journal of perinatology Contag, C. H., Stevenson, D. K. 2001; 21: S119-24

  Abstract

  Gene fusions composed of specific promoters and bioluminescent reporter genes can be used to assess gene expression patterns using whole-body imaging in living animal models. A transgenic mouse model was developed using the regulatory elements of the heme oxygenase promoter to drive luciferase as the reporter gene. In these transgenic mice, heme oxygenase (HO)-1 expression was apparent in neuronal tissues of neonates but not adults as measured by whole-body imaging, and in adults transcription of the reporter gene was inducible by known inducers of HO-1 transcription. Whole-body imaging of luciferase activity was then used to evaluate the effects of metalloporphyrins (Mps) on the transcription of the reporter gene. Some of the Mps, which are potent inhibitors of HO activity, did not activate the reporter gene above background. These Mps are ideally suited as chemotherapeutics that may target bilirubin production rates by inhibiting HO activity, but not result in a net increase in output from the HO gene. In contrast, known inducers of HO transcription did increase luciferase activity as did some of the other Mps that have been examined. Using whole-body in vivo transcriptional assays may facilitate rapid screening of potential therapeutic compounds for both desired and untoward effects.

  View details for PubMedID 11803432

• Bioluminescence imaging of lymphocyte trafficking in vivo EXPERIMENTAL HEMATOLOGY Hardy, J., Edinger, M., Bachmann, M. H., Negrin, R. S., Fathman, C. G., Contag, C. H. 2001; 29 (12): 1353-1360

  Abstract

  Lymphocytes are highly mobile cells that travel throughout the body in response to a tremendous variety of stimuli. Revealing lymphocyte trafficking patterns in vivo is necessary for a complete understanding of immune function, as well as cell-cell and cell-tissue interactions in immune development and in response to insult. Although the location of cell populations in various tissues at any given point in time may be revealed by techniques such as flow cytometry and immunofluorescence, these methods are not readily amenable to the assessment of dynamic cell migration patterns in vivo. In the past 5 years, technologies for imaging molecular and cellular changes in living animals have advanced to a point where it is possible to reveal the migratory paths of these vitally important cells. Here, we review one advancement in cellular imaging, in vivo bioluminescence imaging, which addresses the problem of lymphocyte tracking. This imaging strategy has the potential to elucidate the temporal patterns of immune responses and the spatial distribution of lymphocytes within the body.

  View details for PubMedID 11750093

• Real time in vivo imaging of syngeneic and allogeneic T cells after bone marrow transplantation. Edinger, M., Verneris, M. R., Cao, Y., Bachmann, M., Contag, C. H., Negrin, R. S. AMER SOC HEMATOLOGY. 2001: 384A–384A

  View details for Web of Science ID 000172134101622

• Monitoring the anti tumor activity of expanded CD8(+) NKT cells after allogeneic bone marrow transplantation using bioluminescent imaging. Edinger, M., Verneris, M. R., Cao, Y., Bachmann, M., Contag, C. H., Negrin, R. S. AMER SOC HEMATOLOGY. 2001: 433A–433A

  View details for Web of Science ID 000172134101821

• Adoptive immunotherapy of experimental autoimmune encephalomyelitis via T cell delivery of the IL-12 p40 subunit JOURNAL OF IMMUNOLOGY Costa, G. L., Sandora, M. R., Nakajima, A., Nguyen, E. V., Taylor-Edwards, C., Slavin, A. J., Contag, C. H., Fathman, C. G., Benson, J. M. 2001; 167 (4): 2379-2387

