| 1. |
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| 2. |
- Van der Meer, J. M. R., et al.
(author)
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IL-15 superagonist N-803 improves IFNγ production and killing of leukemia and ovarian cancer cells by CD34+ progenitor-derived NK cells
- 2020
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In: Cancer Immunology and Immunotherapy. - : Springer Science and Business Media Deutschland GmbH. - 0340-7004 .- 1432-0851.
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Journal article (peer-reviewed)abstract
- Allogeneic natural killer (NK) cell transfer is a potential immunotherapy to eliminate and control cancer. A promising source are CD34 + hematopoietic progenitor cells (HPCs), since large numbers of cytotoxic NK cells can be generated. Effective boosting of NK cell function can be achieved by interleukin (IL)-15. However, its in vivo half-life is short and potent trans-presentation by IL-15 receptor α (IL-15Rα) is absent. Therefore, ImmunityBio developed IL-15 superagonist N-803, which combines IL-15 with an activating mutation, an IL-15Rα sushi domain for trans-presentation, and IgG1-Fc for increased half-life. Here, we investigated whether and how N-803 improves HPC-NK cell functionality in leukemia and ovarian cancer (OC) models in vitro and in vivo in OC-bearing immunodeficient mice. We used flow cytometry-based assays, enzyme-linked immunosorbent assay, microscopy-based serial killing assays, and bioluminescence imaging, for in vitro and in vivo experiments. N-803 increased HPC-NK cell proliferation and interferon (IFN)γ production. On leukemia cells, co-culture with HPC-NK cells and N-803 increased ICAM-1 expression. Furthermore, N-803 improved HPC-NK cell-mediated (serial) leukemia killing. Treating OC spheroids with HPC-NK cells and N-803 increased IFNγ-induced CXCL10 secretion, and target killing after prolonged exposure. In immunodeficient mice bearing human OC, N-803 supported HPC-NK cell persistence in combination with total human immunoglobulins to prevent Fc-mediated HPC-NK cell depletion. Moreover, this combination treatment decreased tumor growth. In conclusion, N-803 is a promising IL-15-based compound that boosts HPC-NK cell expansion and functionality in vitro and in vivo. Adding N-803 to HPC-NK cell therapy could improve cancer immunotherapy.
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| 3. |
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| 4. |
- Benninger, Richard K. P., et al.
(author)
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Live Cell Linear Dichroism Imaging Reveals Extensive Membrane Ruffling within the Docking Structure of Natural Killer Cell Immune Synapses
- 2009
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In: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 96:2, s. L13-L15
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Journal article (peer-reviewed)abstract
- We have applied fluorescence imaging of two-photon linear dichroism to measure the subresolution organization of the cell membrane during formation of the activating (cytolytic) natural killer (NK) cell immune synapse (IS). This approach revealed that the NK cell plasma membrane is convoluted into ruffles at the periphery, but not in the center of a mature cytolytic NK cell IS. Time-lapse imaging showed that the membrane ruffles formed at the initial point of contact between NK cells and target cells and then spread radialy across the intercellular contact as the size of the IS increased, becoming absent from the center of the mature synapse. Understanding the role of such extensive membrane ruff ling in the assembly of cytolytic synapses is an intriguing new goal.
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| 5. |
- Dunsby, C., et al.
(author)
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An electronically tunable ultrafast laser source applied to fluorescence imaging and fluorescence lifetime imaging microscopy
- 2004
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In: Journal of Physics D. - : IOP Publishing. - 0022-3727 .- 1361-6463. ; 37:23, s. 3296-3303
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Journal article (peer-reviewed)abstract
- Fluorescence imaging is used widely in microscopy and macroscopic imaging applications for fields ranging from biomedicine to materials science. A critical component for any fluorescence imaging system is the excitation source. Traditionally, wide-field systems use filtered thermal or arc-generated white light sources, while point scanning confocal microscope systems require spatially coherent (point-like) laser sources. Unfortunately, the limited range of visible wavelengths available from conventional laser sources constrains the design and usefulness of fluorescent probes in confocal microscopy. A 'hands-off' laser-like source, electronically tunable across the visible spectrum, would be invaluable for fluorescence imaging and provide new opportunities, e.g. automated excitation fingerprinting and in situ measurement of excitation cross-sections. Yet more information can be obtained using fluorescence lifetime imaging (FLIM), which requires that the light source be pulsed or rapidly modulated. We show how a white light continuum, generated by injecting femtosecond optical radiation into a micro-structured optical fibre, coupled with a simple prism-based tunable filter arrangement, can fulfil all these roles as a continuously electronically tunable (435-1150 nm) visible ultrafast light source in confocal, wide-field and FLIM systems.
