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Biomedical subjects

Christopher H Contag

Publications and source records attributed to Christopher H Contag.

At least 19 recordsLinked to original sources
PubMed2007

Miniature near-infrared dual-axes confocal microscope utilizing a two-dimensional microelectromechanical systems scanner.

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.

Computer Systems↗
PubMed2007

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.

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.

Animals↗
PubMed2006

In vivo dynamics of regulatory T-cell trafficking and survival predict effective strategies to control graft-versus-host disease following allogeneic transplantation.

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.

Animals↗
PubMed2006

Early CD30 signaling is critical for adoptively transferred CD4+CD25+ regulatory T cells in prevention of acute graft-versus-host disease.

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.

Acute Disease↗
PubMed2006

Luciferin derivatives for enhanced in vitro and in vivo bioluminescence assays.

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.

Animals↗
PubMed2006

Expression and regulation of heme oxygenase isozymes in the developing mouse cortex.

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.

Animals↗
PubMed2006

Releasable luciferin-transporter conjugates: tools for the real-time analysis of cellular uptake and release.

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.

2,2'-Dipyridyl↗
PubMed2006

Synergistic antitumor effects of immune cell-viral biotherapy.

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.

Animals↗
PubMed2006

Enhanced killing of primary ovarian cancer by retargeting autologous cytokine-induced killer cells with bispecific antibodies: a preclinical study.

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.

Animals↗
PubMed2006

Inhibition of CD4+CD25+ regulatory T-cell function by calcineurin-dependent interleukin-2 production.

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.

Animals↗
PubMed2006

Selective intratumoral amplification of an antiangiogenic vector by an oncolytic virus produces enhanced antivascular and anti-tumor efficacy.

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.

Adenocarcinoma↗
PubMed2006

Spatiotemporal expression of heme oxygenase-1 detected by in vivo bioluminescence after hepatic ischemia in HO-1/Luc mice.

Upregulation of heme oxygenase-1 (HO-1) has been proposed as a critical mechanism protecting against cellular stress during liver transplantation, providing a potential target for new therapeutic interventions. We investigated the feasibility of in vivo bioluminescence imaging (BLI) to noninvasively quantify the spatiotemporal expression of HO-1 after warm hepatic ischemia in living animals. Luciferase activity was measured by BLI as an index of HO-1 transcription in transgenic reporter mice (Ho1-luc) at standardized time points after 60 minutes of warm hepatic ischemia. HO-1 mRNA levels were measured in postischemic livers of mice sacrificed at the same time points in separate experiments. Bioluminescent signals from postischemic liver lobes were first detected at 3 hours after reperfusion. Peak levels were reached at 9 hours, after which bioluminescent activity declined and returned to baseline values at 48 hours after reperfusion. Upregulation of HO-1 as detected by in vivo BLI was preceded by increased HO-1 mRNA expression and confirmed by enhanced immunohistochemical staining of hepatocytes. In conclusion, this study shows that in vivo BLI allows a sensitive assessment of HO-1 expression after hepatic ischemia in living animals. The capability of whole-body temporal imaging of HO-1 expression provides a valuable tool in the development of novel strategies to modulate HO-1 expression in liver transplantation.

Animals↗
PubMed2006

Molecular imaging using visible light to reveal biological changes in the brain.

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.

Animals↗
PubMed2006

In vivo imaging using bioluminescence: a tool for probing graft-versus-host disease.

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.

Animals↗
PubMed2006

Induced biliary excretion of Listeria monocytogenes.

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.

Animals↗
PubMed2006

New enzyme for reductive cancer chemotherapy, YieF, and its improvement by directed evolution.

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.

Antineoplastic Agents↗
PubMed2006

Options for visualizing metastatic disease in the living body.

Detection and observation of primary tumor growth and metastasis in living subjects is an important task in clinical and basic cancer research. Recently several approaches and techniques emerged which offer a huge variety of options with respect to the specific objectives and questions of a given study. Recent developments in the field of in vivo imaging not only allow the assessment of anatomic information but also functional processes with cellular resolution and molecular sensitivity. This chapter will provide an overview of the most common imaging techniques which are currently available for the detection and observation of metastasizing tumor cells. General capacities, advantages, limitations and drawbacks will be discussed. These techniques include computed tomography (CT), molecular resonance imaging (MRI), positron emission tomography (PET), single photon emission computed tomography (SPECT), fluorescence imaging (FI), and bioluminescent imaging (BLI). The objective is to provide the cancer researcher with information that will help solve the dilemma of how best to apply the latest imaging tools for studying biological questions in the context of the living body.

Animals↗