  Abstract

  CD4+ T cells are believed to play a central role in the initiation and perpetuation of autoimmune diseases such as multiple sclerosis. In the murine model for multiple sclerosis, experimental autoimmune encephalomyelitis, pathogenic T cells exhibit a Th1-like phenotype characterized by heightened expression of proinflammatory cytokines. Systemic administration of "regulatory" cytokines, which serve to counter Th1 effects, has been shown to ameliorate autoimmune responses. However, the inherent problems of nonspecific toxicity limit the usefulness of systemic cytokine delivery as a potential therapy. Therefore, we used the site-specific trafficking properties of autoantigen-reactive CD4+ T cells to develop an adoptive immunotherapy protocol that provided local delivery of a Th1 cytokine antagonist, the p40 subunit of IL-12. In vitro analysis demonstrated that IL-12 p40 suppressed IFN-gamma production in developing and effector Th1 populations, indicating its potential to modulate Th1-promoted inflammation. We have previously demonstrated that transduction of myelin basic protein-specific CD4+ T cells with pGC retroviral vectors can result in efficient and stable transgene expression. Therefore, we adoptively transferred myelin basic protein-specific CD4+ T cells transduced to express IL-12 p40 into mice immunized to develop experimental autoimmune encephalomyelitis and demonstrated a significant reduction in clinical disease. In vivo tracking of bioluminescent lymphocytes, transduced to express luciferase, using low-light imaging cameras demonstrated that transduced CD4+ T cells trafficked to the central nervous system, where histological analysis confirmed long-term transgene expression. These studies have demonstrated that retrovirally transduced autoantigen-specific CD4+ T cells inhibited inflammation and promoted immunotherapy of autoimmune disorders.

  View details for Web of Science ID 000170949600070

  View details for PubMedID 11490028

• Antigen-specific T cell-mediated gene therapy in collagen-induced arthritis JOURNAL OF CLINICAL INVESTIGATION Nakajima, A., Seroogy, C. M., Sandora, M. R., Tarner, I. H., Costa, G. L., Taylor-Edwards, C., Bachmann, M. H., Contag, C. H., Fathman, C. G. 2001; 107 (10): 1293-1301

  Abstract

  Autoantigen-specific T cells have tissue-specific homing properties, suggesting that these cells may be ideal vehicles for the local delivery of immunoregulatory molecules. We tested this hypothesis by using type II collagen-specific (CII-specific) CD4(+) T hybridomas or primary CD4(+) T cells after gene transfer, as vehicles to deliver an immunoregulatory protein for the treatment of collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis (RA). CII-specific T cells or hybridomas were transduced using retroviral vectors to constitutively express the IL-12 antagonist, IL-12 p40. Transfer of engineered CD4(+) T cells after immunization significantly inhibited the development of CIA, while cells transduced with vector control had no effect. The beneficial effect on CIA of IL-12 p40-transduced T cells required TCR specificity against CII, since transfer of T cells specific for another antigen producing equivalent amounts of IL-12 p40 had no effect. In vivo cell detection using bioluminescent labels and RT-PCR showed that transferred CII-reactive T-cell hybridomas accumulated in inflamed joints in mice with CIA. These results indicate that the local delivery of IL-12 p40 by T cells inhibited CIA by suppressing autoimmune responses at the site of inflammation. Modifying antigen-specific T cells by retroviral transduction for local expression of immunoregulatory proteins thus offers a promising strategy for treating RA.

  View details for Web of Science ID 000168867400014

  View details for PubMedID 11375419

• Carbon monoxide and bilirubin production in neonates SEMINARS IN PERINATOLOGY Stevenson, D. K., Vreman, H. J., Wong, R. J., Contag, C. H. 2001; 25 (2): 85-93

  Abstract

  Neonatal hyperbilirubinemia is a normal postnatal phenomenon resulting from a transitional imbalance between the production and elimination of bilirubin in the neonate. Bilirubin has been shown to be not only a potent antioxidant, but also toxic at excessive concentrations. As a result, the biology of bilirubin, its production, regulation, and measurements have been the focus of extensive studies. Bilirubin, carbon monoxide, and iron are derived from the degradation of heme, a ubiquitous two-step pathway catalyzed by the enzyme, heme oxygenase. It has been shown that these metabolically active products from the heme catabolic pathway may, in turn, influence many other biologic processes. This report provides a brief overview of these interrelationships in the hope that it may provide insight into the central role this pathway plays in the existence of most organisms.

