Internists, Oncology Specialist (cancer), Obstetrician & Gynecologist (OB/GYN)
16 years of experience

Accepting new patients
Fenway - Kenmore - Audubon Circle - Longwood
Beth Israel Deaconess Medical Center
330 Brookline Ave
Boston, MA 02215
617-667-0642
Locations and availability (2)

Education ?

Medical School Score Rankings
Harvard University (1994)
  • Currently 4 of 4 apples
Top 25%

Awards & Distinctions ?

Appointments
Wmdhs

Affiliations ?

Dr. Frangioni is affiliated with 1 hospitals.

Hospital Affilations

Score

Rankings

  • Beth Israel Deaconess Medical Center
    Medical Oncology
    330 Brookline Ave, Boston, MA 02215
    • Currently 3 of 4 crosses
    Top 50%
  • Publications & Research

    Dr. Frangioni has contributed to 84 publications.
    Title Nerve-highlighting Fluorescent Contrast Agents for Image-guided Surgery.
    Date October 2011
    Journal Molecular Imaging
    Excerpt

    Nerve damage is the major morbidity of many surgeries, resulting in chronic pain, loss of function, or both. The sparing of nerves during surgical procedures is a vexing problem because surrounding tissue often obscures them. To date, systemically administered nerve-highlighting contrast agents that can be used for nerve-sparing image-guided surgery have not been reported. In the current study, physicochemical and optical properties of 4,4'-[(2-methoxy-1,4-phenylene)di-(1E)-2,1-ethenediyl]bis-benzenamine (BMB) and a newly synthesized, red-shifted derivative 4-[(1E)-2-[4-[(1E)-2-[4-aminophenyl]ethenyl]-3-methoxyphenyl]ethenyl]-benzonitrile (GE3082) were characterized in vitro and in vivo. Both agents crossed the blood-nerve barrier and blood-brain barrier and rendered myelinated nerves fluorescent after a single systemic injection. Although both BMB and GE3082 also exhibited significant uptake in white adipose tissue, GE3082 underwent a hypsochromic shift in adipose tissue that provided a means to eliminate the unwanted signal using hyperspectral deconvolution. Dose and kinetic studies were performed in mice to determine the optimal dose and drug-imaging interval. The results were confirmed in rat and pig, with the latter used to demonstrate, for the first time, simultaneous fluorescence imaging of blood vessels and nerves during surgery using the FLARE™ (Fluorescence-Assisted Resection and Exploration) imaging system. These results lay the foundation for the development of ideal nerve-highlighting fluorophores for image-guided surgery.

    Title Ease of Synthesis, Controllable Sizes, and in Vivo Large-animal-lymph Migration of Polymeric Nanoparticles.
    Date January 2011
    Journal Chemmedchem
    Title Intraoperative Localization of Insulinoma and Normal Pancreas Using Invisible Near-infrared Fluorescent Light.
    Date June 2010
    Journal Annals of Surgical Oncology
    Excerpt

    Neuroendocrine tumors of the pancreas, such as insulinoma, are difficult to localize, and complete resection is essential for cure. Our hypothesis is that a near-infrared (NIR) fluorophore exhibiting uptake in insulinoma could provide high-sensitivity detection intraoperatively.

    Title Nanotechnology in Thoracic Surgery.
    Date June 2010
    Journal The Annals of Thoracic Surgery
    Excerpt

    Nanotechnology is an exciting and rapidly progressing field offering potential solutions to multiple challenges in the diagnosis and treatment of lung cancer, with the potential for improving imaging and mapping techniques, drug delivery, and ablative therapy. With promising preclinical results in many applications directly applicable to thoracic oncology, it is possible that the frontiers of minimally invasive thoracic surgery will eventually be explored at the nanoscale.

    Title Image-guided Sentinel Lymph Node Mapping and Nanotechnology-based Nodal Treatment in Lung Cancer Using Invisible Near-infrared Fluorescent Light.
    Date June 2010
    Journal Seminars in Thoracic and Cardiovascular Surgery
    Excerpt

    Current methods for sentinel lymph node (SLN) mapping and nodal treatment in lung cancer remain inadequate for routine clinical use. In this study, we discuss the potential for using the combination of invisible near-infrared (NIR) fluorescent light and nanotechnology for these applications. NIR fluorescence imaging has recently received significant attention for in vivo imaging applications because of its low tissue autofluorescence, high photon penetration into living tissue, and high signal-to-background ratio. Our large animal in vivo studies have been able to successfully identify SLNs in lung tissue, and several clinical studies have examined the use of NIR fluorescence imaging systems for SLN mapping in breast and gastric cancer. Promising new nanoparticle technologies, when combined with NIR fluorescence imaging, offer the potential for image-guided treatment of lymph nodes at high risk for tumor recurrence. This review provides a theoretic and empiric framework for developing the next generation of diagnostic and therapeutic agents for lung cancer.

    Title Annexin A2 is a Molecular Target for Tm601, a Peptide with Tumor-targeting and Anti-angiogenic Effects.
    Date May 2010
    Journal The Journal of Biological Chemistry
    Excerpt

    TM601 is a synthetic form of chlorotoxin, a 36-amino acid peptide derived from the venom of the Israeli scorpion, Leirius quinquestriatus, initially found to specifically bind and inhibit the migration of glioma cells in culture. Subsequent studies demonstrated specific in vitro binding to additional tumor cell lines. Recently, we demonstrated that proliferating human vascular endothelial cells are the only normal cell line tested that exhibits specific binding to TM601. Here, we identify annexin A2 as a novel binding partner for TM601 in multiple human tumor cell lines and human umbilical vein endothelial cell (HUVEC). We demonstrate that the surface binding of TM601 to the pancreatic tumor cell line Panc-1 is dependent on the expression of annexin A2. Identification of annexin A2 as a binding partner for TM601 is also consistent with the anti-angiogenic effects of TM601. Annexin A2 functions in angiogenesis by binding to tissue plasminogen activator and regulating plasminogen activation on vascular endothelial cells. We demonstrate that in HUVECs, TM601 inhibits both vascular endothelial growth factor- and basic fibroblast growth factor-induced tissue plasminogen activator activation, which is required for activation of plasminogen to plasmin. Consistent with inhibition of cell surface protease activity, TM601 also inhibits platelet-derived growth factor-C induced trans-well migration of both HUVEC and U373-MG glioma cells.

    Title First-in-human Clinical Trials of Imaging Devices: an Example from Optical Imaging.
    Date March 2010
    Journal Conference Proceedings : ... Annual International Conference of the Ieee Engineering in Medicine and Biology Society. Ieee Engineering in Medicine and Biology Society. Conference
    Excerpt

    Clinical translation of scientific discoveries is often the long-term goal of academic medical research. However, this goal is not always realized due to the complicated path between bench research and clinical use. In this review, we outline the fundamental steps required for first-in-human testing of a new imaging device, and use the FLARE() (Fluorescence-Assisted Resection and Exploration) near-infrared fluorescence optical imaging platform as an example.

    Title In Vivo Myocardial Distribution of Multipotent Progenitor Cells Following Intracoronary Delivery in a Swine Model of Myocardial Infarction.
    Date March 2010
    Journal European Heart Journal
    Excerpt

    There are few data comparing the fate of multipotent progenitor cells (MPCs) used in cardiac cell therapy after myocardial infarction (MI). To document in vivo distribution of MPCs delivered by intracoronary (IC) injection.

    Title Real-time Assessment of Cardiac Perfusion, Coronary Angiography, and Acute Intravascular Thrombi Using Dual-channel Near-infrared Fluorescence Imaging.
    Date July 2009
    Journal The Journal of Thoracic and Cardiovascular Surgery
    Excerpt

    We have developed an image-guided surgical system based on invisible near-infrared fluorescent light. Presently, the only clinically available near-infrared fluorophore is indocyanine green, which fluoresces at approximately 800 nm and is used for coronary angiography. Our objective was to determine whether methylene blue, already US Food and Drug Administration approved for other indications, has useful near-infrared fluorescence properties for image-guided cardiac surgery.

    Title Class 1a Pi3k Regulates Vessel Integrity During Development and Tumorigenesis.
    Date September 2008
    Journal Proceedings of the National Academy of Sciences of the United States of America
    Excerpt

    PI3K is important in the regulation of growth, proliferation, and survival of tumor cells. We show that class 1A PI3K is also critical in the tumor microenvironment by regulating the integrity of the tumor vasculature. Using Tie2Cre-mediated deletion of the PI3K regulatory subunits (p85alpha, p55alpha, p50alpha, and p85beta), we generated mice with endothelial cell-specific loss of class 1A PI3K. Complete loss of all subunits caused acute embryonic lethality at E11.5 due to hemorrhaging, whereas retention of a single p85alpha allele yielded viable mice that survived to adulthood. These heterozygous mice exhibited no vascular defects until challenged with a pathological insult, such as tumor cells or high levels of VEGF. Under these pathological conditions, heterozygous mice exhibited localized vascular abnormalities, including vessel leakage and the inability to maintain large vessels, which caused a deceleration of tumorigenesis. Furthermore, we show that a PI3K inhibitor can mimic the effects of class 1A PI3K loss, which suggests that targeting class 1A PI3K may be a promising therapy for blocking tumor angiogenesis.

    Title New Technologies for Human Cancer Imaging.
    Date September 2008
    Journal Journal of Clinical Oncology : Official Journal of the American Society of Clinical Oncology
    Excerpt

    Despite technical advances in many areas of diagnostic radiology, the detection and imaging of human cancer remains poor. A meaningful impact on cancer screening, staging, and treatment is unlikely to occur until the tumor-to-background ratio improves by three to four orders of magnitude (ie, 10(3)- to 10(4)-fold), which in turn will require proportional improvements in sensitivity and contrast agent targeting. This review analyzes the physics and chemistry of cancer imaging and highlights the fundamental principles underlying the detection of malignant cells within a background of normal cells. The use of various contrast agents and radiotracers for cancer imaging is reviewed, as are the current limitations of ultrasound, x-ray imaging, magnetic resonance imaging (MRI), single-photon emission computed tomography, positron emission tomography (PET), and optical imaging. Innovative technologies are emerging that hold great promise for patients, such as positron emission mammography of the breast and spectroscopy-enhanced colonoscopy for cancer screening, hyperpolarization MRI and time-of-flight PET for staging, and ion beam-induced PET scanning and near-infrared fluorescence-guided surgery for cancer treatment. This review explores these emerging technologies and considers their potential impact on clinical care. Finally, those cancers that are currently difficult to image and quantify, such as ovarian cancer and acute leukemia, are discussed.

    Title Quantitation of Cxcr4 Expression in Myocardial Infarction Using 99mtc-labeled Sdf-1alpha.
    Date July 2008
    Journal Journal of Nuclear Medicine : Official Publication, Society of Nuclear Medicine
    Excerpt

    The chemokine stromal-derived factor-1alpha (SDF-1alpha, CXCL12) and its receptor CXCR4 are implicated as key mediators of hematopoietic stem cell retention, cancer metastasis, and HIV infection. Their role in myocardial infarction (MI) is not as well defined. The noninvasive in vivo quantitation of CXCR4 expression is central to understanding its importance in these diverse processes as well in the cardiac response to injury. METHODS: Recombinant SDF-1alpha was radiolabeled under aprotic conditions and purified by gel-filtration chromatography (GFC) using high-specific-activity 99mTc-S-acetylmercaptoacetyltriserine-N-hydroxysuccinimide ([99mTc-MAS3]-NHS) prepared by solid-phase preloading. Radiotracer stability and transmetallation under harsh conditions were quantified by GFC. Affinity, specificity, and maximum number of binding sites (Bmax) were quantified, with adenoviral-expressed CXCR4 on nonexpressing cells and endogenous receptor on rat neonatal cardiomyocytes, using a high-throughput live-cell-binding assay. Blood half-life, biodistribution, and clearance of intravenously injected [99mTc-MAS3]-SDF-1alpha were quantified in Sprague-Dawley rats before and after experimentally induced MI. RESULTS: [99mTc-MAS3]-SDF-1alpha could be prepared in 2 h total with a specific activity of 8.0 x 10(7) MBq/mmol (2,166 Ci/mmol) and a radiochemical purity greater than 98%. Degradation of the radiotracer after boiling for 5 min, with and without 1 mM dithiothreitol, and transmetallation in 100% serum at 37 degrees C for 4 h were negligible. [99mTc-MAS3]-SDF-1alpha exhibits high specificity for CXCR4 on the surface of living rat neonatal cardiomyocytes, with an affinity of 2.7 +/- 0.9 nM and a Bmax of 4.8 x 10(4) binding sites per cell. After intravenous injection, 99mTc-labeled SDF-1alpha displays a blood half-life of 25.8 +/- 4.6 min, rapid renal clearance with only 26.2 +/- 6.1 percentage injected dose remaining in the carcass at 2 h, consistently low uptake in most organs (<0.1 percentage injected dose per gram), and no evidence of blood-brain barrier penetration. After MI was induced, CXCR4 expression levels in the myocardium increased more than 5-fold, as quantified using [99mTc-MAS3]-SDF-1alpha and confirmed using confocal immunofluorescence. CONCLUSION: We describe a 99mTc-labeled SDF-1alpha radiotracer that can be used as a sensitive and specific probe for CXCR4 expression in vivo and demonstrate that this radiotracer is able to quantify changes in CXCR4 expression under different physiologic and pathologic states. Taken together, CXCR4 levels should now be quantifiable in vivo in a variety of animal model systems of human diseases.

