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Biomedical subjects

Ralph Weissleder

Publications and source records attributed to Ralph Weissleder.

At least 37 records · Page 2Linked to original sources
PubMed2006

The impact of human EGFR kinase domain mutations on lung tumorigenesis and in vivo sensitivity to EGFR-targeted therapies.

To understand the role of human epidermal growth factor receptor (hEGFR) kinase domain mutations in lung tumorigenesis and response to EGFR-targeted therapies, we generated bitransgenic mice with inducible expression in type II pneumocytes of two common hEGFR mutants seen in human lung cancer. Both bitransgenic lines developed lung adenocarcinoma after sustained hEGFR mutant expression, confirming their oncogenic potential. Maintenance of these lung tumors was dependent on continued expression of the EGFR mutants. Treatment with small molecule inhibitors (erlotinib or HKI-272) as well as prolonged treatment with a humanized anti-hEGFR antibody (cetuximab) led to dramatic tumor regression. These data suggest that persistent EGFR signaling is required for tumor maintenance in human lung adenocarcinomas expressing EGFR mutants.

Adenocarcinoma↗
PubMed2006

Both p16(Ink4a) and the p19(Arf)-p53 pathway constrain progression of pancreatic adenocarcinoma in the mouse.

Activating KRAS mutations and p16(Ink4a) inactivation are near universal events in human pancreatic ductal adenocarcinoma (PDAC). In mouse models, Kras(G12D) initiates formation of premalignant pancreatic ductal lesions, and loss of either Ink4a/Arf (p16(Ink4a)/p19(Arf)) or p53 enables their malignant progression. As recent mouse modeling studies have suggested a less prominent role for p16(Ink4a) in constraining malignant progression, we sought to assess the pathological and genomic impact of inactivation of p16(Ink4a), p19(Arf), and/or p53 in the Kras(G12D) model. Rapidly progressive PDAC was observed in the setting of homozygous deletion of either p53 or p16(Ink4a), the latter with intact germ-line p53 and p19(Arf) sequences. Additionally, Kras(G12D) in the context of heterozygosity either for p53 plus p16(Ink4a) or for p16(Ink4a)/p19(Arf) produced PDAC with longer latency and greater propensity for distant metastases relative to mice with homozygous deletion of p53 or p16(Ink4a)/p19(Arf). Tumors from the double-heterozygous cohorts showed frequent p16(Ink4a) inactivation and loss of either p53 or p19(Arf). Different genotypes were associated with specific histopathologic characteristics, most notably a trend toward less differentiated features in the homozygous p16(Ink4a)/p19(Arf) mutant model. High-resolution genomic analysis revealed that the tumor suppressor genotype influenced the specific genomic patterns of these tumors and showed overlap in regional chromosomal alterations between murine and human PDAC. Collectively, our results establish that disruptions of p16(Ink4a) and the p19(ARF)-p53 circuit play critical and cooperative roles in PDAC progression, with specific tumor suppressor genotypes provocatively influencing the tumor biological phenotypes and genomic profiles of the resultant tumors.

Adenocarcinoma↗
PubMed2006

Identification of inhibitors of ribozyme self-cleavage in mammalian cells via high-throughput screening of chemical libraries.

We have recently described an RNA-only gene regulation system for mammalian cells in which inhibition of self-cleavage of an mRNA carrying ribozyme sequences provides the basis for control of gene expression. An important proof of principle for that system was provided by demonstrating the ability of one specific small molecule inhibitor of RNA self-cleavage, toyocamycin, to control gene expression in vitro and vivo. Here, we describe the development of the high-throughput screening (HTS) assay that led to the identification of toyocamycin and other molecules capable of inhibiting RNA self-cleavage in mammalian cells. To identify small molecules that can serve as inhibitors of ribozyme self-cleavage, we established a cell-based assay in which expression of a luciferase (luc) reporter is controlled by ribozyme sequences, and screened 58,076 compounds for their ability to induce luciferase expression. Fifteen compounds able to inhibit ribozyme self-cleavage in cells were identified through this screen. The most potent of the inhibitors identified were toyocamycin and 5-fluorouridine (FUR), nucleoside analogs carrying modifications of the 7-position and 5-position of the purine or pyrimidine bases. Individually, these two compounds were able to induce gene expression of the ribozyme-controlled reporter approximately 365-fold and 110-fold, respectively. Studies of the mechanism of action of the ribozyme inhibitors indicate that the compounds must be incorporated into RNA in order to inhibit RNA self-cleavage.