  View details for PubMedID 11339670

• In utero delivery of adeno-associated viral vectors: Intraperitoneal gene transfer produces long-term expression MOLECULAR THERAPY Lipshutz, G. S., Gruber, C. A., Cao, Y. A., Hardy, J., Contag, C. H., Gaensler, K. M. 2001; 3 (3): 284-292

  Abstract

  Recombinant adeno-associated viruses (rAAV) are promising gene transfer vectors that produce long-term expression without toxicity. To investigate future approaches for in utero gene delivery, the efficacy and safety of prenatal administration of rAAV were determined. Using luciferase as a reporter, expression was assessed by whole-body imaging and by analysis of luciferase activity in tissue extracts, at the time of birth and monthly thereafter. Transgene expression was detected in all injected animals. Highest levels of luciferase activity were detected at birth in the peritoneum and liver, while the heart, brain, and lung demonstrated low-level expression. In vivo luciferase imaging revealed persistent peritoneal expression for 18 months after in utero injection and provided a sensitive whole-body assay, useful in identifying tissues for subsequent analyses. There was no detectable hepatocellular injury. Antibodies that reacted with either luciferase or rAAV were not found. AAV sequences were not detected in germ-line tissues of injected animals or in tissues of their progeny. In utero AAV-mediated gene transfer in this animal model demonstrates that novel therapeutic vectors and strategies can be rapidly tested in vivo and that rAAV may be developed to ameliorate genetic diseases with perinatal morbidity and mortality.

  View details for Web of Science ID 000167967900003

  View details for PubMedID 11273769

• Revealing the spatiotemporal patterns of bacterial infectious diseases using bioluminescent pathogens and whole body imaging. Contributions to microbiology Burns, S. M., Joh, D., Francis, K. P., Shortliffe, L. D., Gruber, C. A., Contag, P. R., Contag, C. H. 2001; 9: 71-88

  View details for PubMedID 11764723

• Rapid in vivo functional analysis of transgenes in mice using whole body imaging of luciferase expression TRANSGENIC RESEARCH Zhang, W. S., Feng, J. Q., Harris, S. E., Contag, P. R., Stevenson, D. K., Contag, C. H. 2001; 10 (5): 423-434

  Abstract

  The use of transgenic animals in biomedical research is increasing rapidly and may be the best means of determining gene function. Generating transgenic animals typically requires time-consuming screening processes, and gene function is assessed by an array of difficult phenotypic and biochemical assays performed ex vivo. To address the unmet need in transgenic research for functional assays performed with ease in living animals, we demonstrate here that in vivo detection of luciferase enzyme as a transcriptional reporter facilitates rapid screening for both the presence and function of transgenes in intact living mice. Using this approach we identified three bioluminescent transgenic founders where the transgene consisted of the heme oxygenase promoter fused to the modified coding sequence of the luciferase gene. These founders were identified from 183 pups and confirmed by PCR analysis. Identification of HO-1-luc homozygotes from back- crossed F2 littermates was then accelerated by in vivo imaging. In another transgenic mouse line, where the transgene was comprised of the bone morphogenic-4 (BMP4) promoter fused to the modified luciferase gene, we were able to identify transgenic animals and in each line we were able to visualize patterns of expression in living animals over time. The light production from these transgenic mice indicated that the desired DNA fragment was functional and different expression profiles apparent at different ages and after gene induction.

  View details for PubMedID 11708652

• Prenatal transmission of subtype CHIV-1 in Zimbabwe: HIV-1 RNA and DNA in maternal and cord blood 13th International AIDS Conference Guevara, H., Johnston, E., Zijenah, L., Tobaiwa, O., Mason, P., Contag, C., Mahomed, K., Hendry, M., Katzenstein, D. LIPPINCOTT WILLIAMS & WILKINS. 2000: 390–97

  Abstract

  Maternal and cord samples from HIV-seropositive women and their infants in Zimbabwe, where subtype C is the predominant strain of HIV, were analyzed to determine the frequency of detection of HIV RNA and DNA. HIV RNA was detected in 90% of maternal and in 38% of cord plasma at levels at least 25% of maternal plasma. Heteroduplex mobility assays and sequencing of virus envelope (C2-V5) demonstrated closely related, but unique, subtype C viruses in maternal and cord RNA, and a significantly greater frequency of cord viremia among women with homogenous, compared with heterogeneous viral envelope RNA. Quantification of RNA, measures of envelope viral diversity, and phylogenetic analysis of maternal and cord plasma RNA provide evidence for the frequent exposure and potential transmission of HIV from mother to infant before birth.