    Title Real-time Intraoperative Assessment of the Extrahepatic Bile Ducts in Rats and Pigs Using Invisible Near-infrared Fluorescent Light.
    Date July 2008
    Journal Surgery
    Excerpt

    BACKGROUND: Currently, only x-ray fluoroscopy is available for visualization of the extrahepatic bile ducts intraoperatively. We hypothesized that with an appropriate fluorophore and imaging system, invisible near-infrared (NIR) light could be used for image-guided procedures on the extrahepatic bile ducts. METHODS: We quantified the performance of three 800 nm NIR fluorophores, differing primarily in their degree of hydrophilicity, for real-time imaging of the extrahepatic bile ducts in rats and pigs: IR-786, indocyanine green (ICG), and the carboxylic form of IRDyetrade mark 800CW (CW800-CA). The signal-to-background ratio (SBR) of the common bile duct relative to liver and pancreas was measured as a function of the dose of contrast agent, injection site, and kinetics using an intraoperative NIR fluorescence imaging system described previously. Bile samples were examined by high performance liquid chromatography tandem mass spectrometry (HPLC/MS) to determine the chemical form of fluorophores in bile. RESULTS: Non-sulfonated (IR-786) and di-sulfonated (ICG) NIR fluorophores had poor efficiency and kinetics of excretion into bile. Tetra-sulfonated CW800-CA, however, provided sensitive, specific, and real-time visualization of the extrahepatic bile ducts after a single low-dose given either intraportally or intravenously via systemic vein. A SBR >/=2 provided sensitive assessment of extrahepatic bile duct anatomy and function for over 30 min post-injection, including the detection of millimeter-sized, radiolucent inclusions in pigs. CW800-CA remained intact chemically after secretion into bile. CONCLUSION: The combination of invisible NIR light and an IV injection of CW800-CA provides prolonged, real-time visualization of the extrahepatic bile duct, without ionizing radiation and without changing the look of the operative field.

    Title The Impact of Greed on Academic Medicine and Patient Care.
    Date June 2008
    Journal Nature Biotechnology
    Excerpt

    To what extent is the increasing emphasis on profit generation at US academic institutions shackling intellectual freedom and compromising healthcare?

    Title Rigid Multivalent Scaffolds Based on Adamantane.
    Date April 2008
    Journal The Journal of Organic Chemistry
    Excerpt

    We present two new synthetic strategies to rigid multivalent scaffolds of the general structure 1 based on adamantane. Both routes start from arylated adamantane derivatives and give the target compounds 12 and 18 in 5 and 7 steps, respectively. These scaffolds have been designed for the assembly of multivalent binders for cell surface epitopes. The adamantane nucleus exposes three carboxylic acid groups in a well-defined tripodal geometry for conjugation of targeting ligands. In addition, an amino group at the fourth bridgehead position provides a flexible linker for attachment of effector molecules such as contrast agents, radiotracers, or cytotoxins without interfering with the cell binding process.

    Title Sentinel Lymph Node Mapping of Invasive Urinary Bladder Cancer in Animal Models Using Invisible Light.
    Date January 2008
    Journal European Urology
    Excerpt

    OBJECTIVES: With conventional methodology, sentinel lymph node (SLN) mapping of invasive urinary bladder cancer is technically challenging. This study was performed to determine the utility of invisible, near-infrared fluorescent (NIRF) light for patient-specific SLN mapping, in real time under complete image guidance. METHODS: Lymphatic tracers, injection volume, NIRF excitation fluence rate, light collection of emitted fluorescence, and degree of bladder distension were systematically optimized in normal dogs and pigs. SLN mapping was then performed in pet dogs with naturally occurring invasive transitional cell carcinoma (InvTCC) of the urinary bladder, which closely mimics the human disease. RESULTS: NIRF albumin (hydrodynamic diameter [HD], 7.4 nm) and NIRF quantum dots (15-20 nm HD) injected into the bladder wall resulted in identification of draining lymph nodes (LNs) in under 3 min. In both species, considerable variability in the lymphatic drainage was observed among individuals. Optimal SLN mapping was achieved with the use of superficial, serosal injection of NIRF tracer, with the bladder distended to an intraluminal pressure of 20-40 cm H(2)O. In dogs with InvTCC, NIRF tracers identified SLNs that were confirmed histologically to harbor metastases. CONCLUSIONS: The use of invisible NIRF light permits real-time, patient-specific identification of SLNs that drain bladder cancer. Intraluminal bladder pressure is a key parameter that needs to be controlled for optimal results.

    Title Renal Clearance of Quantum Dots.
    Date January 2008
    Journal Nature Biotechnology
    Excerpt

    The field of nanotechnology holds great promise for the diagnosis and treatment of human disease. However, the size and charge of most nanoparticles preclude their efficient clearance from the body as intact nanoparticles. Without such clearance or their biodegradation into biologically benign components, toxicity is potentially amplified and radiological imaging is hindered. Using intravenously administered quantum dots in rodents as a model system, we have precisely defined the requirements for renal filtration and urinary excretion of inorganic, metal-containing nanoparticles. Zwitterionic or neutral organic coatings prevented adsorption of serum proteins, which otherwise increased hydrodynamic diameter by >15 nm and prevented renal excretion. A final hydrodynamic diameter <5.5 nm resulted in rapid and efficient urinary excretion and elimination of quantum dots from the body. This study provides a foundation for the design and development of biologically targeted nanoparticles for biomedical applications.

    Title Compact Cysteine-coated Cdse(zncds) Quantum Dots for in Vivo Applications.
    Date January 2008
    Journal Journal of the American Chemical Society
    Excerpt

    We have developed a versatile nanoparticle construct using a compact cysteine coating on a CdSe(ZnCdS) core(shell) nanocrystal (QD-Cys) that is biologically compatible, exceptionally compact, highly fluorescent, and easily functionalized. The small hydrodynamic diameter of QD-Cys ( approximately 6 nm) allows for renal clearance of these nanoparticles in rat models. Moreover, the ability to directly conjugate to QD-Cys opens up the possibility of functionalized nanocrystals for in vivo targeted imaging, in which small targeting molecules can be appended to QD-Cys, and unbound QDs can be rapidly cleared to achieve high signal/noise ratios and to reduce background toxicity.

    Title Synthesis of Conjugatable Bisphosphonates for Molecular Imaging of Large Animals.
    Date January 2008
    Journal Angewandte Chemie (international Ed. in English)
    Title Sentinel Lymph Node Mapping with Type-ii Quantum Dots.
    Date December 2007
    Journal Methods in Molecular Biology (clifton, N.j.)
    Excerpt

    Sentinel lymph node (SLN) mapping is an important cancer surgery during which the first lymph node draining the site of a tumor is identified, resected, and analyzed for the presence or absence of malignant cells. Fluorescent semiconductor nanocrystals (quantum dots [QDs]) of the appropriate size, charge, and emission wavelength permit this surgery to be performed rapidly, with high sensitivity and under complete image guidance. We describe the materials and methods necessary for the production and characterization of type-II near-infrared fluorescent QDs, which have been optimized for SLN mapping. They contain a CdTe core, CdSe shell, and a highly anionic, oligomeric phosphine organic coating. We also describe how to utilize such QDs in animal model systems of SLN mapping.

    Title Image-guided Quantification of Cardioplegia Delivery During Cardiac Surgery.
    Date November 2007
    Journal The Heart Surgery Forum
    Excerpt

    BACKGROUND: Homogenous distribution of cardioplegia delivered to the myocardium has been identified as an important predictor of post-cardiopulmonary bypass ventricular recovery and function. Presently, a method to determine adequate distribution of cardioplegia in patients during cardiac surgery does not exist. The goal of this study was to evaluate the feasibility of quantifying cardioplegia delivery using a novel, noninvasive optical method. Such a system would permit instantaneous imaging of jeopardized myocardium and allow immediate, intraoperative corrective measures. METHODS: We have previously developed a portable, intraoperative near-infrared (NIR) fluorescence imaging system for use in large animal cardiac surgery that simultaneously displays color video and NIR fluorescent images of the surgical field. By introducing exogenous, NIR fluorophores, specific cardiac functions can be visualized in real-time. RESULTS: In a porcine cardiopulmonary bypass model, we demonstrate that the FDA-approved intravascular fluorophore indocyanine green (ICG) permits real-time assessment of cardioplegia delivery. ICG was injected into an aortic root and/or transatrial coronary sinus catheter during delivery of crystalloid cardioplegia solution. Segmental distribution was immediately noted at the time of injection. In a subset of animals, simulated coronary occlusions resulted in imaging defects consistent with poor cardioplegia delivery and jeopardized myocardium. Videodensitometric analysis was performed on-line to quantify distribution to the right ventricle and left ventricle. CONCLUSION: We report the development of a novel, noninvasive, intraoperative technique that can easily and safely provide a visual assessment of cardioplegia delivery (antegrade and/or retrograde) and that offers the potential to quantify the relative segmental distribution during cardiac surgical procedures.

    Title Real-time Intraoperative Ureteral Guidance Using Invisible Near-infrared Fluorescence.
    Date November 2007
    Journal The Journal of Urology
    Excerpt

    PURPOSE: Invisible near-infrared light is safe and it penetrates relatively deeply through tissue and blood without altering the surgical field. Our hypothesis was that near-infrared fluorescence imaging would enable visualization of the ureteral anatomy and flow intraoperatively and in real time. MATERIALS AND METHODS: CW800-CA (LI-COR, Lincoln, Nebraska), the carboxylic acid form of near-infrared fluorophore IRDye 800CW, was injected intravenously, and its renal clearance kinetics and imaging performance were quantified in 350 gm rats and 35 kg pigs. High performance liquid chromatography and electrospray time-of-flight mass spectrometry were used to characterize CW800-CA metabolism in urine. The clinically available near-infrared fluorophore indocyanine green was also used via retrograde injection into the ureter. Using the 2 near-infrared fluorophores the ureters were imaged under the conditions of steady state, intraluminal foreign bodies and injury. RESULTS: In rat models the highest signal-to-background ratio for visualization occurred after intravenous injection of 7.5 microg/kg CW800-CA with values of 4.0 or greater and 2.3 or greater at 10 and 30 minutes, respectively. In pig models 7.5 microg/kg CW800-CA clearly visualized the normal ureter and intraluminal foreign bodies as small as 2.5 mm in diameter. Retrograde injection of 10 microM indocyanine green also permitted the detection of normal ureter and pinpointed urine leakage caused by injury. Electrospray time-of-flight mass spectrometry, and absorbance and fluorescence spectral analysis confirmed that the fluorescent material in urine was chemically identical to CW800-CA. CONCLUSIONS: A convenient intravenous injection of CW800-CA or direct injection of indocyanine green permits high sensitivity visualization of the ureters under steady state and abnormal conditions using invisible light.