Acridine Orange↗
PubMed2006

Molecular and cellular imaging of atherosclerosis: emerging applications.

Molecular imaging studies have shed light on important biological aspects of atherosclerosis, and are now entering the clinical arena for the detection of clinical atheroma. This review first discusses fundamental principles regarding the rationale for and development of molecular imaging technologies for investigating atherosclerosis. Next, we highlight clinically promising imaging strategies that illuminate key biological aspects of atherosclerosis, including macrophage activity, protease activity, lipoprotein presence, apoptosis, and angiogenesis. We envision that several molecular imaging approaches will become important adjuncts to the clinical management of high-risk atherosclerosis.

Atherosclerosis↗
PubMed2006

Development of a dual fluorogenic and chromogenic dipeptidyl peptidase IV substrate.

A new far-red dual fluorogenic and chromogenic substrate, 5-glycylprolylglycylprolyl-9-di-3-sulfonyl-propylaminobenza[a]phenoxazonium perchlorate (GPGP-2SBPO), was developed for dipeptidyl peptidase IV (DPP-IV) sensing. The glycylprolylglycylprolyl tetrapeptide was chosen as the recognition sequence due to its stability under physiological conditions. In contrast, the truncated substrate, GP-2SBPO, containing only a glycylprolyl peptide, is unstable. Proteolysis of GPGP-2SBPO was assayed by monitoring the absorbance and fluorescence signals from the released fluorochrome, 2SBPO, at 625 and 670nm, respectively.

Dipeptidyl Peptidase 4↗
PubMed2006

Murine B16 melanomas expressing high levels of the chemokine stromal-derived factor-1/CXCL12 induce tumor-specific T cell chemorepulsion and escape from immune control.

The chemokine, stromal-derived factor-1/CXCL12, is expressed by normal and neoplastic tissues and is involved in tumor growth, metastasis, and modulation of tumor immunity. T cell-mediated tumor immunity depends on the migration and colocalization of CTL with tumor cells, a process regulated by chemokines and adhesion molecules. It has been demonstrated that T cells are repelled by high concentrations of the chemokine CXCL12 via a concentration-dependent and CXCR4 receptor-mediated mechanism, termed chemorepulsion or fugetaxis. We proposed that repulsion of tumor Ag-specific T cells from a tumor expressing high levels of CXCL12 allows the tumor to evade immune control. Murine B16/OVA melanoma cells (H2b) were engineered to constitutively express CXCL12. Immunization of C57BL/6 mice with B16/OVA cells lead to destruction of B16/OVA tumors expressing no or low levels of CXCL12 but not tumors expressing high levels of the chemokine. Early recruitment of adoptively transferred OVA-specific CTL into B16/OVA tumors expressing high levels of CXCL12 was significantly reduced in comparison to B16/OVA tumors, and this reduction was reversed when tumor-specific CTLs were pretreated with the specific CXCR4 antagonist, AMD3100. Memory OVA-specific CD8+ T cells demonstrated antitumor activity against B16/OVA tumors but not B16/OVA.CXCL12-high tumors. Expression of high levels of CXCL12 by B16/OVA cells significantly reduced CTL colocalization with and killing of target cells in vitro in a CXCR4-dependent manner. The repulsion of tumor Ag-specific T cells away from melanomas expressing CXCL12 confirms the chemorepellent activity of high concentrations of CXCL12 and may represent a novel mechanism by which certain tumors evade the immune system.

Animals↗
PubMed2006

Factor XIII deficiency causes cardiac rupture, impairs wound healing, and aggravates cardiac remodeling in mice with myocardial infarction.

