Biomedical subjects

S S Gambhir

Publications and source records attributed to S S Gambhir.

At least 19 recordsLinked to original sources

PubMed2006

Diagnosis of aseptic deep venous thrombosis of the upper extremity in a cancer patient using fluorine-18 fluorodeoxyglucose positron emission tomography/computerized tomography (FDG PET/CT).

We describe a patient with a history of recurrent squamous cell carcinoma of the tongue and abnormal FDG uptake in the left arm during a re-staging FDG PET/CT. After revision of the patient's clinical history, tests and physical exam, the abnormal FDG uptake was found to correspond to an extensive aseptic deep venous thrombosis of the upper extremity.

Carcinoma, Squamous Cell↗

PubMed2005

Quantum dots for live cells, in vivo imaging, and diagnostics.

Research on fluorescent semiconductor nanocrystals (also known as quantum dots or qdots) has evolved over the past two decades from electronic materials science to biological applications. We review current approaches to the synthesis, solubilization, and functionalization of qdots and their applications to cell and animal biology. Recent examples of their experimental use include the observation of diffusion of individual glycine receptors in living neurons and the identification of lymph nodes in live animals by near-infrared emission during surgery. The new generations of qdots have far-reaching potential for the study of intracellular processes at the single-molecule level, high-resolution cellular imaging, long-term in vivo observation of cell trafficking, tumor targeting, and diagnostics.

Animals↗

PubMed2005

Non-invasive imaging of a transgenic mouse model using a prostate-specific two-step transcriptional amplification strategy.

Non-invasive assessment of transgenic animals using bioluminescence imaging offers a rapid means of evaluating disease progression in animal models of disease. One of the challenges in the field is to develop models with robust expression to image repetitively live intact animals through solid tissues. The prostate-specific antigen (PSA) promoter is an attractive model for studying gene regulation due to its hormonal response and tissue-specificity permitting us to measure signaling events that occur within the native tissues. The use of the GAL4-VP16 activator offers a powerful means to augment gene expression levels driven by a weak promoter. We have used a two-step transcriptional amplification (TSTA) system to develop a transgenic mouse model to investigate the tissue-specificity and developmental regulation of firefly luciferase (fl) gene expression in living mice using bioluminescence imaging. We employed an enhanced prostate-specific promoter to drive the yeast transcriptional activator, GAL4-VP16 (effector). The reporter construct carries five Gal4 binding sites upstream of the fl gene. We generated a transgenic mouse model using a single vector carrying the effector and reporter constructs. The transgenic mice show prostate-specific expression as early as three weeks of age. The bioluminescence signal in the prostate is significantly higher than in other organs. We also demonstrate that blocking androgen availability can downregulate the fl expression in the prostate. The transgenic mice display normal physical characteristics and developmental behavior, indicating that the high level of GAL4 driven expression is well tolerated. These findings suggest that the GAL4-VP16 transactivator can be used to amplify reporter gene expression from a relatively weak promoter in a transgenic mouse model. The transgenic TSTA model in conjunction with other transgenic cancer models should also help to detect and track malignancies. The strategies developed will be useful for transgenic research in general by allowing for amplified tissue specific gene expression.

Animals↗

PubMed2004

Molecular imaging of gene expression in living subjects by spliceosome-mediated RNA trans-splicing.

Spliceosome-mediated RNA trans-splicing (SMaRT) provides an effective means to reprogram mRNAs and the proteins they encode. SMaRT technology has a broad range of applications, including RNA repair and molecular imaging, each governed by the nature of the sequences delivered by the pre-trans-splicing molecule. Here, we show the ability of SMaRT to optically image the expression of an exogenous gene at the level of pre-mRNA splicing in cells and living animals. Because of the modular design of pre-trans-splicing molecules, there is great potential to employ SMaRT to image the expression of any arbitrary gene of interest in living subjects. In this report, we describe a model system that demonstrates the feasibility of imaging gene expression by transsplicing in small animals. This represents a previously undescribed approach to molecular imaging of mRNA levels in living subjects.

Animals↗

PubMed2004

Gene therapy progress and prospects: noninvasive imaging of gene therapy in living subjects.

Recent progress in the development of noninvasive imaging technologies should allow molecular imaging to play a major role in the field of gene therapy. These tools have recently been validated in gene therapy models for continuous quantitative monitoring of the location(s), magnitude, and time variation of gene delivery and/or expression. This article reviews the use of radionuclide, magnetic resonance, and optical imaging technologies, as they have been used in imaging gene delivery and gene expression for gene therapy applications. The studies published to date lend support that noninvasive imaging tools will help to accelerate preclinical model validation, as well as allow for clinical monitoring of human gene therapy.