    Title Production of Multimeric Prostate-specific Membrane Antigen Small-molecule Radiotracers Using a Solid-phase 99mtc Preloading Strategy.
    Date September 2007
    Journal Journal of Nuclear Medicine : Official Publication, Society of Nuclear Medicine
    Excerpt

    Small-molecule ligands specific for prostate-specific membrane antigen (PSMA) have the potential to improve prostate cancer imaging. However, highly charged ligands are difficult to label with 99mTc and to purify. In this study, we present an adamantane-trimerized small molecule that has nanomolar binding to PSMA and also has 12 negative charges. METHODS: To convert this molecule into a clinically viable SPECT diagnostic, we have developed a simple, cartridge-based, solid-phase prelabeling strategy that, within 25 min, converts readily available and inexpensive 99mTc-pertechnetate into a chemically pure complex, with a reactive N-hydroxysuccinimide (NHS) ester, in neat organic solvent. This stable intermediate can label any amine-containing small molecule or peptide with 99mTc in 1 step, with high specific activity and without the need for high-performance liquid chromatography (HPLC). RESULTS: Solid-phase conversion of 99mTc-pertechnetate to 99mTc-MAS3-NHS (MAS3 is S-acetylmercaptoacetyltriserine) could be completed in 25 min, with >99% radiochemical purity and with no coligands present. This intermediate was then conjugated to adamantane-trimerized GPI (2[(3-amino-3-carboxypropyl)(hydroxy)(phosphinyl)-methyl]pentane-1,5-dioic acid) in 1 step with >95% yield and no need for HPLC purification. The final molecule bound specifically to living human tumor cells expressing PSMA on their surface. Quantitative comparison was made among GPI monomer, GPI trimer, and their 99mTc-derivatives. CONCLUSION: Our study describes a simple cartridge-based conversion of 99mTc-pertechnetate to a useful, preloaded NHS ester intermediate that takes only 25 min to prepare and results in >99% radiochemical purity. Using this chemistry, we produced a high-specific-activity, 99mTc-labeled, PSMA-targeted small molecule and demonstrate gamma-ray radioscintigraphic imaging of living human prostate cancer cells.

    Title In Vivo Tracking in Cardiac Stem Cell-based Therapy.
    Date July 2007
    Journal Progress in Cardiovascular Diseases
    Excerpt

    Cell-based therapy has been heralded as a promising, novel therapeutic strategy for cardiovascular diseases. Despite a rapid transition from animal studies to clinical trials, there remain numerous unresolved, and at times, controversial issues with respect to underlying molecular mechanisms. In parallel, recent advances in the field of molecular imaging has provided a means to bridge the gap in knowledge through in vivo stem cells tracking. Herein, we review current in vivo imaging techniques and future directions for tracking the effects of cell-based therapy.

    Title Three Catheter-based Strategies for Cardiac Delivery of Therapeutic Gelatin Microspheres.
    Date April 2007
    Journal Gene Therapy
    Excerpt

    Gelatin hydrogel microspheres (GHMs) have been reported as novel non-viral vectors for gene or protein delivery (GHM therapy). However, the components of an effective catheter-based delivery strategy for GHM therapy are unknown. We evaluated the effectiveness of three catheter-based strategies for cardiac GHM therapy: (1) antegrade injection (AI) via coronary arteries; (2) retrograde injection (RI) via coronary veins; and (3) direct myocardial injection (DI) via the coronary sinus. AI distributed microspheres homogeneously throughout the target area with 73+/-11% retention. RI scattered microspheres non-homogenously with 22+/-8% retention. DI distributed microspheres in the needle-advanced area with 47+/-14% retention. However, despite high efficiency, AI did not show biological effects of inducing angiogenesis from basic fibroblast growth factor bound to GHMs. Furthermore, focal micro-infarctions, owing to micro-embolism of aggregated GHMs into small coronary arterioles, were detected in the AI group. Conversely, only RI and DI groups displayed increased coronary flow reserve. DI groups also demonstrated increased capillary density. These results suggest that RI and DI are effective for cardiac GHM therapy, while AI appears inappropriate owing to the risk of focal infarctions.

    Title Lymphatic Drainage of the Peritoneal Space: a Pattern Dependent on Bowel Lymphatics.
    Date April 2007
    Journal Annals of Surgical Oncology
    Excerpt

    BACKGROUND: Understanding lymph drainage patterns of the peritoneum could assist in staging and treatment of gastrointestinal and ovarian malignancies. Sentinel lymph nodes (SLNs) have been identified for solid organs and the pleural space. Our purpose was to determine whether the peritoneal space has a predictable lymph node drainage pattern. METHODS: Rats received intraperitoneal injections of near-infrared (NIR) fluorescent tracers: namely, quantum dots (designed for retention in SLNs) or human serum albumin conjugated with IRDye800 (HSA800; designed for lymphatic flow beyond the SLN). A custom imaging system detected NIR fluorescence at 10 and 20 minutes and 1, 4, and 24 hours after injection. To determine the contribution of viscera to peritoneal lymphatic flow, additional cohorts received bowel resection before NIR tracer injection. Associations with appropriate controls were assessed with the chi(2) test. RESULTS: Quantum dots drained to the celiac, superior mesenteric, and periportal lymph node groups. HSA800 drained to these same groups at early time points but continued flowing to the mediastinal lymph nodes via the thoracic duct. After bowel resection, both tracers were found in the thoracic, not abdominal, lymph node groups. Additionally, HSA800 was no longer found in the thoracic duct but in the anterior chest wall and diaphragmatic lymphatics. CONCLUSIONS: The peritoneal space drains to the celiac, superior mesenteric, and periportal lymph node groups first. Lymph continues via the thoracic duct to the mediastinal lymph nodes. Bowel lymphatics are a key determinant of peritoneal lymph flow, because bowel resection shifts lymph flow directly to the intrathoracic lymph nodes via chest wall lymphatics.

    Title An Hplc/mass Spectrometry Platform for the Development of Multimodality Contrast Agents and Targeted Therapeutics: Prostate-specific Membrane Antigen Small Molecule Derivatives.
    Date March 2007
    Journal Contrast Media & Molecular Imaging
    Excerpt

    The production of disease-targeted agents requires the covalent conjugation of a targeting molecule with a contrast agent or therapeutic, followed by purification of the product to homogeneity. Typical targeting molecules, such as small molecules and peptides, often have high charge-to-mass ratios and/or hydrophobicity. Contrast agents and therapeutics themselves are also diverse, and include lanthanide chelates for MRI, (99m)Tc chelates for SPECT, (90)Y chelates for radiotherapy, (18)F derivatives for PET, and heptamethine indocyanines for near-infrared fluorescent optical imaging. We have constructed a general-purpose HPLC/mass spectrometry platform capable of purifying virtually any targeted agent for any modality. The analytical sub-system is composed of a single dual-head pump that directs mobile phase to either a hot cell for the purification of radioactive agents or to an ES-TOF MS for the purification of nonradioactive agents. Nonradioactive agents are also monitored during purification by ELSD, absorbance and fluorescence. The preparative sub-system is composed of columns and procedures that permit rapid scaling from the analytical system. To demonstrate the platform's utility, we describe the preparation of five small molecule derivatives specific for prostate-specific membrane antigen (PSMA): a gadolinium derivative for MRI, indium, rhenium and technetium derivatives for SPECT, and an yttrium derivative for radiotherapy. All five compounds are derived from a highly anionic targeting ligand engineered to have a single nucleophile for N-hydroxysuccinimide-based conjugation. We also describe optimized column/mobile phase combinations and mass spectrometry settings for each class of agent, and discuss strategies for purifying molecules with extreme charge and/or hydrophobicity. Taken together, our study should expedite the development of disease-targeted, multimodality diagnostic and therapeutic agents.

    Title Endogenous N-acetylaspartylglutamate Reduced Nmda Receptor-dependent Current Neurotransmission in the Ca1 Area of the Hippocampus.
    Date March 2007
    Journal Journal of Neurochemistry
    Excerpt

    N-Acetylaspartylglutamate (NAAG) is a neuropeptide found in high concentrations in the brain. Using whole-cell recordings of CA1 pyramidal neurons in acute hippocampal slices, we found that either (i) the application of exogenous NAAG or (ii) an increase of endogenous extracellular NAAG, caused by the inhibition of its catabolic enzyme glutamate carboxypeptidase II (GCP II), resulted in a significant reduction in the amplitude of the isolated NMDA receptor (NMDAR) component of the evoked excitatory postsynaptic current (EPSC). Conversely, reduction of endogenous extracellular NAAG caused by either (i) perfusion with a soluble form of pure human GCP II or (ii) affinity purified antibodies against NAAG, enhanced the amplitude of the isolated NMDAR current. Bath application of GCP II inhibitor induced a progressive loss of spontaneous NMDAR miniatures. Furthermore, NAAG blocked the induction of long-term potentiation at Schaffer collateral axons-CA1 pyramidal neuron synapses. All together, these results suggest that NAAG acts as an endogenous modulator of NMDARs in the CA1 area of the hippocampus.

    Title Image-guided Oncologic Surgery Using Invisible Light: Completed Pre-clinical Development for Sentinel Lymph Node Mapping.
    Date February 2007
    Journal Annals of Surgical Oncology
    Excerpt

    BACKGROUND: Invisible near-infrared (NIR) fluorescent light permits high sensitivity, real-time image-guidance during oncologic surgery without changing the look of the surgical field. In this study, we complete pre-clinical development of the technology for sentinel lymph node (SLN) mapping using a large animal model of spontaneous melanoma. METHODS: Sinclair swine with spontaneous melanoma metastatic to regional lymph nodes were used because of their similarity to human melanoma. Organic lymphatic tracers tested included FDA-approved indocyanine green adsorbed non-covalently to human serum albumin (HSA), and NIR fluorophore CW800 conjugated covalently to HSA (HSA800). The inorganic/organic hybrid tracer tested was type II NIR quantum dots with an anionic coating. Primary tumors received four peri-tumoral injections of each tracer, with a fluorophore dose of 100 pmol to 1 nmol per injection. SLN mapping and image-guided resection were performed in real-time. RESULTS: Each of the 3 lymphatic tracers was injected into n = 4 separate primary melanomas in a total of 6 animals. All 12 injections resulted in identification of the SLN(s) and their associated lymphatic channels within 1 minute in 100% of cases, despite highly pigmented skin and black fur. Hydrodynamic diameter had a profound impact on tracer behavior in vivo. CONCLUSIONS: This study completes the pre-clinical development of NIR fluorescence-guided SLN mapping and provides insight into imaging system optimization and tracer choice for future human clinical trials. The technology is likely to eliminate the need for radioactive and colored tracers, permits real-time image guidance throughout the procedure, and assists the pathologist in tissue analysis.

    Title Localization and Quantification of Platelet-rich Thrombi in Large Blood Vessels with Near-infrared Fluorescence Imaging.
    Date January 2007
    Journal Circulation
    Excerpt

    BACKGROUND: Imaging of thrombus formation in vivo has been limited by the inability to directly visualize and measure thrombi in large blood vessels in real time. Near-infrared light, with its superior tissue penetration and reduced scatter, could potentially solve this problem. METHODS AND RESULTS: Platelets were labeled with the near-infrared fluorophore IR-786. Optimal total fluorescence yield occurred at 6 attomoles of IR-786 per platelet. IR-786-labeled platelets were tested for their ability to detect thrombus formation in large animal model systems relevant to common human vascular procedures. Invisible near-infrared light did not distort the surgical field in any way, and even after optimization of per-platelet fluorescent yield, platelets remained fully functional. Intravenous infusion of just 3.6x10(10) labeled platelets into a 35-kg Yorkshire pig permitted thrombus visualization, with a signal-to-background ratio > or = 2, for at least 2 hours in coronary, carotid, and femoral vessels. Platelet-rich, actively growing clots were monitored in real time and quantified with respect to size and kinetics after injury to vessels, cutaneous incisions, intravascular stent insertion, or introduction of embolic coils. Similarly, formed clots were monitored in real time during thrombolysis with streptokinase and heparin. Vessel patency was assessed independently with a second near-infrared fluorescent blood pool agent. CONCLUSIONS: IR-786-labeled platelets provide sensitive, specific, and real-time visualization of thrombi in thick-walled blood vessels. In addition to immediate application in cardiac, transplant, and vascular surgery, the mechanisms that underlie thrombus formation in large blood vessels can now be investigated.