BACKGROUND: Identification of key molecular players in myocardial healing could lead to improved therapies, reduction of scar formation, and heart failure after myocardial infarction (MI). We hypothesized that clotting factor XIII (FXIII), a transglutaminase involved in wound healing, may play an important role in MI given prior clinical and mouse model data. METHODS AND RESULTS: To determine whether a truly causative relationship existed between FXIII activity and myocardial healing, we prospectively studied myocardial repair in FXIII-deficient mice. All FXIII(-/-) and FXIII(-)(/+) (FXIII activity <5% and 70%) mice died within 5 days after MI from left ventricular rupture. In contradistinction, FXIII(-/-) mice that received 5 days of intravenous FXIII replacement therapy had normal survival rates; however, cardiac MRI demonstrated worse left ventricular remodeling in these reconstituted FXIII(-/-) mice. Using a FXIII-sensitive molecular imaging agent, we found significantly greater FXIII activity in wild-type mice and FXIII(-/-) mice receiving supplemental FXIII than in FXIII(-/-) mice (P<0.05). In FXIII(-/-) but not in reconstituted FXIII(-/-) mice, histology revealed diminished neutrophil migration into the MI. Reverse transcriptase-polymerase chain reaction studies suggested that the impaired inflammatory response in FXIII(-/-) mice was independent of intercellular adhesion molecule and lipopolysaccharide-induced CXC chemokine, both important for cell migration. After MI, expression of matrix metalloproteinase-9 was 650% higher and collagen-1 was 53% lower in FXIII(-/-) mice, establishing an imbalance in extracellular matrix turnover and providing a possible mechanism for the observed cardiac rupture in the FXIII(-/-) mice. CONCLUSIONS: These data suggest that FXIII has an important role in murine myocardial healing after infarction.

Animals↗
PubMed2006

Particularities of the vasculature can promote the organ specificity of autoimmune attack.

How certain autoimmune diseases target specific organs remains obscure. In the 'K/BxN' arthritis model, autoantibodies to a ubiquitous antigen elicit joint-restricted pathology. Here we have used intravital imaging to demonstrate that transfer of arthritogenic antibodies caused macromolecular vasopermeability localized to sites destined to develop arthritis, augmenting its severity. Vasopermeability depended on mast cells, neutrophils and FcgammaRIII but not complement, tumor necrosis factor or interleukin 1. Unexpectedly, radioresistant FcRgamma-expressing cells in an organ distant from the joint were required. Histamine and serotonin were critical, and systemic administration of these vasoactive amines recapitulated the joint localization of immune complex-triggered vasopermeability. We propose that regionally distinct vascular properties 'interface' with immune effector pathways to foster organ-specific autoimmune damage, perhaps explaining why arthritis accompanies many human infectious and autoimmune disorders.

Animals↗
PubMed2006

An X-ray computed tomography imaging agent based on long-circulating bismuth sulphide nanoparticles.

Nanomaterials have become increasingly important in the development of new molecular probes for in vivo imaging, both experimentally and clinically. Nanoparticulate imaging probes have included semiconductor quantum dots, magnetic and magnetofluorescent nanoparticles, gold nanoparticles and nanoshells, among others. However, the use of nanomaterials for one of the most common imaging techniques, computed tomography (CT), has remained unexplored. Current CT contrast agents are based on small iodinated molecules. They are effective in absorbing X-rays, but non-specific distribution and rapid pharmacokinetics have rather limited their microvascular and targeting performance. Here we propose the use of a polymer-coated Bi(2)S(3) nanoparticle preparation as an injectable CT imaging agent. This preparation demonstrates excellent stability at high concentrations (0.25 M Bi(3+)), high X-ray absorption (fivefold better than iodine), very long circulation times (>2 h) in vivo and an efficacy/safety profile comparable to or better than iodinated imaging agents. We show the utility of these polymer-coated Bi(2)S(3) nanoparticles for enhanced in vivo imaging of the vasculature, the liver and lymph nodes in mice. These nanoparticles and their bioconjugates are expected to become an important adjunct to in vivo imaging of molecular targets and pathological conditions.

Animals↗
PubMed2006

Wortmannin-C20 conjugates generate wortmannin.

We report on C20-6-(N-methylamino)hexanoic conjugates of wortmannin featuring a tertiary enamine attached to the C20 that inhibit phosphoinositol-3-OH kinase (PI3K) by producing wortmannin (Wm) through an intramolecular attack. The generation of Wm by these conjugates permits the design of Wm based PI3K inhibitors that need not fit into the ATP pocket of PI3K, including Wm conjugates of BSA, IgG, or beads. Wm generating WmC20-N(Me)-hexanoate conjugates offer an approach to the design of targeted or slow release forms of Wm which may inhibit PI3K in tissues more selectively than the parent Wm, a compound which has desirable anti-inflammatory and anti-proliferative activities but which also has a variety of toxic effects.

Androstadienes↗
PubMed2006

Identification of the target self-antigens in reperfusion injury.