Cardiovascular Diseases↗

PubMed2004

MicroPET imaging of Cre-loxP-mediated conditional activation of a herpes simplex virus type 1 thymidine kinase reporter gene.

Site-specific recombination tools such as the Cre-loxP system are used to create animal models where conditional gene deletion/activation studies are required. In the current proof of principle study, we have demonstrated that a PET reporter gene (PRG), the herpes simplex virus type 1 thymidine kinase (HSV1-tk), can be made to remain silent and can be activated by Cre-loxP-mediated recombination in cell culture and in living mice. An adenovirus carrying a silent HSV1-tk was tail-vein injected (1 x 10(9) PFU) in six transgenic mice that express Cre recombinase in their liver (Cre+) and in four control mice (Cre-). The liver-specific expression of the PRG in Cre+ mice was detected in the microPET following injection of the reporter probe, 9-[4-fluoro-3-(hydroxymethyl)butyl]guanine ([(18)F]-FHBG). The [(18)F]-FHBG accumulation in the liver in terms of percent-injected dose per gram of tissue was 7.72+/-1.13 for the Cre+ mice and 0.10+/-0.02 for the Cre- mice (P<0.05) 48 h after adenoviral injection. These results were further validated by quantitative RT-PCR, western blotting and by in vitro assays for herpes simplex virus type 1 thymidine kinase enzyme activity. Thus by using the Cre-loxP system it is possible to modulate a PRG and noninvasively monitor the extent of Cre-loxP-mediated gene activation by imaging in a microPET scanner.

Adenoviridae↗

PubMed2003

Monitoring protein-protein interactions using split synthetic renilla luciferase protein-fragment-assisted complementation.

In this study we developed an inducible synthetic renilla luciferase protein-fragment-assisted complementation-based bioluminescence assay to quantitatively measure real time protein-protein interactions in mammalian cells. We identified suitable sites to generate fragments of N and C portions of the protein that yield significant recovered activity through complementation. We validate complementation-based activation of split synthetic renilla luciferase protein driven by the interaction of two strongly interacting proteins, MyoD and Id, in five different cell lines utilizing transient transfection studies. The expression level of the system was also modulated by tumor necrosis factor alpha through NFkappaB-promoter/enhancer elements used to drive expression of the N portion of synthetic renilla luciferase reporter gene. This new system should help in studying protein-protein interactions and when used with other split reporters (e.g., split firefly luciferase) should help to monitor different components of an intracellular network.

Amino Acid Sequence↗

PubMed2003

Optical imaging of transferrin targeted PEI/DNA complexes in living subjects.

Noninvasive optical bioluminescence imaging systems are important tools for evaluating gene expression in vivo for study of individual and temporal variation in a living animal. In this report, we demonstrate that expression of the firefly luciferase reporter gene (fl) delivered by transferrin (Tf) targeted polyethylenimine (PEI) complexes with, or without, poly(ethylene glycol) (PEG) modifications can be imaged in living A/J mice bearing N2A tumors using a cooled charged coupled device (CCD) camera. Tf-PEI-PEG, Tf-PEI, and PEI (positive control) complexes were tail-vein injected and mice were imaged at 5, 24, 48, and 72 h after complex injection. After imaging, the organs were analyzed ex vivo for firefly luciferase protein (FL) activity. The Tf and PEG modified formulations show significantly (P<0.05) higher FL activity in vivo and ex vivo at the tumor as compared to other organs, including the lungs (a site of high expression with PEI, the positive control). Furthermore, the in vivo bioluminescent signal correlated well (R(2)=0.83) with ex vivo FL activity. These data support that noninvasive imaging of fl reporter expression can be used to monitor the specificity of Tf-PEI and Tf-PEI-PEG polyplex targeting of N2A tumors in A/J mice.

Animals↗

PubMed2002

Noninvasive imaging of protein-protein interactions in living subjects by using reporter protein complementation and reconstitution strategies.

In this study we have developed bioluminescence-imaging strategies to noninvasively and quantitatively image protein-protein interactions in living mice by using a cooled charge-coupled device camera and split reporter technology. We validate both complementation and intein-mediated reconstitution of split firefly luciferase proteins driven by the interaction of two strongly interacting proteins, MyoD and Id. We use transient transfection of cells and image MyoD-Id interaction after induction of gene expression in cell culture and in cells implanted into living mice. Techniques to study protein-protein interactions in living subjects will allow the study of cellular networks, including signal transduction pathways, as well as development and optimization of pharmaceuticals for modulating protein-protein interactions.