    Title Intraoperative Detection of Cell Injury and Cell Death with an 800 Nm Near-infrared Fluorescent Annexin V Derivative.
    Date December 2006
    Journal American Journal of Transplantation : Official Journal of the American Society of Transplantation and the American Society of Transplant Surgeons
    Excerpt

    The intraoperative detection of cell injury and cell death is fundamental to human surgeries such as organ transplantation and resection. Because of low autofluorescence background and relatively high tissue penetration, invisible light in the 800 nm region provides sensitive detection of disease pathology without changing the appearance of the surgical field. In order to provide surgeons with real-time intraoperative detection of cell injury and death after ischemia/reperfusion (I/R), we have developed a bioactive derivative of human annexin V (annexin800), which fluoresces at 800 nm. Total fluorescence yield, as a function of bioactivity, was optimized in vitro, and final performance was assessed in vivo. In liver, intestine and heart animal models of I/R, an optimal signal to background ratio was obtained 30 min after intravenous injection of annexin800, and histology confirmed concordance between planar reflectance images and actual deep tissue injury. In summary, annexin800 permits sensitive, real-time detection of cell injury and cell death after I/R in the intraoperative setting, and can be used during a variety of surgeries for rapid assessment of tissue and organ status.

    Title Radiolabeled and Near-infrared Fluorescent Fibrinogen Derivatives Create a System for the Identification and Repair of Obscure Gastrointestinal Bleeding.
    Date December 2006
    Journal Surgery
    Excerpt

    BACKGROUND: Gastrointesintal hemorrhage is often difficult to localize. A test that does not depend on active bleeding might prove clinically useful. We tested 2 novel fibrinogen (FBG)-based contrast agents for their ability to localize gastrointestinal (GI) hemorrhage after bleeding stopped. 125I-FBG permits gamma ray-based preoperative or intraoperative scanning, and near-infrared (NIR) flourescent FBG (FBG800) permits real-time intraoperative visualization of active clot. METHODS: Bovine FBG was radiolabeled with 125I or conjugated to the NIR fluorophore CW800. Sites of bleeding were created by gastrotomy, mucosal resection of the stomach, or laceration of a mesenteric vessel; then 1.7 mg/kg FBG800 or 15 microCi/kg 125I-FBG was injected intravenously into mice, rabbits, or pigs 30 minutes before or after injury. Sites of active clot were quantified by using gamma counting and were also imaged by using invisible NIR light intraoperatively, for up to 3 hours postinjection. RESULTS: After an injection of either 125I-FBG or FBG800, sites of prior bleeding could be identified in the absence of active bleeding. Blood clearance was such that a signal-to-background ratio of 2.0 or greater could be achieved within 20 minutes after injection. A similarly labeled human serum albumin did not accumulate at any site, with an SBR of 1.0 or less. CONCLUSIONS: Both radiolabeled (preoperative gamma scanning) and NIR fluorescent (intraoperative real-time imaging) FBG can be used in experimental situations to identify the location of prior bleeding in the absence of active bleeding. Taken together, these contrast agents create a system for the identification and control of obscure GI bleeding.

    Title A New Approach to Drug Development.
    Date November 2006
    Journal The New England Journal of Medicine
    Title Translating in Vivo Diagnostics into Clinical Reality.
    Date October 2006
    Journal Nature Biotechnology
    Title Sentinel Lymph Node Mapping of the Gastrointestinal Tract by Using Invisible Light.
    Date June 2006
    Journal Annals of Surgical Oncology
    Excerpt

    BACKGROUND: Because many gastrointestinal (GI) tumors spread by way of lymphatics, histological assessment of the first draining lymph nodes has both prognostic and therapeutic significance. However, sentinel lymph node mapping of the GI tract by using available techniques is limited by unpredictable drainage patterns, high background signal, and the inability to image lymphatic tracers relative to surgical anatomy in real time. Our goal was to develop a method for patient-specific intraoperative sentinel lymph node mapping of the GI tract by using invisible near-infrared light. METHODS: We developed an intraoperative near-infrared fluorescence imaging system that simultaneously displays surgical anatomy and otherwise invisible near-infrared fluorescence images of the surgical field. Near-infrared fluorescent quantum dots were injected intraparenchymally into the stomach, small bowel, and colon, and draining lymphatic channels and sentinel lymph nodes were visualized. Dissection was performed under real-time image guidance. RESULTS: In 10 adult pigs, we demonstrated that 200 pmol of quantum dots quickly and accurately map lymphatic drainage and sentinel lymph nodes. Injection into the mid jejunum and colon results in fluorescence of a single lymph node at the root of the bowel mesentery. Injection into the stomach resulted in identification of a retrogastric node. Histological analysis in all cases confirmed the presence of nodal tissue. CONCLUSIONS: We report the use of invisible near-infrared light for intraoperative sentinel lymph node mapping of the GI tract. This technology overcomes the limitations of currently available methods, permits patient-specific imaging of lymphatic flow and sentinel nodes, and provides highly sensitive, real-time image-guided dissection.

    Title Self-illuminating Quantum Dots Light the Way.
    Date June 2006
    Journal Nature Biotechnology
    Title Tissue-like Phantoms for Near-infrared Fluorescence Imaging System Assessment and the Training of Surgeons.
    Date May 2006
    Journal Journal of Biomedical Optics
    Excerpt

    We demonstrate how to construct calibrated, stable, and inexpensive tissue-like phantoms for near-IR (NIR) fluorescence imaging applications. The bulk phantom material is composed of gelatin, intralipid, hemoglobin, and indocyanine green (ICG). Absorbance, scatter, background fluorescence, and texture can be tuned as desired. NIR fluorescent inclusions are comprised of ICG-labeled polystyrene divinylbenzene beads and Pam78-labeled hydroxyapatite crystals. The former mimic tumor masses of controllable size and contrast agent concentration, and the latter mimic microcalcifications in breast cancer. NIR-fluorescent inclusions can be positioned precisely in phantoms, with one or more regions having different optical properties, and their position can be verified independently using microcomputed tomography. We demonstrate how these phantoms can be used to calibrate and compare imaging systems, and to train surgeons to operate under NIR fluorescence image guidance.

    Title Optical Imaging of Hydroxyapatite in the Calcified Vasculature of Transgenic Animals.
    Date May 2006
    Journal Arteriosclerosis, Thrombosis, and Vascular Biology
    Excerpt

    OBJECTIVE: To detect the hydroxyapatite component of vascular calcification in vivo so that the process of calcium deposition can be studied in transgenic model systems. METHODS AND RESULTS: We have previously developed a near-infrared fluorescent bisphosphonate derivative that binds with high affinity and specificity to hydroxyapatite, and an intraoperative near-infrared fluorescence imaging system for small animals. Using these tools, and a transgenic mouse strain with homozygous deletion of the matrix GLA protein (Mgp(-/-)), we demonstrate that the hydroxyapatite component of vascular calcification can be detected in vivo with high sensitivity, specificity, and resolution. CONCLUSIONS: The hydroxyapatite component of vascular calcification can be detected optically, in real-time, without sacrifice of the animal. It is now possible to study the earliest events associated with vascular mineralization, at the cell and organ level, and to monitor the process in living animals.

    Title Size Series of Small Indium Arsenide-zinc Selenide Core-shell Nanocrystals and Their Application to in Vivo Imaging.
    Date April 2006
    Journal Journal of the American Chemical Society
    Excerpt

    We have developed a size series of unusually small, water-soluble (InAs)ZnSe (core)shell quantum dots (QDs) that emit in the near-infrared and exhibit new behavior in vivo, including multiple sequential lymph node mapping and extravasation from the vasculature. The biological utility of these fluorescent probes resulted from our intentional choice to match the semiconductor material and water-soluble ligand with a desired final hydrodynamic diameter and emission wavelength.

    Title Tracking Stem Cells in the Cardiovascular System.
    Date February 2006
    Journal Trends in Cardiovascular Medicine
    Excerpt

    Stem cells are a promising approach to cardiovascular therapeutics. Animal experiments have assessed the fate of injected stem cells through ex vivo methods on sacrificed animals. Approaches are needed for in vivo tracking of stem cells. Various imaging techniques and contrast agents for stem cell tracking will be reviewed.

    Title High-affinity Near-infrared Fluorescent Small-molecule Contrast Agents for in Vivo Imaging of Prostate-specific Membrane Antigen.
    Date January 2006
    Journal Molecular Imaging
    Excerpt

    Surgical resection remains a definitive treatment for prostate cancer. Yet, prostate cancer surgery is performed without image guidance for tumor margin, extension beyond the capsule and lymph node positivity, and without verification of other occult metastases in the surgical field. Recently, several imaging systems have been described that exploit near-infrared (NIR) fluorescent light for sensitive, real-time detection of disease pathology intraoperatively. In this study, we describe a high-affinity (9 nM), single nucleophile-containing, small molecule specific for the active site of the enzyme PSMA. We demonstrate production of a tetra-sulfonated heptamethine indocyanine NIR fluorescent derivative of this molecule using a high-yield LC/MS purification strategy. Interestingly, NIR fluorophore conjugation improves affinity over 20-fold, and we provide mechanistic insight into this observation. We describe the preparative production of enzymatically active PSMA using a baculovirus expression system and an adenovirus that co-expresses PSMA and GFP. We demonstrate sensitive and specific in vitro imaging of endogenous and ectopically expressed PSMA in human cells and in vivo imaging of xenograft tumors. We also discuss chemical strategies for improving performance even further. Taken together, this study describes nearly complete preclinical development of an optically based small-molecule contrast agent for image-guided surgery.

    Title Synthesis of Rigid Multivalent Scaffolds Based on Adamantane.
    Date January 2006
    Journal Organic Letters
    Excerpt

    An efficient route to novel 1,3,5,7-tetrasubstituted derivatives of adamantane is described. This route starts from adamantane and gives the tetrafunctionalized derivative 9 in eight steps with an overall yield of 23%. These tetrahedrally shaped molecules possess three identical arms terminated by an activated carboxylic acid derivative and a protected amino function in the 1-position. We propose these tetravalent cage compounds such as 9 as scaffolds for the assembly of ligand/marker conjugates for studies of multivalent ligand receptor interactions. [reaction: see text]

    Title Organic Alternatives to Quantum Dots for Intraoperative Near-infrared Fluorescent Sentinel Lymph Node Mapping.
    Date October 2005
    Journal Molecular Imaging : Official Journal of the Society for Molecular Imaging
    Excerpt

    Intraoperative near-infrared (NIR) fluorescence imaging provides the surgeon with real-time image guidance during cancer and other surgeries. We have previously reported the use of NIR fluorescent quantum dots (QDs) for sentinel lymph node (SLN) mapping. However, because of concerns over potential toxicity, organic alternatives to QDs will be required for initial clinical studies. We describe a family of 800 nm organic heptamethine indocyanine-based contrast agents for SLN mapping spanning a spectrum from 775 Da small molecules to 7 MDa nanocolloids. We provide a detailed characterization of the optical and physical properties of these contrast agents and discuss the advantages and disadvantages of each. We present robust methods for the covalent conjugation, purification, and characterization of proteins with tetra-sulfonated heptamethine indocyanines, including mass spectroscopic site mapping of highly substituted molecules. One contrast agent, NIR fluorescent human serum albumin (HSA800), emerged as the molecule with the best overall performance with respect to entry to lymphatics, flow to the SLN, retention in the SLN, fluorescence yield and reproducibility. This preclinical study, performed on large animals approaching the size of humans, should serve as a foundation for future clinical studies.