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| 6. |
- Eleme, K., et al.
(author)
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Cell surface organization of stress-inducible proteins ULBP and MICA that stimulate human NK cells and T cells via NKG2D
- 2004
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In: Journal of Experimental Medicine. - : Rockefeller University Press. - 0022-1007 .- 1540-9538. ; 199:7, s. 1005-1010
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Journal article (peer-reviewed)abstract
- Cell surface proteins major histocompatibility complex (MHC) class I-related chain A (MICA) and UL16-binding proteins (ULBP) 1, 2, and 3 are up-regulated upon infection or tumor transformation and can activate human natural killer (NK) cells. Patches of cross-linked raft resident ganglioside GM1 colocalized with ULBP1, 2, 3, or MICA, but not CD45. Thus, ULBPs and MICA are expressed in lipid rafts at the cell surface. Western blotting revealed that glycosylphosphatidylinositol (GPI)-anchored ULBP3 but not transmembrane MICA, MHC class I protein, or transferrin receptor, accumulated in detergent-resistant membranes containing GM1. Thus, MICA may have a weaker association with lipid rafts than ULBP3, yet both proteins accumulate at an activating human N-K cell immune synapse. Target cell lipid rafts marked by green fluorescent protein-tagged GPI also accumulate with ULBP3 at some synapses. Electron microscopy reveals constitutive clusters of ULBP at the cell surface. Regarding a specific molecular basis for the organization of these proteins, ULBP1, 2, and 3 and MICA are lipid modified. ULBP1, 2, and 3 are GPI anchored, and we demonstrate here that MICA is S-acylated. Finally, expression of a truncated form of MICA that lacks the putative site for S-acylation and the cytoplasmic tall can be expressed at the cell surface, but is unable to activate NK cells.
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| 7. |
- Frisk, T., et al.
(author)
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Live-cell imaging of natural killer cell mediated tumor rejection in arrays of microwells
- 2010
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In: 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2010, MicroTAS 2010. - 9781618390622 ; , s. 950-952
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Conference paper (peer-reviewed)abstract
- Due to the inherent heterogeneity of immune cell populations a comprehensive view of immune functions can only be achieved by collecting data from many individual cells. We here demonstrate a novel multi-well microchip platform for cell analysis, which we use to study the interaction between natural killer (NK) cells and their target cells.
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| 8. |
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| 9. |
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| 10. |
- Olofsson, J., et al.
(author)
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Picosecond Kerr-gated time-resolved resonance Raman spectroscopy of the Ru(phen)(2)dppz (2+) interaction with DNA
- 2002
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In: Journal of Inorganic Biochemistry. - 0162-0134 .- 1873-3344. ; 91:1, s. 286-297
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Journal article (peer-reviewed)abstract
- To investigate the basis of the 'light-switch' effect, the solvent dependence of the Kerr-gated picosecond-time resolved resonance Raman (TR3) spectra of [Ru(bpy),dppz](2+), [Ru(phen)(2)dppz](2+), and the modified complex [Ru(phen)(2)cpdppzOMe](2+) and a dimer [mu-C4(cpdppz)(2)-(phen)(4)Ru-2](4+) were studied. The investigation focussed on comparing the behaviour of [Ru(phen)(2)dppz](2+) in acetonitrile, ethanol, H2O, D2O, and DNA. The data are consistent with a model wherein excitation induces metal-to-ligand charge transfer (MLCT) to any of the ligands (termed the 'precursor' state) which, by interligand electron transfer (ILET), produces an excited state localised on the dppz ligand, MLCT1. In water this state relaxes with a characteristic time of similar to6 ps to a non-emissive state (MLCT2). The TR3 spectra in water, acetonitrile and DNA are all distinctly different. However. the early (4 ps) water spectrum resembles the spectrum in DNA. This interesting observation suggests that the DNA-bound excited state of the complex can be thought of as a model for the initial, poorly solvated state in water.
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| 11. |
- Olofsson, K., et al.