Animals↗

PubMed2002

Noninvasive quantitative imaging of protein-protein interactions in living subjects.

We are developing methods to image molecular and cellular events in living subjects. In this study, we validate imaging of protein-protein interactions in living mice by using bioluminescent optical imaging. We use the well studied yeast two-hybrid system adapted for mammalian cells and modify it to be inducible. We employ the NF-kappaB promoter to drive expression of two fusion proteins (VP16-MyoD and GAL4-ID). We modulate the NF-kappaB promoter through tumor necrosis factor alpha. Firefly luciferase reporter gene expression is driven by the interaction of MyoD and ID through a transcriptional activation strategy. We demonstrate the ability to detect this induced protein-protein interaction in cell culture and image this induced interaction in living mice by using transiently transfected cells. The current approach will be a valuable and potentially generalizable tool to noninvasively and quantitatively image protein-protein interactions in living subjects. The approaches validated should have important implications for the study of protein-protein interactions in cells maintained in their natural in vivo environment as well as for the in vivo evaluation of new pharmaceuticals targeted to modulate protein-protein interactions.

Animals↗

PubMed2002

Monitoring adenoviral DNA delivery, using a mutant herpes simplex virus type 1 thymidine kinase gene as a PET reporter gene.

Current gene therapy protocols often suffer from an inability to monitor the site, level and persistence of gene expression following somatic DNA delivery. Herpes simplex virus 1 thymidine kinase (HSV1-tk) is currently under intensive investigation as a reporter gene for in vivo imaging of reporter gene expression. The presence of the HSV1-tk reporter gene is repetitively and non-invasively monitored by systemic injection of positron-emitting, radionuclide-labeled thymidine analogues or acycloguanosine HSV1-TK substrates and subsequent detection, by positron emission tomography, of trapped, phosphorylated product. To improve the efficacy of the HSV1-tk PET reporter gene system, both alternative substrates and mutations in the HSV1-tk gene have been described. We used a replication defective adenovirus to deliver the HSV1-sr39tk mutant enzyme and the wild-type HSV1-tk enzyme to mice. HSV1-sr39TK demonstrates greater sensitivity than wild-type HSV1-TK enzyme in vivo, using 9-[(4-[(18)F]fluoro-3-hydroxymethylbutyl)guanine as probe, following adenovirus-mediated hepatic expression in mice. Using this adenoviral delivery system, the location, magnitude and duration of HSV1-sr39tk PET reporter gene expression could be non-invasively, quantitatively and repetitively monitored for over 3 months by microPET.

Adenoviridae↗

PubMed2001

Optical imaging of Renilla luciferase reporter gene expression in living mice.

Imaging reporter gene expression in living subjects is a rapidly evolving area of molecular imaging research. Studies have validated the use of reporter genes with positron emission tomography (PET), single photon emission computed tomography (SPECT), MRI, fluorescence with wild-type and mutants of green fluorescent protein, as well as bioluminescence using Firefly luciferase enzyme/protein (FL). In the current study, we validate for the first time the ability to image bioluminescence from Renilla luciferase enzyme/protein (RL) by injecting the substrate coelenterazine in living mice. A highly sensitive cooled charge-coupled device camera provides images within a few minutes of photon counting. Cells, transiently expressing the Rluc were imaged while located in the peritoneum, s.c. layer, as well as in the liver and lungs of living mice tail-vein injected with coelenterazine. Furthermore, d-luciferin (a substrate for FL) does not serve as a substrate for RL, and coelenterazine does not serve as a substrate for FL either in cell culture or in living mice. We also show that both Rluc and Fluc expression can be imaged in the same living mouse and that the kinetics of light production are distinct. The approaches validated will have direct applications to various studies where two molecular events need to be tracked, including cell trafficking of two cell populations, two gene therapy vectors, and indirect monitoring of two endogenous genes through the use of two reporter genes.

Animals↗

PubMed2001

Two-step transcriptional amplification as a method for imaging reporter gene expression using weak promoters.