    Title Engineering Inas(x)p(1-x)/inp/znse Iii-v Alloyed Core/shell Quantum Dots for the Near-infrared.
    Date September 2005
    Journal Journal of the American Chemical Society
    Excerpt

    Quantum dots with a core/shell/shell structure consisting of an alloyed core of InAs(x)P(1-x), an intermediate shell of InP, and an outer shell of ZnSe were developed. The InAs(x)P(1-x) alloyed core has a graded internal composition with increasing arsenic content from the center to the edge of the dots. This compositional gradient results from two apparent effects: (1) the faster reaction kinetics of the phosphorus precursor compared to the arsenic precursor, and (2) a post-growth arsenic-phosphorus exchange reaction that increases the arsenic content. The cores have a zinc blend structure for all compositions and show tunable emission in the near-infrared (NIR) region. A first shell of InP leads to a red-shift and an increase in quantum yield. The final shell of ZnSe serves to stabilize the dots for applications in aqueous environments, including NIR biomedical fluorescence imaging. These NIR-emitting core/shell/shell InAs(x)P(1-x)/InP/ZnSe were successfully used in a sentinel lymph node mapping experiment.

    Title Intraoperative Sentinel Lymph Node Mapping of the Lung Using Near-infrared Fluorescent Quantum Dots.
    Date August 2005
    Journal The Annals of Thoracic Surgery
    Excerpt

    BACKGROUND: The presence of lymph node metastases is an important prognostic marker with regard to non-small-cell lung cancer (NSCLC). Assessment of the sentinel lymph node (SLN) for the presence of tumor may improve staging. Our objective was to develop an optical noninvasive imaging tool that would permit intraoperative SLN mapping and provide real-time visual feedback for image-guided localization and resection. METHODS: Invisible near-infrared (NIR) light penetrates relatively deeply into tissue and background autofluorescence is low. We have developed a NIR fluorescence imaging system that simultaneously displays color video and NIR images of the surgical field. We recently engineered 15 nm nonradioactive NIR fluorescent quantum dots (QDs) as optimal lymphotrophic optical probes. The introduction of these QDs into lung tissue allows real-time visualization of draining lymphatic channels and nodes. RESULTS: In 12 Yorkshire pigs (mean weight 35 kg) we demonstrated that 200 pmol of NIR QDs injected into lobar parenchyma accurately maps lymphatic drainage and the SLN. All SLNs were strongly fluorescent and easily visualized within 5 minutes of injection. In 14 separate injections QDs localized to a mediastinal node, whereas in 2 injections QDs localized to a hilar intraparenchymal node. Histologic analysis in all cases confirmed the presence of nodal tissue. CONCLUSIONS: We report a highly sensitive rapid technique for SLN mapping of the lung. This technique permits precise real-time imaging and therefore overcomes many limitations of currently available techniques.

    Title Catheter-based Antegrade Intracoronary Viral Gene Delivery with Coronary Venous Blockade.
    Date July 2005
    Journal American Journal of Physiology. Heart and Circulatory Physiology
    Excerpt

    The purpose of this study is to evaluate the feasibility of percutaneous antegrade myocardial gene transfer (PAMGT). A consistent and safe technique for in vivo gene transfer is required for clinical application of myocardial gene therapy. PAMGT with concomitant coronary venous blockade was performed in 12 swine. The myocardium was preconditioned with 1 min of occlusion of the left anterior descending and left circumflex arteries. The anterior interventricular vein was occluded during left anterior descending artery delivery, and the great cardiac vein at the entrance of the middle cardiac vein was occluded during left circumflex artery delivery. With arterial and venous balloons inflated (3 min) and after adenosine (25 mug) injection, PAMGT was performed by antegrade injection of an adenoviral solution (1 ml of 10(11) plaque-forming units in each coronary artery) carrying beta-galactosidase or saline through the center lumen of the angioplasty balloon. In one set of animals, PAMGT was performed with selective coronary vein blockade (n = 9); in another set of animals, PAMGT was performed without coronary vein blockade (n = 5). At 1 wk after gene delivery, the animals were killed. Quantitative beta-galactosidase analysis was performed in the left and right ventricular walls. PAMGT was successfully performed in all animals with and without concomitant occlusion of the coronary veins. Quantitative beta-galactosidase analysis showed that PAMGT with coronary blockade was superior to PAMGT without coronary blockade. beta-Galactosidase activity increased significantly in the beta-galactosidase group compared with the saline group: 1.34 +/- 0.18 vs. 0.81 +/- 0.1 ng (P </= 0.01) in the left ventricular wall and 0.91 +/- 0.1 vs. 0.66 +/- 0.07 ng (P </= 0.05) in the right ventricular wall. PAMGT with selective coronary venous blockade is feasible, reproducible, and safely achieved in a large-animal model.

    Title In Vivo Tracking of Stem Cells for Clinical Trials in Cardiovascular Disease.
    Date June 2005
    Journal Circulation
    Excerpt

    Various stem cells hold promise for the treatment of human cardiovascular disease. Regardless of stem cell origin, future clinical trials will require that the location and number of such cells be tracked in vivo, over long periods of time. The problem of tracking small numbers of cells in the body is a difficult one, and an optimal solution does not yet exist. We review the many contrast agents and detectors that have been proposed for stem cell tracking during clinical trials, define the characteristics of an ideal imaging technology, and suggest future directions for research.

    Title Sentinel Lymph Node Mapping of the Pleural Space.
    Date June 2005
    Journal Chest
    Excerpt

    STUDY OBJECTIVES: Although the sentinel lymph node (SLN) concept has traditionally been applied to solid organs, we hypothesized that the pleural space might drain into a specific SLN group. The identification of such a nodal group could assist in the staging and treatment of pleural-based diseases, such as mesothelioma, or other lung cancers with visceral pleural invasion. The purpose of this study was to determine whether the pleural space has an SLN group. DESIGN: Sixteen rats underwent right or left pleural space injection of a novel lymph tracer, quantum dots (QDs), which have a hydrodynamic diameter of 15 nm and fluoresce in the near-infrared (NIR) spectrum. Nodal uptake of the entire thorax was imaged with a custom system that simultaneously acquired color video, NIR fluorescence of the QDs, and a merged picture of the two in real-time. Six pigs underwent right or left pleural space injection of QDs and similar imaging. MEASUREMENTS AND RESULTS: In the rat, the QDs drained solely to the highest superior mediastinal lymph node group, corresponding to lymph node station 1, according the regional lymph node classification for lung of the American Joint Committee on Cancer. In one rat, the injection of QDs in the left pleural space resulted in migration to the contralateral station 1 lymph node group. The injection of QDs in the right or left pleural space of the pig resulted in migration solely to the ipsilateral highest superior mediastinal lymph node group. CONCLUSIONS: NIR fluorescence imaging in two species demonstrated that the highest superior mediastinal lymph nodes of station 1 are the SLNs of the pleural space. This study also provides intraoperative feasibility and proof of the concept for identifying lymph nodes communicating with the pleural space on a patient-specific basis, in real-time, and with high sensitivity.

    Title Intraoperative Identification of Esophageal Sentinel Lymph Nodes with Near-infrared Fluorescence Imaging.
    Date May 2005
    Journal The Journal of Thoracic and Cardiovascular Surgery
    Excerpt

    OBJECTIVE: In esophageal cancer, selective removal of involved lymph nodes could improve survival and limit complications from extended lymphadenectomy. Mapping with vital blue dyes or technetium Tc-99m often fails to identify intrathoracic sentinel lymph nodes. Our purpose was to develop an intraoperative method for identifying sentinel lymph nodes of the esophagus with high-sensitivity near-infrared fluorescence imaging. METHODS: Six Yorkshire pigs underwent thoracotomy and received submucosal, esophageal injection of quantum dots, a novel near-infrared fluorescent lymph tracer designed for retention in sentinel lymph nodes. Six additional pigs underwent thoracotomy and received submucosal esophageal injection of CW800 conjugated to human serum albumin, another novel lymph tracer designed for uptake into distant lymph nodes. Finally, 6 pigs received submucosal injection of the fluorophore-conjugated albumin with an endoscopic needle through an esophagascope. These lymph tracers fluoresce in the near-infrared, permitting visualization of migration to sentinel lymph nodes with a custom intraoperative imaging system. RESULTS: Injection of the near-infrared fluorescent lymph tracers into the esophagus revealed communicating lymph nodes within 5 minutes of injection. In all 6 pigs that received quantum dot injection, only a single sentinel lymph node was identified. Among pigs that received fluorophore-conjugated albumin injection, in 5 of 12 a single sentinel lymph node was revealed, but in 7 of 12 two sentinel lymph nodes were identified. There was no dominant pattern in the appearance of the sentinel lymph nodes either cranial or caudal to the injection site. CONCLUSION: Near-infrared fluorescence imaging of sentinel lymph nodes is a novel and reliable intraoperative technique with the power to assist with identification and resection of esophageal sentinel lymph nodes.

    Title In Vivo Optical Imaging of Pleural Space Drainage to Lymph Nodes of Prognostic Significance.
    Date March 2005
    Journal Annals of Surgical Oncology
    Excerpt

    BACKGROUND: Understanding the spatial and temporal drainage patterns of the pleural space could have profound impact on the treatment of lung cancer and mesothelioma. The purpose of this study was to identify the in vivo pattern of drainage from the pleural space to prognostic lymph node stations. METHODS: Fifty-six rats underwent pleural space injection of a novel lymph tracer composed of recombinant human serum albumin (HSA) covalently conjugated to the near-infrared (NIR) fluorophore IRDye78 via an amide bond (HSA-78). Nodal uptake was imaged at 10, 20, 30, and 60 minutes and 4, 12, and 24 hours after injection with a custom system that simultaneously acquires color video, NIR fluorescence of HSA-78, and a merged picture of the two. Six pigs underwent the same procedure with imaging at 30 minutes, 1 hour, and 24 hours. RESULTS: In both the rat model and the pig model, HSA-78 drained from the pleural space to superior mediastinal lymph nodes first, followed by other intrathoracic and then extrathoracic lymph nodes over the course of 24 hours. CONCLUSION: NIR fluorescence imaging in two species shows that the superior mediastinal lymph nodes are the first to drain the pleural space. Over the course of 24 hours, the pleural space also communicates with other intrathoracic and then extrathoracic lymph nodes. This study also demonstrates an intraoperative method for identifying nodes communicating with the pleural space, with potential utility in the staging and/or resection of lung cancer and mesothelioma.

    Title In Vivo Near-infrared Fluorescence Imaging.
    Date November 2004
    Journal Current Opinion in Chemical Biology
    Excerpt

    Photon penetration into living tissue is highly dependent on the absorption and scattering properties of tissue components. The near-infrared region of the spectrum offers certain advantages for photon penetration, and both organic and inorganic fluorescence contrast agents are now available for chemical conjugation to targeting molecules. This review focuses on those parameters that affect image signal and background during in vivo imaging with near-infrared light and exogenous contrast agents. Recent examples of in vivo near-infrared fluorescence imaging of animals and humans are presented, including imaging of normal and diseased vasculature, tissue perfusion, protease activity, hydroxyapatite and cancer.

    Title Near-infrared Fluorescent Type Ii Quantum Dots for Sentinel Lymph Node Mapping.
    Date August 2004
    Journal Nature Biotechnology
    Excerpt

    The use of near-infrared or infrared photons is a promising approach for biomedical imaging in living tissue. This technology often requires exogenous contrast agents with combinations of hydrodynamic diameter, absorption, quantum yield and stability that are not possible with conventional organic fluorophores. Here we show that the fluorescence emission of type II quantum dots can be tuned into the near infrared while preserving absorption cross-section, and that a polydentate phosphine coating renders them soluble, disperse and stable in serum. We then demonstrate that these quantum dots allow a major cancer surgery, sentinel lymph node mapping, to be performed in large animals under complete image guidance. Injection of only 400 pmol of near-infrared quantum dots permits sentinel lymph nodes 1 cm deep to be imaged easily in real time using excitation fluence rates of only 5 mW/cm(2). Taken together, the chemical, optical and in vivo data presented in this study demonstrate the potential of near-infrared quantum dots for biomedical imaging.

