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      1. Author :
        Noberini, R.; Rubio de la Torre, E.; Pasquale, E. B.
      2. Title :
      3. Type :
        Journal Article
      4. Year :
        2012
      5. Publication :
        Cell Adh Migr
      6. Products :
      7. Volume :
        6
      8. Issue :
        N/A
      9. Page Numbers :
        N/A
      10. Research Area :
        N/A
      11. Keywords :
        PC-3M-luc-C6, PC-3M-luc, IVIS, Bioware, Prostate cancer, Bioluminescence, Animals; Chromatography, Liquid; Enzyme-Linked Immunosorbent Assay; Ephrins/genetics/*metabolism; Humans; Mass Spectrometry/*methods; Mice; Receptor, EphA1/genetics/*metabolism
      12. Abstract :
        The Eph receptor tyrosine kinase family includes many members, which are often expressed together in various combinations and can promiscuously interact with multiple ephrin ligands, generating intricate networks of intracellular signals that control physiological and pathological processes. Knowing the entire repertoire of Eph receptors and ephrins expressed in a biological sample is important when studying their biological roles. Moreover, given the correlation between Eph receptor/ephrin expression and cancer pathogenesis, their expression patterns could serve important diagnostic and prognostic purposes. However, profiling Eph receptor and ephrin expression has been challenging. Here we describe a novel and straightforward approach to catalog the Eph receptors present in cultured cells and tissues. By measuring the binding of ephrin Fc fusion proteins to Eph receptors in ELISA and pull-down assays, we determined that a mixture of four ephrins is suitable for isolating both EphA and EphB receptors in a single pull-down. We then used mass spectrometry to identify the Eph receptors present in the pull-downs and estimate their relative levels. This approach was validated in cultured human cancer cell lines, human tumor xenograft tissue grown in mice, and mouse brain tissue. The new mass spectrometry approach we have developed represents a useful tool for the identification of the spectrum of Eph receptors present in a biological sample and could also be extended to profiling ephrin expression.
      13. URL :
        http://www.ncbi.nlm.nih.gov/pubmed/22568954
      14. Call Number :
        PKI @ kd.modi @ 8
      15. Serial :
        10538
      1. Author :
        Christoph Bremer; Ching-Hsuan Tung; Ralph Weissleder
      2. Title :
      3. Type :
        Journal Article
      4. Year :
        2001
      5. Publication :
        Nature Medicine
      6. Products :
      7. Volume :
        7
      8. Issue :
        6
      9. Page Numbers :
        N/A
      10. Research Area :
        Cancer
      11. Keywords :
        near-infrared; near infrared; matrix metalloproteinase; MMP; in vivo imaging; near-infrared fluorescence imaging
      12. Abstract :
        A number of different matrix metalloproteinase (MMP) inhibitors have been developed as cytostatic and anti-angiogenic agents and are currently in clinical testing. One major hurdle in assessing the efficacy of such drugs has been the inability to sense or image anti-proteinase activity directly and non-invasively in vivo. We show here that novel, biocompatible near-infrared fluorogenic MMP substrates can be used as activatable reporter probes to sense MMP activity in intact tumors in nude mice. Moreover, we show for the first time that the effect of MMP inhibition can be directly imaged using this approach within hours after initiation of treatment using the potent MMP inhibitor, prinomastat (AG3340). The developed probes, together with novel near-infrared fluorescence imaging technology will enable the detailed analysis of a number of proteinases critical for advancing the therapeutic use of clinical proteinase inhibitors.