We are developing assays to image tissue-specific reporter gene expression in living mice by using optical methods and positron emission tomography. Approaches for imaging reporter gene expression depend on robust levels of mRNA and reporter protein. Attempts to image reporter gene expression driven by weak promoters are often hampered by the poor transcriptional activity of such promoters. Most tissue-specific promoters are weak relative to stronger but constitutively expressing viral promoters. In this study, we have validated methods to enhance the transcriptional activity of the prostate-specific antigen promoter for imaging by using a two-step transcriptional amplification (TSTA) system. We used the TSTA system to amplify expression of firefly luciferase (fl) and mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk) in a prostate cancer cell line (LNCaP). We demonstrate approximately 50-fold (fl) and approximately 12-fold (HSV1-sr39tk) enhancement by using the two-step approach. The TSTA system is observed to retain tissue selectivity. A cooled charge-coupled device optical imaging system was used to visualize the amplified fl expression in living mice implanted with LNCaP cells transfected ex vivo. These imaging experiments reveal a approximately 5-fold gain in imaging signal by using the TSTA system over the one-step system. The TSTA approach will be a valuable and generalizable tool to amplify and noninvasively image reporter gene expression in living animals by using tissue-specific promoters. The approaches validated should have important implications for study of gene therapy vectors, cell trafficking, transgenic models, as well as studying development of eukaryotic organisms.

Animals↗

PubMed2001

Positron emission tomography in evaluation of dementia: Regional brain metabolism and long-term outcome.

CONTEXT: Deficits in cerebral glucose utilization have been identified in patients with cognitive dysfunction attributed to various disease processes, but their prognostic and diagnostic value remains to be defined. OBJECTIVE: To assess the sensitivity and specificity with which cerebral metabolic patterns at a single point in time forecast subsequent documentation of progressive dementia. DESIGN, SETTING, AND PATIENTS: Positron emission tomography (PET) studies of [(18)F]fluorodeoxyglucose in 146 patients undergoing evaluation for dementia with at least 2 years' follow-up for disease progression at the University of California, Los Angeles, from 1991 to 2000, and PET studies in 138 patients undergoing evaluation for dementia at an international consortium of facilities, with histopathological diagnoses an average of 2.9 years later, conducted from 1984 to 2000. MAIN OUTCOME MEASURES: Regional distribution of [(18)F]fluorodeoxyglucose in each patient, classified by criteria established a priori as positive or negative for presence of a progressive neurodegenerative disease in general and of Alzheimer disease (AD) specifically, compared with results of longitudinal or neuropathologic analyses. RESULTS: Progressive dementia was detected by PET with a sensitivity of 93% (191/206) and a specificity of 76% (59/78). Among patients with neuropathologically based diagnoses, PET identified patients with AD and patients with any neurodegenerative disease with a sensitivity of 94% and specificities of 73% and 78%, respectively. The negative likelihood ratio of experiencing a progressive vs nonprogressive course over the several years following a single negative brain PET scan was 0.10 (95% confidence interval, 0.06-0.16), and the initial pattern of cerebral metabolism was significantly associated with the subsequent course of progression overall (P<.001). CONCLUSION: In patients presenting with cognitive symptoms of dementia, regional brain metabolism was a sensitive indicator of AD and of neurodegenerative disease in general. A negative PET scan indicated that pathologic progression of cognitive impairment during the mean 3-year follow-up was unlikely to occur.

Aged↗

PubMed2001

Noninvasive optical imaging of firefly luciferase reporter gene expression in skeletal muscles of living mice.

The ability to monitor reporter gene expression noninvasively offers significant advantages over current techniques such as postmortem tissue staining or enzyme activity assays. Here we demonstrate a novel method of repetitively tracking in vivo gene expression of firefly luciferase (FL) in skeletal muscles of mice using a cooled charged coupled device (CCD) camera. We first show that the cooled CCD camera provides consistent and reproducible results within +/-8% standard deviation from mean values, and a detection sensitivity (range tested: 1 x 10(4) - 1 x 10(9) plaque form-ing units (pfu)) of 1 x 10(6) pfu of E1-deleted adenovirus expressing FL driven by a cytomegalovirus promoter (Ad-CMV-FL). The duration and magnitude of adenoviral mediated (1 x 10(9) pfu) FL gene expression were then followed over time. FL gene expression in immunocompetent Swiss Webster mice peaks within the first 48 hours, falls by 98% after 20 days, and persists for >150 days. In contrast, FL activity in nude mice remains elevated for >110 days. Finally, transduced Swiss Webster and nude mice were sacrificed to show that the in vivo CCD signals correlate well with in vitro luciferase enzyme assays (r(2)=0.91 and 0.96, respectively). Our findings demonstrate the ability of the cooled CCD camera to sensitively and noninvasively track the location, magnitude, and persistence of FL gene expression. Monitoring of gene therapy studies in small animals may be aided considerably with further extensions of this technique.

