| 1. |
- Amin, Risul, et al.
(författare)
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The kidney injury caused by the onset of acute graft-versus-host disease is associated with down-regulation of alpha Klotho
- 2020
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Ingår i: International Immunopharmacology. - : Elsevier BV. - 1567-5769 .- 1878-1705. ; 78
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Tidskriftsartikel (refereegranskat)abstract
- Acute graft-versus-host disease (aGVHD) and kidney injury are the major complications after allogeneic hematopoietic stem cell transplantation (HSCT). Although the underlying mechanisms for the development of these complications are not yet fully understood, it has been proposed that emergence of aGVHD contributes to the development of kidney injury after HSCT. We have shown previously that aGVHD targets the kidney in a biphasic manner: at the onset, inflammatory genes are up-regulated, while when aGVHD becomes established, donor lymphocytes infiltrate the kidney. Here, we characterize renal manifestations at the onset of aGVHD. Mice receiving allogeneic bone marrow and spleen cells displayed symptoms of aGVHD and elevated serum levels of tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) within 4 days. There was concurrent kidney injury with the following characteristics: (1) elevated expression of the kidney injury biomarker, neutrophil gelatinase-associated lipocalin (NGAL), (2) accumulation of hetero-lysosomes in proximal tubule epithelial cells, and (3) reductions in alpha Klotho mRNA and protein and increased serum levels of fibroblast growth factor 23 (Fgf23), phosphate and urea. This situation resembled acute renal injury caused by bacterial lipopolysaccharide. We conclude that the onset of aGVHD is associated with kidney injury involving down-regulation of alpha Klotho, a sight that may inspire novel therapeutic approaches.
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| 2. |
- Bost, Jeremy P., et al.
(författare)
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Novel endosomolytic compounds enable highly potent delivery of antisense oligonucleotides
- 2022
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Ingår i: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 5:1
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Tidskriftsartikel (refereegranskat)abstract
- The therapeutic and research potentials of oligonucleotides (ONs) have been hampered in part by their inability to effectively escape endosomal compartments to reach their cytosolic and nuclear targets. Splice-switching ONs (SSOs) can be used with endosomolytic small molecule compounds to increase functional delivery. So far, development of these compounds has been hindered by a lack of high-resolution methods that can correlate SSO trafficking with SSO activity. Here we present in-depth characterization of two novel endosomolytic compounds by using a combination of microscopic and functional assays with high spatiotemporal resolution. This system allows the visualization of SSO trafficking, evaluation of endosomal membrane rupture, and quantitates SSO functional activity on a protein level in the presence of endosomolytic compounds. We confirm that the leakage of SSO into the cytosol occurs in parallel with the physical engorgement of LAMP1-positive late endosomes and lysosomes. We conclude that the new compounds interfere with SSO trafficking to the LAMP1-positive endosomal compartments while inducing endosomal membrane rupture and concurrent ON escape into the cytosol. The efficacy of these compounds advocates their use as novel, potent, and quick-acting transfection reagents for antisense ONs.
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| 3. |
- Cavallaro, Sara, et al.
(författare)
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Label-Free Surface Protein Profiling of Extracellular Vesicles by an Electrokinetic Sensor
- 2019
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Ingår i: ACS Sensors. - : AMER CHEMICAL SOC. - 2379-3694. ; 4:5, s. 1399-1408
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Tidskriftsartikel (refereegranskat)abstract
- Small extracellular vesicles (sEVs) generated from the endolysosomal system, often referred to as exosomes, have attracted interest as a suitable biomarker for cancer diagnostics, as they carry valuable biological information and reflect their cells of origin. Herein, we propose a simple and inexpensive electrical method for label-free detection and profiling of sEVs in the size range of exosomes. The detection method is based on the electrokinetic principle, where the change in the streaming current is monitored as the surface markers of the sEVs interact with the affinity reagents immobilized on the inner surface of a silica microcapillary. As a proof-of-concept, we detected sEVs derived from the non-small-cell lung cancer (NSCLC) cell line H1975 for a set of representative surface markers, such as epidermal growth factor receptor (EGFR), CD9, and CD63. The detection sensitivity was estimated to be similar to 175000 sEVs, which represents a sensor surface coverage of only 0.04%. We further validated the ability of the sensor to measure the expression level of a membrane protein by using sEVs displaying artificially altered expressions of EGFR and CD63, which were derived from NSCLC and human embryonic kidney (HEK) 293T cells, respectively. The analysis revealed that the changes in EGFR and CD63 expressions in sEVs can be detected with a sensitivity in the order of 10% and 3%, respectively, of their parental cell expressions. The method can be easily parallelized and combined with existing microfluidic-based EV isolation technologies, allowing for rapid detection and monitoring of sEVs for cancer diagnosis.
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| 4. |
- Cavallaro, Sara, et al.