(author)
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Unanchored micro-tumors in an ultrasonic actuated multi-well microplate with protein repellent coating
- 2016
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In: 20th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2016. - : Chemical and Biological Microsystems Society. - 9780979806490 ; , s. 409-410
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Conference paper (peer-reviewed)abstract
- In this paper we demonstrate an improved tissue engineering method producing 100 three-dimensional (3D) HepG2 cell structures in parallel based on a combination of ultrasonic actuation and polymer coating in a multi-well microplate. By the use of a polymer coating in the plates, the method creates non-adherent tumor models of controlled size and shape which introduces both a more flexible 3D culture system as well as improved quality of the 3D tumor relative to previous studies [1].
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| 12. |
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| 13. |
- Sandström, Niklas, 1981-, et al.
(author)
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Live single cell imaging assays in glass microwells produced by laser-induced deep etching
- 2022
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In: Lab on a Chip. - : Royal Society of Chemistry (RSC). - 1473-0197 .- 1473-0189. ; 22:11, s. 2107-2121
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Journal article (peer-reviewed)abstract
- Miniaturization of cell culture substrates enables controlled analysis of living cells in confined micro-scale environments. This is particularly suitable for imaging individual cells over time, as they can be monitored without escaping the imaging field-of-view (FoV). Glass materials are ideal for most microscopy applications. However, with current methods used in life sciences, glass microfabrication is limited in terms of either freedom of design, quality, or throughput. In this work, we introduce laser-induced deep etching (LIDE) as a method for producing glass microwell arrays for live single cell imaging assays. We demonstrate novel microwell arrays with deep, high-aspect ratio wells that have rounded, dimpled or flat bottom profiles in either single-layer or double-layer glass chips. The microwells are evaluated for microscopy-based analysis of long-term cell culture, clonal expansion, laterally organized cell seeding, subcellular mechanics during migration and immune cell cytotoxicity assays of both adherent and suspension cells. It is shown that all types of microwells can support viable cell cultures and imaging with single cell resolution, and we highlight specific benefits of each microwell design for different applications. We believe that high-quality glass microwell arrays enabled by LIDE provide a great option for high-content and high-resolution imaging-based live cell assays with a broad range of potential applications within life sciences.
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| 14. |
- Taner, S. B., et al.
(author)
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Control of immune responses by trafficking cell surface proteins, vesicles and lipid rafts to and from the immunological synapse
- 2004
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In: Traffic. - : Wiley. - 1398-9219 .- 1600-0854. ; 5:9, s. 651-661
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Research review (peer-reviewed)abstract
- Supramolecular clusters at the immunological synapse provide a mechanism for structuring complex communication networks between cells of the immune system. Regulating intra- and intercellular trafficking of proteins and lipids to and from the immunological synapse provides an additional level of complexity in determining the functional outcome of immune cell interactions. An emergent principle is that molecules requiring tightly regulated cell surface expression, e.g. negative regulators of cell activation or molecules promoting cytotoxicity, are trafficked to the immunological synapse from intracellular secretory as required lysosomes. Many molecules required for the early stages of the intercellular communication are already present at the cell surface, sometimes in lipid rafts, and are rapidly translocated laterally to the intercellular contact. Our understanding of these events critically depends on utilizing appropriate technologies for probing supramolecular recognition in live cells. Thus, we also present here a critical discussion of the technologies used to study lipid rafts and, more broadly, a map of the spatial and temporal dimensions covered by current live cell physical techniques, highlighting where advances are needed to exceed current spatial and temporal boundaries.
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| 15. |
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| 16. |
- Wiklund, Martin, et al.
(author)
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Ultrasonic manipulation of single cells
- 2012
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In: Single-Cell Analysis. - Totowa, NJ : Springer Science+Business Media B.V.. - 9781617795664 ; 853, s. 177-96
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Book chapter (peer-reviewed)abstract
- Ultrasonic manipulation has emerged as a simple and powerful tool for trapping, aggregation, and separation of cells. During the last decade, an increasing amount of applications in the microscale format has been demonstrated, of which the most important is acoustophoresis (continuous acoustic cell or particle separation). Traditionally, the technology has proven to be suitable for treatment of high-density cell and particle suspensions, where large cell and particle numbers are handled simultaneously. In this chapter, we describe how ultrasound can be combined with microfluidics and microplates for particle and cell manipulation approaching the single-cell level. We demonstrate different cell handling methods with the purpose to select, trap, aggregate, and position individual cells in microdevices based on multifrequency ultrasonic actuation, and we discuss applications of the technology involving immune cell interaction studies.