Reperfusion injury (RI), a potential life-threatening disorder, represents an acute inflammatory response after periods of ischemia resulting from myocardial infarction, stroke, surgery, or trauma. The recent identification of a monoclonal natural IgM that initiates RI led to the identification of nonmuscle myosin heavy chain type II A and C as the self-targets in two different tissues. These results identify a novel pathway in which the innate response to a highly conserved self-antigen expressed as a result of hypoxic stress results in tissue destruction.

Animals↗
PubMed2006

Conjugation of a photosensitizer to an oligoarginine-based cell-penetrating peptide increases the efficacy of photodynamic therapy.

To improve the efficiency of intracellular delivery of photosensitizers and the efficacy of photodynamic therapy, a membrane-penetrating arginine oligopeptide (R7) was conjugated to 5-[4-carboxyphenyl]-10,15,20-triphenyl-2,3-dihydroxychlorin (TPC). The resulting conjugate (R7-TPC) enhanced intracellular TPC uptake, which increased proportionally with the incubation time of the conjugate. The water solubility of the highly hydrophobic TPC photosensitizer was also improved after conjugation. Increased phototoxicity of R7-TPC was observed after an incubation time of only 30 min. Tumor cells mainly underwent apoptosis at lower concentrations of the photosensitizer-polyarginine conjugate, whereas necrotic cell damage became prevalent at higher concentrations.

Arginine↗
PubMed2006

Simultaneous fluorescence imaging of protease expression and vascularity during murine colonoscopy for colonic lesion characterization.

BACKGROUND: Molecularly targeted fluorescent probes are currently being developed to improve the endoscopic detection of intestinal pathologic conditions. OBJECTIVE: We report on the development and testing of a novel multichannel microendoscope capable of quantitatively reporting such probes simultaneously at different wavelengths in real time. We assessed the feasibility of detecting and quantifying beacons that can be activated by protease and correlating imaging with disease state. DESIGN: The microendoscope consisted of a 20-gauge fiberoptic catheter and dichroic beam splitters that simultaneously display visible light, 700 nm and 800 nm near infrared (NIR) fluorescent light. NIR interchannel separation was tested on in vitro phantoms. Two mouse models were used (Apcmin(+/-) mice for colonic adenomas and CT26 murine colon cancer). A perfusion probe and one activated by protease at a separate wavelength were injected before endoscopic evaluation. RESULTS: The microendoscope fluorochrome detection limit was approximately 10 fmol; ratio imaging in the NIR was accurate (+/-8% of true probe concentration between 0.3 to 100 microg/ml of a protease sensor). Both colonic adenomas and adenocarcinomas were clearly visible in the NIR channel on protease probe administration in live mice. Ratio imaging of protease activity/perfusion increased from healthy colon to adenomas to adenocarcinomas. LIMITATIONS: Evaluation across additional spontaneous tumor models may provide more data on the translation of these findings. CONCLUSIONS: Our data show the feasibility of multichannel microendoscopic imaging of molecular targets in vivo and that ratio imaging may provide a novel means for characterizing colonic lesions. When scaled up clinically, this could aid in increasing lesion detection and quantitative assessment of distinct molecular markers.

Adenocarcinoma↗
PubMed2006

Regulatory T cells reversibly suppress cytotoxic T cell function independent of effector differentiation.

Mechanisms of dominant tolerance have evolved within the mammalian immune system to prevent inappropriate immune responses. CD4(+)CD25(+) regulatory T (T(reg)) cells have emerged as central constituents of this suppressive activity. By using multiphoton intravital microscopy in lymph nodes (LNs) of anesthetized mice, we have analyzed how cytotoxic T lymphocytes (CTLs) interact with antigen-presenting target cells in the presence or absence of activated T(reg) cells. Nonregulated CTLs killed their targets at a 6.6-fold faster rate than regulated CTLs. In spite of this compromised effector activity, regulated CTLs exhibited no defect in proliferation, induction of cytotoxic effector molecules and secretory granules, in situ motility, or ability to form antigen-dependent conjugates with target cells. Only granule exocytosis by CTLs was markedly impaired in the presence of T(reg) cells. This selective form of regulation did not require prolonged contact between CTLs and T(reg) cells but depended on CTL responsiveness to transforming growth factor-beta. CTLs quickly regained full killing capacity in LNs upon selective removal of T(reg) cells. Thus, T(reg) cells reversibly suppress CTL-mediated immunity by allowing acquisition of full effector potential but withholding the license to kill.

Animals↗