  View details for Web of Science ID 000166017000002

  View details for PubMedID 11141238

• Visualizing leukemia & lymphoma cell homing and quantification of tumor burden in response to therapy in living animals. Edinger, M., Verneris, M. R., Cao, Y., Bachmann, M. H., Costa, G. L., Contag, C. H., Negrin, R. S. AMER SOC HEMATOLOGY. 2000: 123A–123A

  View details for Web of Science ID 000165256100531

• Rapid and quantitative assessment of cancer treatment response using in vivo bioluminescence imaging NEOPLASIA Rehemtulla, A., Stegman, L. D., Cardozo, S. J., Gupta, S., Hall, D. E., Contag, C. H., Ross, B. D. 2000; 2 (6): 491-495

  Abstract

  Current assessment of orthotopic tumor models in animals utilizes survival as the primary therapeutic end point. In vivo bioluminescence imaging (BLI) is a sensitive imaging modality that is rapid and accessible, and may comprise an ideal tool for evaluating antineoplastic therapies. Using human tumor cell lines constitutively expressing luciferase, the kinetics of tumor growth and response to therapy have been assessed in intraperitoneal, and subcutaneous, and intravascular cancer models. However, use of this approach for evaluating orthotopic tumor models has not been demonstrated. In this report, the ability of BLI to noninvasively quantitate the growth and therapeutic-induced cell kill of orthotopic rat brain tumors derived from 9L gliosarcoma cells genetically engineered to stably express firefly luciferase (9LLuc) was investigated. Intracerebral tumor burden was monitored over time by quantitation of photon emission and tumor volume using a cryogenically cooled CCD camera and magnetic resonance imaging (MRI), respectively. There was excellent correlation (r=0.91) between detected photons and tumor volume. A quantitative comparison of tumor cell kill determined from serial MRI volume measurements and BLI photon counts following 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) treatment revealed that both imaging modalities yielded statistically similar cell kill values (P=.951). These results provide direct validation of BLI imaging as a powerful and quantitative tool for the assessment of antineoplastic therapies in living animals.

  View details for Web of Science ID 000166411400002

  View details for PubMedID 11228541

  View details for PubMedCentralID PMC1508085

• Genetic analysis of viral variants selected in transmission of human immunodeficiency viruses to newborns AIDS RESEARCH AND HUMAN RETROVIRUSES Kliks, S., Contag, C. H., Corliss, H., Learn, G., Rodrigo, A., Wara, D., Mullins, J. I., Levy, J. A. 2000; 16 (13): 1223-1233

  Abstract

  Our previous studies have indicated that HIV transmission from infected mothers to infants occurs with viruses showing rapid kinetics of replication, and either resistance to maternal neutralizing antibodies or sensitivity to enhancing antibodies. The genotypic patterns that result in these and other phenotypic viral characteristics may provide clues to the selection pressures exerted during this mode of transmission. For this reason, DNA sequences of the envelope gene (env) were determined for viral isolates obtained from seropositive women who were mothers of either infected or uninfected infants. Sequences of viruses isolated early in life from the infected newborns were also determined, such that diversity both within isolates and between maternal and infant isolates could be assessed. Among isolates obtained from mothers of uninfected infants, the V3 region of env demonstrated a higher degree of heterogeneity than those from mothers of infected infants. Similar to the viruses obtained from the mothers of infected infants, the infant-derived viral sequences were relatively homogeneous. Finally, the reactivity of maternal plasma with infant-derived HIV isolates, whether via neutralizing or enhancing antibodies, appeared to predict the distribution of viral sequences in the infant isolates. These data suggest that selective pressure on HIV-1 during transmission or growth in the infected infant may be mediated by biologic and/or immunologic processes.

  View details for Web of Science ID 000088921400004

  View details for PubMedID 10957720

• HIV type 1 envelope subtype C sequences from recent seroconverters in Zimbabwe AIDS RESEARCH AND HUMAN RETROVIRUSES Batra, M., Tien, P. C., Shafer, R. W., Contag, C. H., Katzenstein, D. A. 2000; 16 (10): 973-979

  Abstract

  HIV-1 envelope sequence patterns have implications for virus cell tropism and for the development of an effective vaccine. To identify the sequence characteristics of recently transmitted HIV-1 isolates in southern Africa, we sequenced the V3-V5 envelope regions of 24 male seroconverters in Harare, Zimbabwe. Each of the sequences clustered with previously reported subtype C isolates and there was a mean 17% intersequence pairwise genetic distance between the Zimbabwean isolates. Three isolates were syncytium inducing (SI). One of the SI isolates had an unusual GIGK crown and a deletion at codon 23; one had the codon 23 deletion alone; and one had a high net positive charge in the V3 loop. The extensive genetic diversity within the envelope of subtype C HIV-1 isolates must be considered in vaccine development. Further analysis of subtype C SI isolates and site-directed mutagenesis experiments are required to determine the molecular basis of SI activity in global HIV-1 isolates.