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| 17. |
- Önfelt, Björn, et al.
(author)
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Cell studies of the DNA bis-intercalator Delta-Delta mu-C4(cpdppz)(2)-(phen)(4)Ru-2 (4+) : toxic effects and properties as a light emitting DNA probe in V79 Chinese hamster cells
- 2002
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In: Mutagenesis. - : Oxford University Press (OUP). - 0267-8357 .- 1464-3804. ; 17:4, s. 317-320
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Journal article (peer-reviewed)abstract
- Coordination complexes of type [Ru(L)(3)](2+), where L is a nitrogen-containing aromatic bidentate ligand, can often be photolytically reduced, making them useful in studies of DNA- or protein-mediated electron transfer and in artifical photosynthesis model systems. Upon binding to DNA some Ru(L) complexes have been found to display strongly increased fluorescence compared with when free in solution, making those compounds interesting to test as DNA probes. Thus, they are becoming widely used in the chemistry community. Here, asynchronous cultures of V79 Chinese hamster cells were exposed to the DNA bis-intercalator DeltaDelta-DeltaDelta [mu-C4(cpdppz)(2)-(phen)(4)Ru-2](4+) at 10(-10)-10(-4) M. The extraordinarily strong binding of the compound to DNA was the reason for testing its possible interference with DNA metabolism in intact mammalian cells. Exposure for 1 h to 10(-10)-10(-4) M did not significantly decrease DNA synthesis. Cells exposed to 10(-5) M for 27 h showed no staining of the nucleus, while DNA was stained in cells electroporated in the presence of the compound. However, the Ru dimer was probably taken up by pinocytosis, because numerous minute precipitates could be observed in the cytoplasm. Treatment for 24 h at concentrations of 10(-10)-10(-5) M did not inhibit growth, as indicated by cell density and mitotic activity. Neither did it affect chromosomal arrangements during mitosis. However, at 10(-4) M the density of cultures was reduced by similar to45% and apoptotic cells were frequent, as opposed to mitoses. We also investigated the properties of the Ru dimer as a fluorescent DNA stain. The compound appears attractive as a red DNA stain when broad excitation in the visible range is desirable and extremely low background staining is essential. The low toxicity of the compound is a favourable trait in this context.
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| 18. |
- Önfelt, Björn, et al.
(author)
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Picosecond and steady-state emission of Ru(phen)(2)dppz (2+) in glycerol : Anomalous temperature dependence
- 2003
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In: Journal of Physical Chemistry A. - : American Chemical Society (ACS). - 1089-5639 .- 1520-5215. ; 107:7, s. 1000-1009
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Journal article (peer-reviewed)abstract
- The excited-state deactivation of the light-switch compound [Ru(phen)(2)dppz](2+), where phen = 1,10-phenanthroline and dppz = dipyrido[3,2-a:2',3'-c]phenazine, has been investigated in glycerol using single-photon counting at picosecond time resolution. Relaxation back to the ground state occurs in about 8 ns at 20 degreesC, which is much faster than previously reported in monohydric alcohols, though still slow compared to that in water. Multivariate kinetic analysis reveals three distinct excited species involved in the relaxation process in glycerol. Using a matrix exponential approach for the kinetic data analysis, including global fitting of the relaxation data collected at many wavelengths, individual emission spectra for all three excited species could be resolved. The resolved emission profile for the most short-lived species was found to resemble the steady-state emission spectrum of [Ru(phen)(3)](2+) in glycerol whereas the emission profile of the intermediate species resembled that of [Ru(phen)(2)dppz](2+) in ethanol. The spectrum of the third species is considerably red-shifted compared to those of the other two. The longest lifetime as well as the emission quantum yield show pronounced nonmonotonic variations with temperature in apparent conflict with the Arrhenius equation. This anomalous temperature dependence can be accounted for by a model based on the equilibrium between two excited species, corresponding to the two resolved emission spectra retrieved at 20 degreesC. Thermodynamic data indicates that transfer to the fast-relaxing, red-shifted species is accompanied by a substantial lowering in enthalpy. The thermodynamic data, as well as an abnormally high preexponential factor for the back reaction from the third to the second excited species, could be explained in terms of the formation of two hydrogen bonds, one to each of the aza nitrogens of the dppz moiety.
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