Adenoviridae↗

PubMed2001

Direct correlation between positron emission tomographic images of two reporter genes delivered by two distinct adenoviral vectors.

Biodistribution, magnitude and duration of a therapeutic transgene's expression may be assessed by linking it to the expression of a positron emission tomography (PET) reporter gene (PRG) and then imaging the PRG's expression by a PET reporter probe (PRP) in living animals. We validate the simple approach of co-administering two distinct but otherwise identical adenoviruses, one expressing a therapeutic transgene and the other expressing the PRG, to track the therapeutic gene's expression. Two PET reporter genes, a mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk) and dopamine-2 receptor (D(2)R), each regulated by the same cytomegalovirus (CMV) promoter, have been inserted into separate adenoviral vectors (Ad). We demonstrate that cells co-infected with equivalent titers of Ad-CMV-HSV1-sr39tk and Ad-CMV-D(2)R express both reporter genes with good correlation (r(2) = 0.93). Similarly, a high correlation (r(2) = 0.97) was observed between the expression of both PRGs in the livers of mice co-infected via tail-vein injection with equivalent titers of these two adenoviruses. Finally, microPET imaging of HSV1-sr39tk and D(2)R expression with 9-(4-[(18)F]fluoro-3-hydroxymethylbutyl) guanine ([(18)F]FHBG) and 3-(2-[(18)F]fluoroethyl)spiperone ([(18)F]FESP), utilizing several adenovirus-mediated delivery routes, illustrates the feasibility of evaluating relative levels of transgene expression in living animals, using this approach.

Adenoviridae↗

PubMed2001

Noninvasive, quantitative imaging in living animals of a mutant dopamine D2 receptor reporter gene in which ligand binding is uncoupled from signal transduction.

The dopamine D2 receptor (D2R) has been used in adenoviral delivery systems and in tumor cell xenografts as an in vivo reporter gene. D2R reporter gene expression has been non-invasively, repetitively and quantitatively imaged by positron emission tomography (PET), following systemic injection of a positron-labeled ligand (3-(2'-[18F]-fluoroethyl)-spiperone; FESP) and subsequent D2R-dependent sequestration. However, dopamine binding to the D2R can modulate cyclic AMP levels. For optimal utilization of D2R as a reporter gene, it is important to uncouple ligand-binding from Gi-protein-mediated inhibition of cAMP production. Mutation of Asp80 or Ser194 produces D2Rs that still bind [3H]spiperone in transfected cells. The D2R80A mutation completely eliminates the ability of the D2R to suppress forskolin-stimulated cAMP accumulation in response to dopamine, in cells transfected with a D2R80A expression plasmid and in cells infected with replication-defective adenovirus expressing D2R80A. The D2R194A mutation substantially reduces, but does not completely eliminate, dopamine modulation of cAMP levels. Cultured cells infected with adenoviruses expressing D2R and D2R80A demonstrated equivalent [3H]spiperone binding activity. Moreover, hepatic FESP sequestration is equivalent, following intravenous injection of adenoviruses expressing D2R and D2R80A. The D2R80A mutant, which can no longer modulate cAMP levels following ligand binding, has full capability as a PET reporter gene.

Adenoviridae↗

PubMed2001

Quantitative imaging of gene induction in living animals.

Methods to repeatedly, non-invasively, and quantitatively image gene expression in living animals are rapidly emerging and should fundamentally change studies of gene expression in vivo. We previously developed assays utilizing positron emission tomography (PET) to image reporter gene expression. In this paper we: (1) describe a new bi-directional, tetracycline-inducible system that can be used to pharmacologically induce target gene expression and to quantitatively image induced expression by using a PET reporter gene; (2) demonstrate the potential of this system in transient and stable cell transfection assays; and (3) demonstrate the ability to repetitively and quantitatively image tetracycline and tetracycline analog induction of gene expression in living animals. We utilize the dopamine type-2 receptor (D(2)R) and the mutant herpes-simplex virus type 1 thymidine kinase (HSV1-sr39tk) reporter genes to validate this system. We utilize microPET technology to show that quantitative tomographic imaging of gene induction is possible. We find a high correlation (r(2) = 0.98) between 'target' and reporter gene expression. This work establishes a new technique for imaging time-dependent variation of gene expression both from vectors with inducible promoters and in transgenic animals in which pharmacologic induction of gene expression must be monitored. These techniques may be applied both in gene therapy and for the study of gene expression in transgenic animals.

Animals↗