    Title Profilin Acts Downstream of Ldl to Mediate Diabetic Endothelial Cell Dysfunction.
    Date July 2004
    Journal The Faseb Journal : Official Publication of the Federation of American Societies for Experimental Biology
    Excerpt

    The changes occurring at the luminal surface of endothelial cells in diabetes and their relevance to endothelial dysfunction are poorly characterized in vivo. In this study, we developed an integrated strategy to discover cell surface proteins associated with diabetes and to test their role in endothelial dysfunction. First, a peptide phage display library was screened over the endothelial surface of the intact aorta or in retinal endothelial cells from diabetic and control rats. Then, we purified profilin-1 as a binding partner for one of the diabetic aorta-specific phage. Profilin was increased in the aortic endothelium of human diabetic individuals and streptozotocin-diabetic rats. Furthermore, overexpressing profilin in rat aortic endothelial cells triggered 3 indicators of endothelial dysfunction: increased apoptosis, elevated expression of ICAM-1, and decreased phosphorylation of the vasodilator-stimulated phosphoprotein, a marker for nitric oxide signaling. The changes in ICAM-1 and vasodilator-stimulated phosphoprotein were recapitulated in the diabetic aorta in vivo. LDL and oxysterols elevated profilin in cultured aortic endothelial cells. Interference with the de novo synthesis of profilin abrogated the LDL-mediated increase in ICAM-1 expression. Finally, profilin expression was markedly elevated in atherosclerotic plaques. These data indicate that profilin contributes to endothelial dysfunction in a pathway that is downstream of LDL.

    Title An Operational Near-infrared Fluorescence Imaging System Prototype for Large Animal Surgery.
    Date July 2004
    Journal Technology in Cancer Research & Treatment
    Excerpt

    Near-infrared (NIR) fluorescence imaging has the potential to revolutionize human cancer surgery by providing sensitive, specific, and real-time intraoperative visualization of normal and disease processes. We have previously introduced the concept of a low-cost, safe, and easy-to-use NIR fluorescence imaging system that permits the surgeon to "see" surgical anatomy and NIR fluorescence simultaneously, non-invasively, with high spatial resolution, in real-time, and without moving parts. In this study, we present an operational prototype designed specifically for use during large animal surgery. Such a system serves as a foundation for future clinical studies. We discuss technical considerations, and provide details of the implementation of subsystems related to excitation light, light collection, computer, and software. Using the prototype, and the clinically available NIR fluorophore indocyanine green, we demonstrate vascular imaging in 35 kg pigs. Cancer-specific applications of this imaging system include image-guided cancer resection with real-time assessment of surgical margins, image-guided sentinel lymph node mapping, intraoperative mapping of tumor and normal vasculature, image-guided avoidance of critical structures such as nerves, and intraoperative detection of occult metastases in the surgical field. Taken together, this study describes an optical imaging system engineered for eventual translation to the clinic.

    Title Functional Analysis of the Mad1-msin3a Repressor-corepressor Interaction Reveals Determinants of Specificity, Affinity, and Transcriptional Response.
    Date May 2004
    Journal Molecular and Cellular Biology
    Excerpt

    The recruitment of corepressors by DNA-bound repressors is likely to be a critical rate-limiting step in the transcriptional regulation of many genes. An excellent paradigm for such an interaction is the association of the basic helix-loop-helix zipper protein Mad1 with the corepressor mSin3A. When bound together, the Sin3 interaction domain (SID) of Mad1 forms extensive hydrophobic contacts with the four-helix bundle formed by the paired amphipathic helix 2 (PAH2) domain of mSin3A. Using the costructure to predict the principle residues required for binding, we have carried out an extensive mutational analysis to examine the Mad1 SID-mSin3A PAH2 interaction in vitro and in vivo. Bulky hydrophobic residues in the alpha1 (I308 and V311) and alpha2 (L329 and L332) helices of the PAH2 domain are necessary to accommodate the precise arrangement of bulky (L12) and short (A15 and A16) hydrophobic residues in the amphipathic Mad1 SID. We have also used phage display to derive an optimal SID, which shows an essentially identical arrangement of key residues. By manipulating these key residues, we have generated altered-specificity Mad1 SID mutants that bind only to a PAH2 domain with a reciprocal mutation, permitting us to demonstrate for the first time that these domains interact directly in vivo. We have also found that the integrity of the PAH1 domain affects the Mad1 SID-PAH2 interaction. It is conceivable that cross talk between different PAH domains and their binding partners helps to determine the subunit composition and order of assembly of mSin3A complexes.

    Title Silencing of Phosphonate-gadolinium Magnetic Resonance Imaging Contrast by Hydroxyapatite Binding.
    Date April 2004
    Journal Investigative Radiology
    Excerpt

    RATIONALE AND OBJECTIVES: GdDOTP5- is a highly charged, bone-seeking paramagnetic complex that could potentially detect bone lesions by magnetic resonance imaging (MRI). To date, its pharmacokinetics, effects on organ relaxivity, and interaction with hydroxyapatite (HA) has not been described. METHODS: Liver, kidney, and bone MRI images were obtained on male white rabbits after the administration of GdDOTP5- or a gold standard MRI contrast agent, GdDTPA2-. Parallel in vitro experiments quantified the effect of HA binding on GdDOTP5- -induced changes in relaxivity. RESULTS: The 2 compounds showed similar MRI enhancements in visceral tissues, but no enhancement of bone was evident with GdDOTP5- despite confirmation of bone and HA binding of the radioactive 153SmDOTP5- and 111InDOTP5- derivatives. In vitro experiments demonstrated that GdDOTP5--induced changes in relaxivity were silenced upon HA binding but could be recovered by acid elution of the complex. CONCLUSIONS: HA binding assays revealed that GdDOTP5- is essentially MR silent when bound to bone, likely because of the exclusion of all outer sphere water molecules from the surface of the complex. These data suggest a novel strategy for creating highly sensitive, switchable MRI contrast agents.

    Title Improved Chemical Strategies for the Targeted Therapy of Cancer.
    Date March 2004
    Journal Angewandte Chemie (international Ed. in English)
    Title Near-infrared Fluorescence Imaging of Microcalcification in an Animal Model of Breast Cancer.
    Date February 2004
    Journal Academic Radiology
    Excerpt

    RATIONALE AND OBJECTIVES: At present, there is no animal model of breast cancer that forms reproducible microcalcification. The aim of this study was to develop a straightforward, reproducible model system that could be used to develop multimodality contrast agents for the identification of breast cancer microcalcification. METHODS: The R3230 mammary adenocarcinoma cell line was implanted in the mammary fat pad of female Fischer 344 rats (two rats with two implanted tumors and two rats with a single implanted tumor). After growth to 1-2 cm in diameter, tumors were implanted with 100 microm hydroxyapatite crystals (positive control) or calcium oxalate crystals (negative control). Twenty-four hours after crystal implantation, rats were injected intravenously with a previously described near-infrared fluorescent bisphosphonate derivative known as Pam78, and the tumors were imaged using a reflectance optical imaging system. RESULTS: Tumors implanted with hydroxyapatite displayed bright, focal, near-infrared fluorescence in the area of crystal implantation. Control tumors, grown in the same animal and implanted with calcium oxalate, did not display any near-infrared fluorescence, even along the needle track used for crystal implantation. CONCLUSIONS: A simple and rapid animal model of focal calcification in breast cancer tumors has been developed and validated. The model used Pam78, a near-infrared fluorescent contrast agent specific for hydroxyapatite. The potential usefulness of the model for developing similar contrast agents for magnetic resonance and other imaging modalities is discussed.

    Title Irdye78 Conjugates for Near-infrared Fluorescence Imaging.
    Date October 2003
    Journal Molecular Imaging : Official Journal of the Society for Molecular Imaging
    Excerpt

    The detection of human malignancies by near-infrared (NIR) fluorescence will require the conjugation of cancer-specific ligands to NIR fluorophores that have optimal photoproperties and pharmacokinetics. IRDye78, a tetra-sulfonated heptamethine indocyanine NIR fluorophore, meets most of the criteria for an in vivo imaging agent, and is available as an N-hydroxysuccinimide ester for conjugation to low-molecular-weight ligands. However, IRDye78 has a high charge-to-mass ratio, complicating purification of conjugates. It also has a potentially labile linkage between fluorophore and ligand. We have developed an ion-pairing purification strategy for IRDye78 that can be performed with a standard C18 column under neutral conditions, thus preserving the stability of fluorophore, ligand, and conjugate. By employing parallel evaporative light scatter and absorbance detectors, all reactants and products are identified, and conjugate purity is maximized. We describe reversible and irreversible conversions of IRDye78 that can occur during sample purification, and describe methods for preserving conjugate stability. Using seven ligands, spanning several classes of small molecules and peptides (neutral, charged, and/or hydrophobic), we illustrate the robustness of these methods, and confirm that IRDye78 conjugates so purified retain bioactivity and permit NIR fluorescence imaging of specific targets.

    Title Quantitation of Brown Adipose Tissue Perfusion in Transgenic Mice Using Near-infrared Fluorescence Imaging.
    Date October 2003
    Journal Molecular Imaging : Official Journal of the Society for Molecular Imaging
    Excerpt

    Brown adipose tissue (BAT; brown fat) is the principal site of adaptive thermogenesis in the human newborn and other small mammals. Of paramount importance for thermogenesis is vascular perfusion, which controls the flow of cool blood in, and warmed blood out, of BAT. We have developed an optical method for the quantitative imaging of BAT perfusion in the living, intact animal using the heptamethine indocyanine IR-786 and near-infrared (NIR) fluorescent light. We present a detailed analysis of the physical, chemical, and cellular properties of IR-786, its biodistribution and pharmacokinetics, and its uptake into BAT. Using transgenic animals with homozygous deletion of Type II iodiothyronine deiodinase, or homozygous deletion of uncoupling proteins (UCPs) 1 and 2, we demonstrate that BAT perfusion can be measured noninvasively, accurately, and reproducibly. Using these techniques, we show that UCP -1/-2 knockout animals, when compared to wild-type animals, have a higher baseline perfusion of BAT but a similar maximal response to beta 3-receptor agonist. These results suggest that compensation for UCP deletion is mediated, in part, by the control of BAT perfusion. Taken together, BAT perfusion can now be measured noninvasively using NIR fluorescent light, and pharmacological modulators of thermogenesis can be screened at relatively high throughput in living animals.

    Title Selection of Quantum Dot Wavelengths for Biomedical Assays and Imaging.
    Date October 2003
    Journal Molecular Imaging : Official Journal of the Society for Molecular Imaging
    Excerpt

    Fluorescent semiconductor nanocrystals (quantum dots [QDs]) are hypothesized to be excellent contrast agents for biomedical assays and imaging. A unique property of QDs is that their absorbance increases with increasing separation between excitation and emission wavelengths. Much of the enthusiasm for using QDs in vivo stems from this property, since photon yield should be proportional to the integral of the broadband absorption. In this study, we demonstrate that tissue scatter and absorbance can sometimes offset increasing QD absorption at bluer wavelengths, and counteract this potential advantage. By using a previously validated mathematical model, we explored the effects of tissue absorbance, tissue scatter, wavelength dependence of the scatter, water-to-hemoglobin ratio, and tissue thickness on QD performance. We conclude that when embedded in biological fluids and tissues, QD excitation wavelengths will often be quite constrained, and that excitation and emission wavelengths should be selected carefully based on the particular application. Based on our results, we produced near-infrared QDs optimized for imaging surface vasculature with white light excitation and a silicon CCD camera, and used them to image the coronary vasculature in vivo. Taken together, our data should prove useful in designing fluorescent QD contrast agents optimized for specific biomedical applications.