  View details for Web of Science ID 000088006300006

  View details for PubMedID 10890359

• HO-1 expression in type II pneumocytes after transpulmonary gene delivery AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY Weng, Y. H., Tatarov, A., Bartos, B. P., Contag, C. H., Dennery, P. A. 2000; 278 (6): L1273-L1279

  Abstract

  Somatic cell gene transfer is a potentially useful strategy to alter lung function. However, achieving efficient transfer to the alveolar epithelium, especially in smaller animals, has not been demonstrated. In this study, the rat heme oxygenase-1 (HO-1) gene was delivered to the lungs of neonatal mice via transpulmonary injection. A bidirectional promoter construct coexpressing both HO-1 and a luciferase reporter gene was used so that in vivo gene expression patterns could be monitored in real time. HO-1 expression levels were also modulated with doxycycline and assessed in vivo with bioluminescent light transmitted through the tissues from the coregulated luciferase reporter. As a model of oxidative stress and HO-1-mediated protection, groups of animals were exposed to hyperoxia. After gene transfer, elevated levels of HO-1 were detected predominantly in alveolar type II cells by immunocytochemistry. With overexpression of HO-1, increased oxidative injury was observed. Furthermore, this model demonstrated a cell-specific effect of lung HO-1 overexpression in oxidative stress. Specific control of expression for therapeutic genes is possible in vivo. The transpulmonary approach may prove useful in targeting gene expression to cells of the alveolar epithelium or to circumscribed areas of the lung.

  View details for Web of Science ID 000087573600020

  View details for PubMedID 10835334

• Use of reporter genes for optical measurements of neoplastic disease in vivo NEOPLASIA Contag, C. H., Jenkins, D., Contag, F. R., Negrin, R. S. 2000; 2 (1-2): 41-52

  Abstract

  Revealing the cellular and molecular changes associated with cancer, as they occur in intact living animal models of human neoplastic disease, holds tremendous potential for understanding disease mechanisms and elucidating effective therapies. Since light is transmitted through mammalian tissues, at a low level, optical signatures conferred on tumor cells by expression of reporter genes encoding bioluminescent and fluorescent proteins can be detected externally using sensitive photon detection systems. Expression of reporter genes, such as the bioluminescent enzyme firefly luciferase (Luc) or variants of green fluorescent protein (GFP) in transformed cells, can effectively be used to reveal molecular and cellular features of neoplasia in vivo. Tumor cell growth and regression in response to various therapies have been evaluated non-invasively in living experimental animals using these reporter genes. Detection of Luc-labeled cells in vivo was extremely sensitive with signals over background from as few as 1000 human tumor cells distributed throughout the peritoneal cavity of a mouse with linear relationships between cell number and signal intensity over five logs. GFP offers the strength of high-resolution ex vivo analyses following in vivo localization of the tumor. The dynamic range of Luc detection allows the full disease course to be monitored since disease progression from small numbers of cells to extensive disease can be assessed. As such, therapies that target minimal disease as well as those designed for late stage disease can be readily evaluated in animal models. Real time spatiotemporal analyses of tumor cell growth can reveal the dynamics of neoplastic disease, and facilitate rapid optimization of effective treatment regimens. Thus, these methods improve the predictability of animal models of human disease as study groups can be followed over time, and can accelerate the development of therapeutic strategies.

  View details for PubMedID 10933067

• Carbon monoxide detection and biological investigations. Transactions of the American Clinical and Climatological Association Stevenson, D. K., Vreman, H. J., Wong, R. J., Dennery, P. A., Contag, C. H. 2000; 111: 61-75

  Abstract

  Even though the heme degradation pathway consists of only two reactions, it and its major enzyme (i.e. HO), nonetheless, impact other processes not only through the removal of excess heme, but also through the production of several metabolically active compounds. Thus CO and biliverdin along with reactive iron, Fe2, are the primordial products of this ancient, highly conserved reaction. That every component of the heme catabolic pathway is directly or indirectly related to other reactions involving oxygen or light is, perhaps, no accident of nature. That a fundamentally destructive event can be linked with a multiplicity of synthetic events and various biological effects, depending on the timing and location of the HO activity, is testament to the economy and the ultimate beauty of nature. Furthermore, the interaction of the heme catabolic pathway with that of the NOS system may lead to even more exciting avenues of research. It may be shown that the integrity of the heme catabolic pathway, which is ever present and plays a role in every tissue, is central to the existence of most complex organisms.