      13. URL :
        http://www.nature.com/nm/journal/v7/n6/abs/nm0601_743.html
      14. Call Number :
        PKI @ sarah.piper @
      15. Serial :
        4509
      1. Author :
        Jenkins, Darlene E; Hornig, Yvette S; Oei, Yoko; Dusich, Joan; Purchio, Tony
      2. Title :
      3. Type :
        Journal Article
      4. Year :
        2005
      5. Publication :
        Breast cancer research: BCR
      6. Products :
      7. Volume :
        7
      8. Issue :
        4
      9. Page Numbers :
        N/A
      10. Research Area :
        N/A
      11. Keywords :
        Animals; Bioware; Breast Neoplasms; Disease Models, Animal; Female; Humans; Luciferases; Mammary Neoplasms, Animal; MDA-MB-231-D3H2LN cells; Mice; Mice, Nude; Neoplasm Metastasis; Plasmids; Transplantation, Heterologous; Tumor Cells, Cultured
      12. Abstract :
        INTRODUCTION Our goal was to generate xenograft mouse models of human breast cancer based on luciferase-expressing MDA-MB-231 tumor cells that would provide rapid mammary tumor growth; produce metastasis to clinically relevant tissues such as lymph nodes, lung, and bone; and permit sensitive in vivo detection of both primary and secondary tumor sites by bioluminescent imaging. METHOD Two clonal cell sublines of human MDA-MB-231 cells that stably expressed firefly luciferase were isolated following transfection of the parental cells with luciferase cDNA. Each subline was passaged once or twice in vivo to enhance primary tumor growth and to increase metastasis. The resulting luciferase-expressing D3H1 and D3H2LN cells were analyzed for long-term bioluminescent stability, primary tumor growth, and distal metastasis to lymph nodes, lungs, bone and soft tissues by bioluminescent imaging. Cells were injected into the mammary fat pad of nude and nude-beige mice or were delivered systemically via intracardiac injection. Metastasis was also evaluated by ex vivo imaging and histologic analysis postmortem. RESULTS The D3H1 and D3H2LN cell lines exhibited long-term stable luciferase expression for up to 4-6 months of accumulative tumor growth time in vivo. Bioluminescent imaging quantified primary mammary fat pad tumor development and detected early spontaneous lymph node metastasis in vivo. Increased frequency of spontaneous lymph node metastasis was observed with D3H2LN tumors as compared with D3H1 tumors. With postmortem ex vivo imaging, we detected additional lung micrometastasis in mice with D3H2LN mammary tumors. Subsequent histologic evaluation of tissue sections from lymph nodes and lung lobes confirmed spontaneous tumor metastasis at these sites. Following intracardiac injection of the MDA-MB-231-luc tumor cells, early metastasis to skeletal tissues, lymph nodes, brain and various visceral organs was detected. Weekly in vivo imaging data permitted longitudinal analysis of metastasis at multiple sites simultaneously. Ex vivo imaging data from sampled tissues verified both skeletal and multiple soft tissue tumor metastasis. CONCLUSION This study characterized two new bioluminescent MDA-MB-231-luc human breast carcinoma cell lines with enhanced tumor growth and widespread metastasis in mice. Their application to current xenograft models of breast cancer offers rapid and highly sensitive detection options for preclinical assessment of anticancer therapies in vivo.
      13. URL :
        http://www.ncbi.nlm.nih.gov/pubmed/15987449
      14. Call Number :
        PKI @ catherine.lautenschlager @
      15. Serial :
        8960
      1. Author :
        Jenkins, Darlene E; Hornig, Yvette S; Oei, Yoko; Dusich, Joan; Purchio, Tony
      2. Title :
      3. Type :
        Journal Article
      4. Year :
        2005
      5. Publication :
        Breast cancer research: BCR
      6. Products :
      7. Volume :
        7
      8. Issue :
        4
      9. Page Numbers :
        N/A
      10. Research Area :
        N/A
      11. Keywords :
        Animals; Bioware; Breast Neoplasms; Disease Models, Animal; Female; Humans; Luciferases; Mammary Neoplasms, Animal; MDA-MB-231-D3H1 cells; Mice; Mice, Nude; Neoplasm Metastasis; Plasmids; Transplantation, Heterologous; Tumor Cells, Cultured
      12. Abstract :
