Surface biotinylation of cytotoxic T lymphocytes for in vivo tracking of tumor immunotherapy in murine models.

Currently, there is no stable and flexible method to label and track cytotoxic T lymphocytes (CTLs) in vivo in CTL immunotherapy. We aimed to evaluate whether the sulfo-hydroxysuccinimide (NHS)-biotin-streptavidin (SA) platform could chemically modify the cell surface of CTLs for in vivo tracking. CD8+ T lymphocytes were labeled with sulfo-NHS-biotin under different conditions and then incubated with SA-Alexa647.

Imaging Tumor Vascularity and Response to Anti-Angiogenic Therapy Using Gaussia Luciferase.

We developed a novel approach to assess tumor vascularity using recombinant Gaussia luciferase (rGluc) protein and bioluminescence imaging. Upon intravenous injection of rGluc followed by its substrate coelenterazine, non-invasive visualization of tumor vascularity by bioluminescence imaging was possible. We applied this method for longitudinal monitoring of tumor vascularity in response to the anti-angiogenic drug tivozanib.

Simultaneous in vivo monitoring of regulatory and effector T lymphocytes using secreted Gaussia luciferase, Firefly luciferase, and secreted alkaline phosphatase.

Regulatory T cells (Tregs) are amongst the most widely studied cells in a variety of immune-mediated conditions, including transplantation and Graft Versus Host Disease (GVHD), cancer and autoimmunity; indeed, there is great interest in the tolerogenic potential of Treg-based therapy. Consequently, the need to establish the mechanisms that determine Treg survival and longevity, in addition to developing new tools to monitor these parameters, is paramount. Using both a mouse model of GVHD and a mouse model of Type 1 Diabetes (T1D), we describe herein a dual reporter system based on Gluc and multiplexed with SEAP and non-secreted Firefly luciferase (Fluc), which permits simultaneous imaging and noninvasive tracking of two different T-cell populations (CD4(+)CD25(+) Tregs and CD4(+)CD25(-) Tcon cells) in vivo by transducing the cells with different lentiviruses bearing distinct color signatures.

Effects of the selective MPS1 inhibitor MPS1-IN-3 on glioblastoma sensitivity to antimitotic drugs.

Background: Glioblastomas exhibit a high level of chemotherapeutic resistance, including to the antimitotic agents vincristine and taxol. During the mitotic agent-induced arrest, glioblastoma cells are able to perform damage-control and self-repair to continue proliferation. Monopolar spindle 1 (MPS1/TTK) is a checkpoint kinase and a gatekeeper of the mitotic arrest.

Directed molecular evolution reveals Gaussia luciferase variants with enhanced light output stability.

Gaussia Luciferase (Gluc) has proven to be a powerful mammalian cell reporter for monitoring numerous biological processes in immunology, virology, oncology, and neuroscience. Current limitations of Gluc as a reporter include its emission of blue light, which is absorbed by mammalian tissues, limiting its use in vivo, and a flash-type bioluminescence reaction, making it unsuited for high-throughput applications. To overcome these limitations, a library of Gluc variants was generated using directed molecular evolution and screened for relative light output, a shift in emission spectrum, and glow-type light emission kinetics.

Single reporter for targeted multimodal in vivo imaging.

We have developed a multifaceted, highly specific reporter for multimodal in vivo imaging and applied it for detection of brain tumors. A metabolically biotinylated, membrane-bound form of Gaussia luciferase was synthesized, termed mbGluc-biotin. We engineered glioma cells to express this reporter and showed that brain tumor formation can be temporally imaged by bioluminescence following systemic administration of coelenterazine.

Evaluation of first-draw whole blood, point-of-care cardiac markers in the context of the universal definition of myocardial infarction: a comparison of a multimarker panel to troponin alone and to testing in the central laboratory.

Context: Previous studies evaluating point-of-care testing (POCT) for cardiac biomarkers did not use current recommendations for troponin cutoff values or recognize the recent universal definition of acute myocardial infarction. Traditionally, achieving optimal sensitivity for the detection of myocardial injury on initial presentation required combining cardiac troponin and/or creatine kinase isoenzyme MB with an early marker, usually myoglobin. In recent years, the performance of central laboratory combining cardiac troponin assays has improved significantly, potentially obviating the need for a multimarker panel to achieve optimum sensitivity.

Multimodal in vivo imaging and blood monitoring of intrinsic and extrinsic apoptosis.

Noninvasive detection and in vivo imaging of apoptosis plays a critical role in the development of therapeutics in many different fields including cancer. We have developed an apoptosis biosensor by fusing green fluorescent protein (GFP) to the N-terminus of the naturally secreted Gaussia luciferase separated by a caspase-3 cleavage peptide consisting of aspartic acid (D), glutamic acid (E), valine (V), and aspartic acid (D) or DEVD. We showed that this fusion is retained in the cytoplasm of cells in an inactive form.