  View details for PubMedID 10881332

• Visualizing the kinetics of tumor-cell clearance in living animals PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sweeney, T. J., Mailander, V., Tucker, A. A., Olomu, A. B., Zhang, W. S., Cao, Y. A., Negrin, R. S., Contag, C. H. 1999; 96 (21): 12044-12049

  Abstract

  Evaluation of potential antineoplastic therapies would be enhanced by noninvasive detection of tumor cells in living animals. Because light is transmitted through mammalian tissues, it was possible to use bioluminescence to monitor (both externally and quantitatively) growth and regression of labeled human cervical carcinoma (HeLa) cells engrafted into immunodeficient mice. The efficacy of both chemotherapy and immunotherapeutic treatment with ex vivo expanded human T cell-derived effector cells was evaluated. In the absence of therapy, animals showed progressive increases in signal intensity over time. Animals treated with cisplatin had marked reductions in tumor signal; 5'-fluorouracil was less effective, and cyclophosphamide was ineffective. Immunotherapy dramatically reduced signals at high effector-to-target cell ratios, and significant decreases were observed with lower ratios. This model system allowed sensitive, quantitative, real-time spatiotemporal analyses of the dynamics of neoplastic cell growth and facilitated rapid optimization of effective treatment regimens.

  View details for PubMedID 10518573

• Noninvasive assessment of tumor cell proliferation in animal models. Neoplasia Edinger, M., Sweeney, T. J., Tucker, A. A., Olomu, A. B., Negrin, R. S., Contag, C. H. 1999; 1 (4): 303-310

  Abstract

  Revealing the mechanisms of neoplastic disease and enhancing our ability to intervene in these processes requires an increased understanding of cellular and molecular changes as they occur in intact living animal models. We have begun to address these needs by developing a method of labeling tumor cells through constitutive expression of an optical reporter gene, and noninvasively monitoring cellular proliferation in vivo using a sensitive photon detection system. A stable line of HeLa cells that expressed a modified firefly luciferase gene was generated, and proliferation of these cells in irradiated severe combined immunodeficiency (SCID) mice was monitored. Tumor cells were introduced into animals via subcutaneous, intraperitoneal and intravenous inoculation and whole body images, that revealed tumor location and growth kinetics, were obtained. The number of photons that were emitted from the labeled tumor cells and transmitted through murine tissues was sufficient to detect 1x10(3) cells in the peritoneal cavity, 1x10(4) cells at subcutaneous sites and 1x10(6) circulating cells immediately following injection. The kinetics of cell proliferation, as measured by photon emission, was exponential in the peritoneal cavity and at subcutaneous sites. Intravenous inoculation resulted in detectable colonies of tumor cells in animals receiving more than 1x10(6) cells. Our demonstrated ability to detect small numbers of tumor cells in living animals noninvasively suggests that therapies designed to treat minimal disease states, as occur early in the disease course and after elimination of the tumor mass, may be monitored using this approach. Moreover, it may be possible to monitor micrometastases and evaluate the molecular steps in the metastatic process. Spatiotemporal analyses of neoplasia will improve the predictability of animal models of human disease as study groups can be followed over time, and this method will accelerate development of novel therapeutic strategies.