        INTRODUCTION Our goal was to generate xenograft mouse models of human breast cancer based on luciferase-expressing MDA-MB-231 tumor cells that would provide rapid mammary tumor growth; produce metastasis to clinically relevant tissues such as lymph nodes, lung, and bone; and permit sensitive in vivo detection of both primary and secondary tumor sites by bioluminescent imaging. METHOD Two clonal cell sublines of human MDA-MB-231 cells that stably expressed firefly luciferase were isolated following transfection of the parental cells with luciferase cDNA. Each subline was passaged once or twice in vivo to enhance primary tumor growth and to increase metastasis. The resulting luciferase-expressing D3H1 and D3H2LN cells were analyzed for long-term bioluminescent stability, primary tumor growth, and distal metastasis to lymph nodes, lungs, bone and soft tissues by bioluminescent imaging. Cells were injected into the mammary fat pad of nude and nude-beige mice or were delivered systemically via intracardiac injection. Metastasis was also evaluated by ex vivo imaging and histologic analysis postmortem. RESULTS The D3H1 and D3H2LN cell lines exhibited long-term stable luciferase expression for up to 4-6 months of accumulative tumor growth time in vivo. Bioluminescent imaging quantified primary mammary fat pad tumor development and detected early spontaneous lymph node metastasis in vivo. Increased frequency of spontaneous lymph node metastasis was observed with D3H2LN tumors as compared with D3H1 tumors. With postmortem ex vivo imaging, we detected additional lung micrometastasis in mice with D3H2LN mammary tumors. Subsequent histologic evaluation of tissue sections from lymph nodes and lung lobes confirmed spontaneous tumor metastasis at these sites. Following intracardiac injection of the MDA-MB-231-luc tumor cells, early metastasis to skeletal tissues, lymph nodes, brain and various visceral organs was detected. Weekly in vivo imaging data permitted longitudinal analysis of metastasis at multiple sites simultaneously. Ex vivo imaging data from sampled tissues verified both skeletal and multiple soft tissue tumor metastasis. CONCLUSION This study characterized two new bioluminescent MDA-MB-231-luc human breast carcinoma cell lines with enhanced tumor growth and widespread metastasis in mice. Their application to current xenograft models of breast cancer offers rapid and highly sensitive detection options for preclinical assessment of anticancer therapies in vivo.
      13. URL :
        http://www.ncbi.nlm.nih.gov/pubmed/15987449
      14. Call Number :
        PKI @ catherine.lautenschlager @
      15. Serial :
        8993
      1. Author :
        Harms, Jerome S; Durward, Marina A; Magnani, Diogo M; Splitter, Gary A
      2. Title :
      3. Type :
        Journal Article
      4. Year :
        2009
      5. Publication :
        Journal of immune based therapies and vaccines
      6. Products :
      7. Volume :
        7
      8. Issue :
        N/A
      9. Page Numbers :
        N/A
      10. Research Area :
        N/A
      11. Keywords :
        Bioware; pXen-13
      12. Abstract :
        BACKGROUND There is no safe, effective human vaccine against brucellosis. Live attenuated Brucella strains are widely used to vaccinate animals. However these live Brucella vaccines can cause disease and are unsafe for humans. Killed Brucella or subunit vaccines are not effective in eliciting long term protection. In this study, we evaluate an approach using a live, non-pathogenic bacteria (E. coli) genetically engineered to mimic the brucellae pathway of infection and present antigens for an appropriate cytolitic T cell response. METHODS E. coli was modified to express invasin of Yersinia and listerialysin O (LLO) of Listeria to impart the necessary infectivity and antigen releasing traits of the intracellular pathogen, Brucella. This modified E. coli was considered our vaccine delivery system and was engineered to express Green Fluorescent Protein (GFP) or Brucella antigens for in vitro and in vivo immunological studies including cytokine profiling and cytotoxicity assays. RESULTS The E. coli vaccine vector was able to infect all cells tested and efficiently deliver therapeutics to the host cell. Using GFP as antigen, we demonstrate that the E. coli vaccine vector elicits a Th1 cytokine profile in both primary and secondary immune responses. Additionally, using this vector to deliver a Brucella antigen, we demonstrate the ability of the E. coli vaccine vector to induce specific Cytotoxic T Lymphocytes (CTLs). CONCLUSION Protection against most intracellular bacterial pathogens can be obtained mostly through cell mediated immunity. Data presented here suggest modified E. coli can be used as a vaccine vector for delivery of antigens and therapeutics mimicking the infection of the pathogen and inducing cell mediated immunity to that pathogen.
      13. URL :
        http://www.ncbi.nlm.nih.gov/pubmed/19126207
      14. Call Number :
        PKI @ catherine.lautenschlager @
      15. Serial :
        9029
      1. Author :
        Pello, O. M.; Chevre, R.; Laoui, D.; De Juan, A.; Lolo, F.; Andres-Manzano, M. J.; Serrano, M.; Van Ginderachter, J. A.; Andres, V.