    Title Functional Near-infrared Fluorescence Imaging for Cardiac Surgery and Targeted Gene Therapy.
    Date October 2003
    Journal Molecular Imaging : Official Journal of the Society for Molecular Imaging
    Excerpt

    Cardiac revascularization is presently performed without real-time visual assessment of myocardial blood flow or perfusion. Moreover, gene therapy of the heart cannot, at present, be directed to specific territories at risk for myocardial infarction. We have developed a surgical imaging system that exploits the low autofluorescence, deep tissue penetration, low tissue scatter, and invisibility of near-infrared (NIR) fluorescent light. By completely isolating visible and NIR light paths, one is able to visualize, simultaneously, the anatomy and/or function of the heart, or any desired tissue. In rat model systems, we demonstrate that the heptamethine indocyanine-type NIR fluorophores IR-786 and the carboxylic acid form of IRDye78 can be injected intravenously in the living animal to provide real-time visual assessment of myocardial blood flow or perfusion intraoperatively. This imaging system may prove useful for the refinement of revascularization techniques, and for the administration of cardiac gene therapy.

    Title Sub-millimeter Technetium-99m Calibration Sources.
    Date October 2003
    Journal Molecular Imaging and Biology : Mib : the Official Publication of the Academy of Molecular Imaging
    Excerpt

    PURPOSE: Small animal radioscintigraphic imaging systems aim to achieve sub-millimeter resolution. At the present time, sub-millimeter calibration sources that can be placed at will within an imaged volume are not readily available. We have developed a method for producing technetium-99m (Tc-99m) sources in less than 15 minutes with readily available reagents. PROCEDURES: Tc-99m pertechnetate [TcO(4)](-) was incubated with 45 microm to 106 microm diameter spherical anion exchange beads, washed, and mounted as desired for instrument calibration. RESULTS: The procedure yields spherical sources having between 6.8 microCi to 11.1 microCi of Tc-99m per source. This work shows that dual imaging of these sources using white light and radioscintigraphy permits measurement of system performance with high precision. CONCLUSION: Easily prepared, sub-millimeter Tc-99m spherical calibration sources are described, and it is demonstrated that such sources are useful for measuring the resolution and sensitivity of radioscintigraphic systems, such as those designed for small animal imaging.

    Title An Integrated Vector System for Cellular Studies of Phage Display-derived Peptides.
    Date April 2003
    Journal Analytical Biochemistry
    Excerpt

    Peptide phage display is a method by which large numbers of diverse peptides can be screened for binding to a target of interest. Even when successful, the rate-limiting step is usually validation of peptide bioactivity using living cells. In this paper, we describe an integrated system of vectors that expedites both the screening and the characterization processes. Library construction and screening is performed using an optimized type 3 phage display vector, mJ(1), which is shown to accept peptide libraries of at least 23 amino acids in length. Peptide coding sequences are shuttled from mJ(1) into one of three families of mammalian expression vectors for cell physiological studies. The vector pAL(1) expresses phage display-derived peptides as Gal4 DNA binding domain fusion proteins for transcriptional activation studies. The vectors pG(1), pG(1)N, and pG(1)C express phage display-derived peptides as green fluorescent protein fusions targeted to the entire cell, nucleus, or cytoplasm, respectively. The vector pAP(1) expresses phage display-derived peptides as fusions to secreted placental alkaline phosphatase. Such enzyme fusions can be used as highly sensitive affinity reagents for high-throughput assays and for cloning of peptide-binding cell surface receptors. Taken together, this system of vectors should facilitate the development of phage display-derived peptides into useful biomolecules.

    Title 14-3-3 Transits to the Nucleus and Participates in Dynamic Nucleocytoplasmic Transport.
    Date April 2002
    Journal The Journal of Cell Biology
    Excerpt

    14-3-3 proteins regulate the cell cycle and prevent apoptosis by controlling the nuclear and cytoplasmic distribution of signaling molecules with which they interact. Although the majority of 14-3-3 molecules are present in the cytoplasm, we show here that in the absence of bound ligands 14-3-3 homes to the nucleus. We demonstrate that phosphorylation of one important 14-3-3 binding molecule, the transcription factor FKHRL1, at the 14-3-3 binding site occurs within the nucleus immediately before FKHRL1 relocalization to the cytoplasm. We show that the leucine-rich region within the COOH-terminal alpha-helix of 14-3-3, which had been proposed to function as a nuclear export signal (NES), instead functions globally in ligand binding and does not directly mediate nuclear transport. Efficient nuclear export of FKHRL1 requires both intrinsic NES sequences within FKHRL1 and phosphorylation/14-3-3 binding. Finally, we present evidence that phosphorylation/14-3-3 binding may also prevent FKHRL1 nuclear reimport. These results indicate that 14-3-3 can mediate the relocalization of nuclear ligands by several mechanisms that ensure complete sequestration of the bound 14-3-3 complex in the cytoplasm.

    Title In Vivo Near-infrared Fluorescence Imaging of Osteoblastic Activity.
    Date February 2002
    Journal Nature Biotechnology
    Excerpt

    In vertebrates, the development and integrity of the skeleton requires hydroxyapatite (HA) deposition by osteoblasts. HA deposition is also a marker of, or a participant in, processes as diverse as cancer and atherosclerosis. At present, sites of osteoblastic activity can only be imaged in vivo using gamma-emitting radioisotopes. The scan times required are long, and the resultant radioscintigraphic images suffer from relatively low resolution. We have synthesized a near-infrared (NIR) fluorescent bisphosphonate derivative that exhibits rapid and specific binding to HA in vitro and in vivo. We demonstrate NIR light-based detection of osteoblastic activity in the living animal, and discuss how this technology can be used to study skeletal development, osteoblastic metastasis, coronary atherosclerosis, and other human diseases.

    Title Minimal Activators That Bind to the Kix Domain of P300/cbp Identified by Phage Display Screening.
    Date November 2000
    Journal Nature Biotechnology
    Excerpt

    Human gene therapy approaches involving transcription factors often rely on artificial activation domains for transcriptional activation. These domains are often large (e.g., 80 amino acids for VP16), recruit multiple co-activation complexes at once, and offer no fine control over the level of transcription. In an attempt to understand the sequence and structural requirements of a minimal mammalian activator, we employed a molecular diversity approach with a peptide phage display library composed of random eight-amino acid peptides. Using the KIX domain of the mammalian co-activators p300 and CBP as target, we discovered a family of synthetic binding peptides. These peptides share significant homology with natural KIX domain ligands, and are shown to bind an overlapping, yet distinct, surface of p300/CREB-binding protein (CBP). When fused to a heterologous DNA binding domain, these synthetic peptides function as titratable, modular, and potent transcriptional activators in living cells through specific recruitment of p300/CBP, with the level of transcriptional activation proportional to the affinity of the synthetic peptide for the KIX domain. Taken together, our data demonstrate that a molecular diversity approach can be used to discover minimal, co-activator domain-specific synthetic activators, and that transcriptional activation can be modulated as desired at the level of co-activator recruitment.

    Title Role of P21ras in Insulin-stimulated Glucose Transport in 3t3-l1 Adipocytes.
    Date September 1994
    Journal The Journal of Biological Chemistry
    Excerpt

    The proto-oncogene p21ras has been implicated as an essential intermediate in several actions of the hormone insulin. This study examines the role of p21ras in the signaling pathways by which insulin increases hexose transport in differentiated 3T3-L1 adipose cells, a model system for study of the metabolic effects of the hormone on a physiological target tissue. Introduction of constitutively activated p21ras(G12V) by microinjection into 3T3-L1 adipocytes stimulated the expression of the ubiquitous glucose transporter, GLUT1, in the absence of insulin. Moreover, introduction of dominant inhibitory forms of p21ras or neutralizing antibodies directed against p21ras blocked the insulin-induced increase in GLUT1 expression. In contrast, microinjection of activating or inhibitory forms of p21ras had no effect on translocation of the "insulin-responsive" glucose transporter, GLUT4, to the cell surface. These results indicate that p21ras mediates the insulin-induced increase in GLUT1 expression in 3T3-L1 adipocytes but is not involved in the translocation of GLUT4 that leads to acute increases in glucose transport.

    Title The Dna Binding Domain of Retinoic Acid Receptor Beta is Required for Ligand-dependent Suppression of Proliferation. Application of General Purpose Mammalian Coexpression Vectors.
    Date September 1994
    Journal Journal of Cell Science
    Excerpt

    We have developed a family of mammalian coexpression vectors that permit identification of living or fixed cells overexpressing a gene of interest by surrogate detection of a coexpressed marker protein. Using these 'pMARK' vectors, a fluorescence-based, single cell proliferation assay was developed and used to study the effect of retinoic acid receptor beta (RAR-beta) on cell cycling. We demonstrate that transient overexpression of RAR-beta in the presence, but not absence, of all-trans retinoic acid results in a dramatic suppression of cell proliferation. We further show that this effect requires the DNA binding (C) domain of RAR-beta. It has been previously shown that RAR-beta expression is markedly altered in a variety of neoplasms and cell lines. Our data support the hypothesis that loss of RAR-beta may contribute to tumor progression by removing normal restraints on proliferation. The pMARK vectors should be useful for studying other genes that putatively suppress or enhance proliferation.

    Title Calpain-catalyzed Cleavage and Subcellular Relocation of Protein Phosphotyrosine Phosphatase 1b (ptp-1b) in Human Platelets.
    Date December 1993
    Journal The Embo Journal
    Excerpt

    The non-transmembrane phosphotyrosine phosphatase 1B (PTP-1B) is an abundant enzyme, normally localized to the cytosolic face of the endoplasmic reticulum via a C-terminal targeting sequence. We have found that agonist-induced platelet activation results in proteolytic cleavage of PTP-1B at a site upstream from this targeting sequence, causing subcellular relocation of its catalytic domain from membranes to the cytosol. PTP-1B cleavage is catalyzed by the calcium-dependent neutral protease calpain and is a general feature of platelet agonist-induced aggregation. Moreover, PTP-1B cleavage correlates with the transition from reversible to irreversible platelet aggregation in platelet-rich plasma. Engagement of gpIIb-IIIa is necessary for inducing PTP-1B cleavage, suggesting that integrins regulate tyrosine phosphatases as well as tyrosine kinases. PTP-1B cleavage is accompanied by a 2-fold stimulation of its enzymatic activity, as measured by immune complex phosphatase assay, and correlates with discrete changes in the pattern of tyrosyl phosphorylation. Cleavage and subcellular relocation of PTP-1B represents a novel mechanism for altering tyrosyl phosphorylation that may have important physiological implications in cell types other than platelets.

    Title Use of a General Purpose Mammalian Expression Vector for Studying Intracellular Protein Targeting: Identification of Critical Residues in the Nuclear Lamin A/c Nuclear Localization Signal.
    Date November 1993
    Journal Journal of Cell Science
    Excerpt

    We have constructed a general purpose mammalian expression vector for the study of intracellular protein targeting. The vector, p3PK, facilitates construction of N- and/or C-terminal fusions of an amino acid sequence of interest to the normally cytosolic protein chicken muscle pyruvate kinase (CMPK). The vector has been engineered such that any fusion construct can be subcloned into the versatile pJx omega family of mammalian expression vectors and into pGEX bacterial expression vectors, for the generation of affinity reagents. In this paper, we demonstrate the general utility of p3PK by redirecting CMPK to mitochondria (using the twelve amino acid pre-sequence of yeast cytochrome c oxidase subunit IV) and to the nucleus (using a putative eight amino acid nuclear localization signal from human nuclear lamins A and C). We also report that, contrary to the predictions of previously published work, substitution of a critical residue in the nuclear lamin A/C nuclear localization signal (the equivalent of lysine 128 in the SV40 large T nuclear localization signal) retains nuclear localization, and discuss how amino acid context might affect targeting to the nucleus.

    Title Solubilization and Purification of Enzymatically Active Glutathione S-transferase (pgex) Fusion Proteins.
    Date June 1993
    Journal Analytical Biochemistry
    Excerpt

    The pGEX glutathione S-transferase (GST) fusion protein system is used extensively for high level expression and rapid purification of fusion proteins from bacterial and eukaryotic cell lysates. Unfortunately, many GST fusion proteins are partially or completely insoluble, and thus cannot be purified efficiently from a crude lysate. We have adapted a protocol, previously used to solubilize actin, for the purification of otherwise insoluble GST fusion proteins. Using a GST fusion of the nontransmembrane protein tyrosine phosphatase 1B, we demonstrate that tyrosine phosphatase enzymatic activity is maintained during the purification process. We provide methods for the purification of GST fusion proteins at analytical and preparative scales, and demonstrate that saturation of glutathione agarose is dependent on fusion protein molecular weight. Finally, we present strategies for eluting purified fusion proteins from glutathione agarose beads, for storing eluted protein, and for preparing covalently coupled affinity matrices.