  View details for PubMedID 10935484

• Illuminating drug discovery CHEMISTRY & INDUSTRY Contag, C. H., Contag, P. R. 1999: 664-666

  View details for Web of Science ID 000082466300025

• Transcriptional regulation of heme oxygenase-1 by metalloporphyrins Zhang, W. S., Contag, P. R., Contag, C. H., Stevenson, D. K. NATURE PUBLISHING GROUP. 1999: 72A–72A

  View details for Web of Science ID 000079476700418

• Real-time monitoring of Escherichia coli O157 : H7 adherence to beef carcass surface tissues with a bioluminescent reporter APPLIED AND ENVIRONMENTAL MICROBIOLOGY Siragusa, G. R., Nawotka, K., Spilman, S. D., Contag, P. R., Contag, C. H. 1999; 65 (4): 1738-1745

  Abstract

  A method for studying bacteria that are attached to carcass surfaces would eliminate the need for exogenous sampling and would facilitate understanding the interaction of potential human food-borne pathogens with food animal tissue surfaces. We describe such a method in which we used a bioluminescent reporter strain of Escherichia coli O157:H7 that was constructed by transformation with plasmid pCGLS1, an expression vector that contains a complete bacterial luciferase (lux) operon. Beef carcass surface tissues were inoculated with the bioluminescent strain, and adherent bacteria were visualized in real time by using a sensitive photon-counting camera to obtain in situ images. The reporter strain was found to luminesce from the tissue surfaces whether it was inoculated as a suspension in buffer or as a suspension in a bovine fecal slurry. With this method, areas of tissues inoculated with the reporter strain could be studied without obtaining, excising, homogenizing, and culturing multiple samples from the tissue surface. Use of the complete lux operon as the bioluminescent reporter eliminated the need to add exogenous substrate. This allowed detection and quantitation of bacterial inocula and rapid evaluation of adherence of a potential human pathogen to tissue surfaces. Following simple water rinses of inoculated carcass tissues, the attachment duration varied with different carcass surface types. On average, the percent retention of bioluminescent signal from the reporter strain was higher on lean fascia-covered tissue (54%) than on adipose fascia-covered tissue (18%) following water washing of the tissues. Bioluminescence and culture-derived viable bacterial counts were highly correlated (r2 = 0.98). Real-time assessment of microbial attachment to this complex menstruum should facilitate evaluation of carcass decontamination procedures and mechanistic studies of microbial contamination of beef carcass tissues.

  View details for Web of Science ID 000079530000053

  View details for PubMedID 10103275

• Primary subtype C HIV-1 infection in Harare, Zimbabwe JOURNAL OF ACQUIRED IMMUNE DEFICIENCY SYNDROMES Tien, P. C., Chiu, T., Latif, A., Ray, S., Batra, M., Contag, C. H., Zejena, L., Mbizvo, M., Delwart, E. L., Mullins, J. I., Katzenstein, D. A. 1999; 20 (2): 147-153

  Abstract

  Heterosexual transmission of HIV-1 is widespread in Southern Africa. Heteroduplex mobility assays (HMA) and phylogenetic analyses of V3-V5 envelope (env) gene sequences demonstrate that subtype C predominates in Zimbabwe. To elucidate factors contributing to the epidemic in Zimbabwe, clinical and virologic characteristics of recently acquired subtype C HIV-1 infection among 21 men and 1 woman were determined. In 12 of 19 men providing clinical histories, a sexually transmitted infection preceded serologic evidence of HIV-1, and 14 of 19 men complained of rash or fever before seroconversion. Quantitative p24 antigen levels, reverse transcriptase activity, and HIV RNA levels of 22 viral isolates correlated with in vitro infectivity in peripheral blood mononuclear cells (p < .05). Biologic phenotype assessed in MT-2 cells demonstrated that 3 of 22 isolates (14%) were syncytia inducing (SI) and the remaining 19 nonsyncytium inducing (NSI). Early growth of virus in culture was associated with increased plasma HIV RNA levels, decreased CD4 cell levels, and SI virus. Recent subtype C HIV-1 infection through heterosexual transmission in Zimbabwe demonstrated clinical and virologic features consistent with reports of seroconversion to subtype B viruses.

  View details for Web of Science ID 000078390800006

  View details for PubMedID 10048901

• Bioluminescence for biological sensing in living mammals 26th Annual Meeting of the International-Society-on-Oxygen-Transport-to-Tissue (ISOTT 98) Zhang, W. S., Contag, P. R., Madan, A., Stevenson, D. K., Contag, C. H. KLUWER ACADEMIC/PLENUM PUBL. 1999: 775–784

  View details for Web of Science ID 000085570200089

  View details for PubMedID 10659213

• Noninvasive monitoring of Salmonella infections in young mice Conference on Biomedical Imaging - Reporters, Dyes, and Instrumentation Olomu, I. N., Contag, P. R., Stevenson, D. K., Contag, C. H. SPIE - INT SOC OPTICAL ENGINEERING. 1999: 125–129