      2. Title :
      3. Type :
        Journal Article
      4. Year :
        2012
      5. Publication :
        PLoS One
      6. Products :
      7. Volume :
        7
      8. Issue :
        N/A
      9. Page Numbers :
        N/A
      10. Research Area :
        N/A
      11. Keywords :
        IntegriSense
      12. Abstract :
        Although tumor-associated macrophages (TAMs) are involved in tumor growth and metastasis, the mechanisms controlling their pro-tumoral activities remain largely unknown. The transcription factor c-MYC has been recently shown to regulate in vitro human macrophage polarization and be expressed in macrophages infiltrating human tumors. In this study, we exploited the predominant expression of LysM in myeloid cells to generate c-Myc(fl/fl) LysM(cre/+) mice, which lack c-Myc in macrophages, to investigate the role of macrophage c-MYC expression in cancer. Under steady-state conditions, immune system parameters in c-Myc(fl/fl) LysM(cre/+) mice appeared normal, including the abundance of different subsets of bone marrow hematopoietic stem cells, precursors and circulating cells, macrophage density, and immune organ structure. In a model of melanoma, however, TAMs lacking c-Myc displayed a delay in maturation and showed an attenuation of pro-tumoral functions (e.g., reduced expression of VEGF, MMP9, and HIF1alpha) that was associated with impaired tissue remodeling and angiogenesis and limited tumor growth in c-Myc(fl/fl) LysM(cre/+) mice. Macrophage c-Myc deletion also diminished fibrosarcoma growth. These data identify c-Myc as a positive regulator of the pro-tumoral program of TAMs and suggest c-Myc inactivation as an attractive target for anti-cancer therapy.
      13. URL :
        http://www.ncbi.nlm.nih.gov/pubmed/23028984
      14. Call Number :
        PKI @ kd.modi @ 33
      15. Serial :
        10376
      1. Author :
        Xie, B. W.; Mol, I. M.; Keereweer, S.; van Beek, E. R.; Que, I.; Snoeks, T. J.; Chan, A.; Kaijzel, E. L.; Lowik, C. W.
      2. Title :
      3. Type :
        Journal Article
      4. Year :
        2012
      5. Publication :
        PLoS One
      6. Products :
      7. Volume :
        7
      8. Issue :
        N/A
      9. Page Numbers :
        N/A
      10. Research Area :
        N/A
      11. Keywords :
        4T1-luc2, ProSense, MMPSense, CRi, Maestro, IVIS Animals; Benzenesulfonates/diagnostic use; Diagnostic Imaging/instrumentation/*methods; Disease Models, Animal; Disease Progression; Fluorescent Dyes/*diagnostic use; Indoles/diagnostic use; Luminescent Measurements/instrumentation/*methods; Mammary Neoplasms, Experimental/*diagnosis/pathology; Mice
      12. Abstract :
        Bioluminescence imaging (BLI) has shown its appeal as a sensitive technique for in vivo whole body optical imaging. However, the development of injectable tumor-specific near-infrared fluorescent (NIRF) probes makes fluorescence imaging (FLI) a promising alternative to BLI in situations where BLI cannot be used or is unwanted (e.g., spontaneous transgenic tumor models, or syngeneic mice to study immune effects).In this study, we addressed the questions whether it is possible to detect tumor progression using FLI with appropriate sensitivity and how FLI correlates with BLI measurements. In addition, we explored the possibility to simultaneously detect multiple tumor characteristics by dual-wavelength FLI (~700 and ~800 nm) in combination with spectral unmixing. Using a luciferase-expressing 4T1-luc2 mouse breast cancer model and combinations of activatable and targeting NIRF probes, we showed that the activatable NIRF probes (ProSense680 and MMPSense680) and the targeting NIRF probes (IRDye 800CW 2-DG and IRDye 800CW EGF) were either activated by or bound to 4T1-luc2 cells. In vivo, we implanted 4T1-luc2 cells orthotopically in nude mice and were able to follow tumor progression longitudinally both by BLI and dual-wavelength FLI. We were able to reveal different probe signals within the tumor, which co-localized with immuno-staining. Moreover, we observed a linear correlation between the internal BLI signals and the FLI signals obtained from the NIRF probes. Finally, we could detect pulmonary metastases both by BLI and FLI and confirmed their presence histologically.Taken together, these data suggest that dual-wavelength FLI is a feasible approach to simultaneously detect different features of one tumor and to follow tumor progression with appropriate specificity and sensitivity. This study may open up new perspectives for the detection of tumors and metastases in various experimental models and could also have clinical applications, such as image-guided surgery.