    Title The Nontransmembrane Tyrosine Phosphatase Ptp-1b Localizes to the Endoplasmic Reticulum Via Its 35 Amino Acid C-terminal Sequence.
    Date March 1992
    Journal Cell
    Excerpt

    We report the first intracellular characterization of an endogenous nontransmembrane protein tyrosine phosphatase (PTP). Using affinity-purified polyclonal antibodies, we have identified PTP-1B as a 50 kd serine phosphoprotein in immunoprecipitation and immunoblotting assays. Surprisingly, indirect immunofluorescence experiments indicate that PTP-1B is localized predominantly in the endoplasmic reticulum (ER). Subcellular fractionation is consistent with this localization and establishes that PTP-1B is tightly associated with microsomal membranes, with its phosphatase domain oriented towards the cytoplasm. The C-terminal 35 amino acids of PTP-1B are both necessary and sufficient for targeting to the ER. The finding of a tyrosine phosphatase on the ER suggests new possibilities for cellular events controlled by tyrosine phosphorylation.

    Title High Frequency of Retinoic Acid Receptor Beta Abnormalities in Human Lung Cancer.
    Date November 1991
    Journal Oncogene
    Excerpt

    One of the three human retinoic acid receptors, RAR-beta, maps to a region on the short arm of chromosome 3 frequently deleted in lung cancer. Because retinoic acid is required for normal epithelial cell growth and regulation, and loss of a retinoic acid receptor might be expected to contribute to oncogenesis, we examined RAR-beta RNA and DNA in normal lung, 33 lung cancer cell lines and nine primary lung tumors. Normally, RAR-beta is expressed as two transcripts, of sizes 3.1 kb and 2.8 kb, which are strongly induced by retinoic acid. At least 50% of the cell lines and 30% of the tumor samples show altered RAR-beta expression and/or inducibility, including examples of absence or specific loss of one of the RAR-beta transcripts. Abnormalities in the expression patterns of RAR-alpha and RAR-gamma also are found, but at a lower frequency than RAR-beta abnormalities. Southern analysis reveals alteration of the RAR-beta gene in three of the cell lines. Our data suggest that abnormalities in structure and expression of the RAR-beta gene may be involved in the pathogenesis of lung cancer.

    Title Control of Intracellular Ph and Growth by Fibronectin in Capillary Endothelial Cells.
    Date June 1990
    Journal The Journal of Cell Biology
    Excerpt

    The aim of this work was to analyze the mechanism by which fibronectin (FN) regulates capillary endothelial cell proliferation. Endothelial cell growth can be controlled in chemically-defined medium by varying the density of FN coated on the substratum (Ingber, D. E., and J. Folkman. J. Cell Biol. 1989. 109:317-330). In this system, DNA synthetic rates are stimulated by FN in direct proportion to its effect on cell extension (projected cell areas) both in the presence and absence of saturating amounts of basic FGF. To investigate direct growth signaling by FN, we carried out microfluorometric measurements of intracellular pH (pHi), a cytoplasmic signal that is commonly influenced by soluble mitogens. pHi increased 0.18 pH units as FN coating densities were raised and cells progressed from round to spread. Intracellular alkalinization induced by attachment to FN was rapid and followed the time course of cell spreading. When measured in the presence and absence of FGF, the effects of FN and FGF on pHi were found to be independent and additive. Furthermore, DNA synthesis correlated with pHi for all combinations of FGF and FN. Ethylisopropylamiloride, a specific inhibitor of the plasma membrane Na+/H+ antiporter, completely suppressed the effects of FN on both pHi and DNA synthesis. However, cytoplasmic pH per se did not appear to be a critical determinant of growth since DNA synthesis was not significantly inhibited when pHi was lowered over the physiological range by varying the pH of the medium. We conclude that FN and FGF exert their growth-modulating effects in part through activation of the Na+/H+ exchanger, although they appear to trigger this system via separate pathways.

    Title Cytoplasmic Ph and Anchorage-independent Growth Induced by V-ki-ras, V-src or Polyoma Middle T.
    Date May 1990
    Journal Oncogene
    Excerpt

    We had previously shown that spreading of normal cells on tissue culture plastic coated with extracellular matrix (ECM) proteins led to an increase in cytoplasmic pH (pHi). Since alkalinization of the cytoplasm is associated with activation of a number of signaling pathways that control growth, and is itself required for cell growth, we proposed that this phenomenon could explain, at least in part, why growth of normal cells is anchorage-dependent. Preliminary results showed that pHi in cells transformed by the ras or src oncogenes had an alkaline pHi even when completely round. To further explore the relationship between pHi and anchorage-independent growth, a series of cells transformed by mutants of the polyoma middle T oncogene, and a series of ras-transformed cells and revertants were examined. Growth in methyl cellulose was assayed, and pHi in both maximally spread and completely round cells was measured for each cell line. We found that all of the normal cells required spreading to maintain an alkaline pHi, whereas transformed cell lines had an alkaline pHi independent of spreading. There was a strong correlation between pHi in round cells and anchorage-independent growth. Thus, some plasma membrane oncogenes can substitute for cell spreading on EMC to raise pHi as well as to promote growth. These results may be relevant to understanding why transformation leads to changes not only in cellular requirements for growth factors, but also for anchorage.

    Title The Mechanism of Low-frequency Sound Production in Muscle.
    Date August 1987
    Journal Biophysical Journal
    Excerpt

    Frog gastrocnemius muscles stimulated isometrically in a saline bath at 20 degrees C were found to produce a single ringing sound event beginning just before the tension record began to rise. The sound event was substantially over by the time the isometric tension began to fall. Results from studies correlating the spatial pattern of the sound, the amplitude and frequency of the sound as a function of the muscle length, and the response of both the passive and active muscle to a transverse pluck were found to be consistent with the conclusion that the sounds in these muscles are caused primarily by transverse resonant vibrations. As the muscle develops force, its lack of cylindrical symmetry gives rise to lateral motions, which are most likely the initiators of the bending vibrations detected as sound.

    Title Imaging in Cardiac Cell-based Therapy: in Vivo Tracking of the Biological Fate of Therapeutic Cells.
    Date
    Journal Nature Clinical Practice. Cardiovascular Medicine
    Excerpt

    Clinical trials in cardiac cell-based therapy (CBT) have demonstrated the immense potential of stem progenitor cells (SPCs) to repair the injured myocardium. The bulk of evidence so far has shown that CBT can lead to structural and functional improvements. Unresolved issues remain, however, including gaps in the understanding of mechanisms and mixed results from CBT trials. To try to provide answers for these issues, assessment of the biological fate of SPCs once delivered to the injured heart has been called for. Advances in contrast agents and imaging modalities have made feasible the objective assessment of the in vivo molecular and cellular evolution of transplanted SPCs. In vivo imaging can target fundamental processes related to SPCs to gain information on their biological activities and outcomes within specific authentic microenvironments. Advantages and inherent drawbacks of imaging techniques, such as reporter-gene systems, optical imaging, radionuclide imaging, and MRI, are discussed in this Review. More than ever, it has become clear to scientists and clinicians that parallel developments in cell-based therapies and in vivo imaging modalities will strengthen this blossoming field.

    Title An Illustration of the Potential for Mapping Mri/mrs Parameters with Genetic Over-expression Profiles in Human Prostate Cancer.
    Date
    Journal Magma (new York, N.y.)
    Excerpt

    INTRODUCTION: Magnetic resonance imaging (MRI) and MR spectroscopy can probe a variety of physiological (e.g. blood vessel permeability) and metabolic characteristics of prostate cancer. However, little is known about the changes in gene expression that underlie the spectral and imaging features observed in prostate cancer. Tumor induced changes in vascular permeability and angiogenesis are thought to contribute to patterns of dynamic contrast enhanced (DCE) MRI images of prostate cancer even though the genetic basis of tumor vasculogenesis is complex and the specific mechanisms underlying these DCEMRI features have not yet been determined. MATERIALS AND METHODS: In order to identify the changes in gene expression that correspond to MRS and DCEMRI patterns in human prostate cancers, we have utilized tissue print micropeel techniques to generate "whole mount" molecular maps of radical prostatectomy specimens that correspond to pre-surgical MRI/MRS studies. These molecular maps include RNA expression profiles from both Affymetrix GeneChip microarrays and quantitative reverse transcriptase PCR (qrt-PCR) analysis, as well as immunohistochemical studies. RESULTS: Using these methods on patients with prostate cancer, we found robust over-expression of choline kinase a in the majority of primary tumors. We also observed overexpression of neuropeptide Y (NPY), a newly identified angiogenic factor, in a subset of prostate cancers, visualized on DCEMRI. CONCLUSION: These studies set the stage for establishing MRI/MRS parameters as validated biomarkers for human prostate cancer.

    Title Microwave-assisted Synthesis of Near-infrared Fluorescent Sphingosine Derivatives.
    Date
    Journal Chemical Communications (cambridge, England)
    Excerpt

    Microwave-assisted synthesis of near-infrared fluorescent sphingosine derivatives is described, and the utility of the probes demonstrated by co-localization studies with visible wavelength fluorescent sphingosine derivatives.

    Title Real-time Visualization and Quantification of Retrograde Cardioplegia Delivery Using Near Infrared Fluorescent Imaging.
    Date
    Journal Journal of Cardiac Surgery
    Excerpt

    BACKGROUND AND AIM: Homogeneous delivery of cardioplegia is essential for myocardial protection during cardiac surgery. Presently, there exist no established methods to quantitatively assess cardioplegia distribution intraoperatively and determine when retrograde cardioplegia is required. In this study, we evaluate the feasibility of near infrared (NIR) imaging for real-time visualization of cardioplegia distribution in a porcine model. METHODS: A portable, intraoperative, real-time NIR imaging system was utilized. NIR fluorescent cardioplegia solution was developed by incorporating indocyanine green (ICG) into crystalloid cardioplegia solution. Real-time NIR imaging was performed while the fluorescent cardioplegia solution was infused via the retrograde route in five ex vivo normal porcine hearts and in five ex vivo porcine hearts status post left anterior descending (LAD) coronary artery ligation. Horizontal cross-sections of the hearts were obtained at proximal, middle, and distal LAD levels. Videodensitometry was performed to quantify distribution of fluorophore content. RESULTS: The progressive distribution of cardioplegia was clearly visualized with NIR imaging. Complete visualization of retrograde distribution occurred within 4 minutes of infusion. Videodensitometry revealed retrograde cardioplegia, primarily distributed to the left ventricle (LV) and anterior septum. In hearts with LAD ligation, antegrade cardioplegia did not distribute to the anterior LV. This deficiency was compensated for with retrograde cardioplegia supplementation. CONCLUSIONS: Incorporation of ICG into cardioplegia allows real-time visualization of cardioplegia delivery via NIR imaging. This technology may prove useful in guiding intraoperative decisions pertaining to when retrograde cardioplegia is mandated.

    Title Rapid Translocation of Nanoparticles from the Lung Airspaces to the Body.
    Date
    Journal Nature Biotechnology
    Excerpt

    Nano-size particles show promise for pulmonary drug delivery, yet their behavior after deposition in the lung remains poorly understood. In this study, a series of near-infrared (NIR) fluorescent nanoparticles were systematically varied in chemical composition, shape, size and surface charge, and their biodistribution and elimination were quantified in rat models after lung instillation. We demonstrate that nanoparticles with hydrodynamic diameter (HD) less than ≈34 nm and a noncationic surface charge translocate rapidly from the lung to mediastinal lymph nodes. Nanoparticles of HD < 6 nm can traffic rapidly from the lungs to lymph nodes and the bloodstream, and then be subsequently cleared by the kidneys. We discuss the importance of these findings for drug delivery, air pollution and carcinogenesis.


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