  View details for Web of Science ID 000081783800015

• Functional imaging: monitoring heme oxygenase-1 gene expression in vivo Conference on Biomedical Imaging - Reporters, Dyes, and Instrumentation Zhang, W. S., Contag, P. R., Stevenson, D. K., Contag, C. H. SPIE - INT SOC OPTICAL ENGINEERING. 1999: 130–135

  View details for Web of Science ID 000081783800016

• Construction and characterization of a red-emitting luciferase Conference on Biomedical Imaging - Reporters, Dyes, and Instrumentation Eames, B. F., Benaron, D. A., Stevenson, D. K., Contag, C. H. SPIE - INT SOC OPTICAL ENGINEERING. 1999: 36–39

  View details for Web of Science ID 000081783800005

• Functional analysis of tumor cell growth and clearance in living animals Conference on Biomedical Imaging - Reporters, Dyes, and Instrumentation Sweeney, T. J., Mailander, V., Tucker, A. A., Olomu, A. B., Zhang, W. S., Negrin, R. S., Contag, C. H. SPIE - INT SOC OPTICAL ENGINEERING. 1999: 136–139

  View details for Web of Science ID 000081783800017

• Visualizing tumor cell growth and response to therapy in the living host. Sweeney, T. J., Olumo, A. B., Mailander, V., Tucker, A. A., Milnick, I., Contag, C. H., Negrin, R. S. AMER SOC HEMATOLOGY. 1998: 643A–643A

  View details for Web of Science ID 000077121302641

• Bioluminescent indicators in living mammals NATURE MEDICINE Contag, P. R., Olomu, I. N., Stevenson, D. K., Contag, C. H. 1998; 4 (2): 245-247

  View details for Web of Science ID 000072249800043

  View details for PubMedID 9461201

• Visualizing tumor dynamics in vivo. Olomu, A. B., Sweeney, T. J., Mailander, V., Eames, B. F., Negrin, R. S., Stevenson, D. K., Benaron, D. A., Contag, C. H. LIPPINCOTT WILLIAMS & WILKINS. 1998: 169A–169A

  View details for Web of Science ID 000071684700907

• In vivo monitoring of antitumor therapies in intact mice. Sweeney, T. J., Olomu, A. B., Mailander, V., Stevenson, D. K., Contag, C. H., Negrin, R. S. LIPPINCOTT WILLIAMS & WILKINS. 1998: 91A–91A

  View details for Web of Science ID 000071684700479

• Mother-to-infant transmission of human immunodeficiency virus type 1 involving five envelope sequence subtypes JOURNAL OF VIROLOGY Contag, C. H., EHRNST, A., Duda, J., Bohlin, A. B., Lindgren, S., Learn, G. H., Mullins, J. I. 1997; 71 (2): 1292-1300

  Abstract

  Genetic analysis of human immunodeficiency virus type 1 (HIV-1) from cases of mother-to-infant transmission were analyzed in an effort to provide insights into the viral selection that may occur during transmission, as well as the timing and source of transmitted viruses. HIV-1 env genes obtained from seven mothers and their perinatally infected infants in Sweden were studied. Five envelope sequence clades (A to E) were found to be represented. We used a heteroduplex tracking assay (HTA) to assess the genetic relatedness between early viral isolates from the infants and serial maternal virus populations taken during pregnancy and at delivery. HTA findings were used to select for DNA sequence analysis maternal virus populations that were either closely or more distantly related to the infant virus. In each case, nucleotide sequence analysis confirmed the genetic relationships inferred by the HTA. Only maternal peripheral blood was sampled, and large sets of maternal specimens throughout pregnancy were generally not available. However, no consistent correlation was found to support the hypothesis that infant viruses should match blood-derived maternal virus genotypes found early in pregnancy if infants were found to be infected at birth or, conversely, that infant viruses should match blood-derived maternal virus genotypes found at delivery if infants were found to be infected only some time later.

  View details for Web of Science ID A1997WC30500053

  View details for PubMedID 8995653

• Simian immunodeficiency virus from Old World monkeys The Human Retroviruses Contag, Christopher H., Stephen Dewhurst, Gregory A. Viglianti, James I. Mullins 1991: 245-276