      13. URL :
        http://www.ncbi.nlm.nih.gov/pubmed/22348134
      14. Call Number :
        PKI @ kd.modi @ 2
      15. Serial :
        10426
      1. Author :
        Cao, L.; Kobayakawa, S.; Yoshiki, A.; Abe, K.
      2. Title :
      3. Type :
        Journal Article
      4. Year :
        2012
      5. Publication :
        PLoS One
      6. Products :
      7. Volume :
        7
      8. Issue :
        N/A
      9. Page Numbers :
        N/A
      10. Research Area :
        N/A
      11. Keywords :
        AngioSense, Abdomen; Animals; Imaging, Three-Dimensional; Liver/cytology; Mice; Mice, Transgenic; Microscopy/*instrumentation/*methods; Molecular Imaging/*instrumentation/*methods; Pancreas/cytology/ultrastructure; Time-Lapse Imaging
      12. Abstract :
        Intravital imaging of brain and bone marrow cells in the skull with subcellular resolution has revolutionized neurobiology, immunology and hematology. However, the application of this powerful technology in studies of abdominal organs has long been impeded by organ motion caused by breathing and heartbeat. Here we describe for the first time a simple device designated 'microstage' that effectively reduces organ motions without causing tissue lesions. Combining this microstage device with an upright intravital laser scanning microscope equipped with a unique stick-type objective lens, the system enables subcellular-level imaging of abdominal organs in live mice. We demonstrate that this technique allows for the quantitative analysis of subcellular structures and gene expressions in cells, the tracking of intracellular processes in real-time as well as three-dimensional image construction in the pancreas and liver of the live mouse. As the aforementioned analyses based on subcellular imaging could be extended to other intraperitoneal organs, the technique should offer great potential for investigation of physiological and disease-specific events of abdominal organs. The microstage approach adds an exciting new technique to the in vivo imaging toolbox.
      13. URL :
        http://www.ncbi.nlm.nih.gov/pubmed/22479464
      14. Call Number :
        PKI @ kd.modi @ 6
      15. Serial :
        10431
      1. Author :
        Kozlowski, C.; Weimer, R. M.
      2. Title :
      3. Type :
        Journal Article
      4. Year :
        2012
      5. Publication :
        PLoS One
      6. Products :
      7. Volume :
        7
      8. Issue :
        N/A
      9. Page Numbers :
        N/A
      10. Research Area :
        N/A
      11. Keywords :
        AngioSense, Animals; Antigens, CD/metabolism; Antigens, Differentiation, Myelomonocytic/metabolism; Calcium-Binding Proteins/metabolism; Central Nervous System/metabolism; Green Fluorescent Proteins/genetics/*metabolism; Immunohistochemistry/*methods; Lipopolysaccharides/pharmacology; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microfilament Proteins/metabolism; Microglia/cytology/drug effects/*metabolism; Microscopy, Confocal/*methods; Receptors, Cytokine/genetics/metabolism; Receptors, HIV/genetics/metabolism; Reproducibility of Results
      12. Abstract :
        Microglia are specialized immune cells of the brain. Upon insult, microglia initiate a cascade of cellular responses including a characteristic change in cell morphology. To study the dynamics of microglia immune response in situ, we developed an automated image analysis method that enables the quantitative assessment of microglia activation state within tissue based solely on cell morphology. Per cell morphometric analysis of fluorescently labeled microglia is achieved through local iterative threshold segmentation, which reduces errors caused by signal-to-noise variation across large volumes. We demonstrate, utilizing systemic application of lipopolysaccharide as a model of immune challenge, that several morphological parameters, including cell perimeter length, cell roundness and soma size, quantitatively distinguish resting versus activated populations of microglia within tissue comparable to traditional immunohistochemistry methods. Furthermore, we provide proof-of-concept data that monitoring soma size enables the longitudinal assessment of microglia activation in the mouse neocortex imaged via 2-photon in vivo microscopy. The ability to quantify microglia activation automatically by shape alone allows unbiased and rapid analysis of both fixed and in vivo central nervous system tissue.
      13. URL :
        http://www.ncbi.nlm.nih.gov/pubmed/22457705
      14. Call Number :
        PKI @ kd.modi @ 8
      15. Serial :
        10435
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