(författare)
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Multiparametric Profiling of Single Nanoscale Extracellular Vesicles by Combined Atomic Force and Fluorescence Microscopy : Correlation and Heterogeneity in Their Molecular and Biophysical Features
- 2021
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Ingår i: Small. - : Wiley. - 1613-6810 .- 1613-6829. ; 17:14
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Tidskriftsartikel (refereegranskat)abstract
- Being a key player in intercellular communications, nanoscale extracellular vesicles (EVs) offer unique opportunities for both diagnostics and therapeutics. However, their cellular origin and functional identity remain elusive due to the high heterogeneity in their molecular and physical features. Here, for the first time, multiple EV parameters involving membrane protein composition, size and mechanical properties on single small EVs (sEVs) are simultaneously studied by combined fluorescence and atomic force microscopy. Furthermore, their correlation and heterogeneity in different cellular sources are investigated. The study, performed on sEVs derived from human embryonic kidney 293, cord blood mesenchymal stromal and human acute monocytic leukemia cell lines, identifies both common and cell line-specific sEV subpopulations bearing distinct distributions of the common tetraspanins (CD9, CD63, and CD81) and biophysical properties. Although the tetraspanin abundances of individual sEVs are independent of their sizes, the expression levels of CD9 and CD63 are strongly correlated. A sEV population co-expressing all the three tetraspanins in relatively high abundance, however, having average diameters of <100 nm and relatively low Young moduli, is also found in all cell lines. Such a multiparametric approach is expected to provide new insights regarding EV biology and functions, potentially deciphering unsolved questions in this field.
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| 5. |
- Gupta, Dhanu, et al.
(författare)
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Amelioration of systemic inflammation via the display of two different decoy protein receptors on extracellular vesicles
- 2021
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Ingår i: Nature Biomedical Engineering. - Stockholm : Karolinska Institutet, Dept of Laboratory Medicine. - 2157-846X.
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Tidskriftsartikel (refereegranskat)abstract
- Extracellular vesicles (EVs) can be functionalized to display specific protein receptors on their surface. However, surface-display technology typically labels only a small fraction of the EV population. Here, we show that the joint display of two different therapeutically relevant protein receptors on EVs can be optimized by systematically screening EV-loading protein moieties. We used cytokine-binding domains derived from tumour necrosis factor receptor 1 (TNFR1) and interleukin-6 signal transducer (IL-6ST), which can act as decoy receptors for the pro-inflammatory cytokines tumour necrosis factor alpha (TNF-α) and IL-6, respectively. We found that the genetic engineering of EV-producing cells to express oligomerized exosomal sorting domains and the N-terminal fragment of syntenin (a cytosolic adaptor of the single transmembrane domain protein syndecan) increased the display efficiency and inhibitory activity of TNFR1 and IL-6ST and facilitated their joint display on EVs. In mouse models of systemic inflammation, neuroinflammation and intestinal inflammation, EVs displaying the cytokine decoys ameliorated the disease phenotypes with higher efficacy as compared with clinically approved biopharmaceutical agents targeting the TNF-α and IL-6 pathways.
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| 6. |
- Gupta, Dhanu
(författare)
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Engineered extracellular vesicles for biomedical applications
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Nature's very own nanoparticle, Extracellular vesicles (EVs), are lipid membrane-enclosed vesicles encapsulated with diverse biomolecules and are actively secreted by all cell types for intercellular communication. The unique properties of EVs, such as stability in circulation, biocompatibility, immune tolerance, and the ability to cross biological barriers, render EVs a next-generation drug delivery tool. Therapeutic EV research has seen tremendous development in the past decade, from in vitro studies towards pre-clinical models to various clinical trials. Even so, the road towards successful clinical translation has faced various hurdles primarily due to the lack of technology to address the knowledge gap in EV biology. Hence, this thesis is focused on addressing some of these critical challenges and exploring novel biomedical applications for EVs. EVs are considered as essential mediators in physiology and disease pathology. However, to elucidate their important role in pathophysiology or as therapeutics, sensitive tools for visualising them are much needed. Here, in paper I, we have developed a sensitive bioluminescent labelling system for tracking EVs in vitro and in vivo. By genetically modifying the producer cells with EV-associated tetraspanins-fusions, we could efficiently load luciferase enzymes (Nanoluciferase and Thermoluciferase) into EVs. Utilising the Nanoluciferase labelling system, we could detect as low as 5000 EVs in a solution, and the naked eye could visualise the luminescence generated from these EVs. With this level of sensitivity, we explored various in vivo applications and observed that exogenous EVs are rapidly distributed throughout the body, primarily to the liver, lung, and spleen. In addition, we identified that EV subpopulations differ in their in vivo biodistribution profile. In summary, this system allows for highly sensitive detection of EVs in vivo and reflects the true fate of EVs. Despite tremendous advancement in understanding EV biology or engineering, techniques to surface engineer EVs with large protein biotherapeutics without altering their innate properties are largely lacking. Here in paper II, we developed a novel surface display technology for EVs, which allows for efficient display of several membrane proteins on the EV surface simultaneously. Using this platform, we decorated EVs' surface with cytokine receptors that can decoy pro-inflammatory cytokines such as TNF-α or IL-6/sIL-6R complexes. These cytokine decoy EVs were more active than a clinically approved biologic against TNF-α in vitro. Importantly, these cytokine decoy EVs ameliorated the disease phenotype in three different mice inflammation models, including neuroinflammation. In paper III, we have applied interleukin 6 signal transducer (IL-6ST) decoy EVs to tackle inflammation in muscle pathologies to enhance the muscle regeneration process. Using decoy EVs as a therapeutic intervention in mdx mice mimicking Duchene Muscular Dystrophy (DMD), we could achieve significant downregulation of phosphorylation of the proinflammatory transcription factor STAT3 in muscles. In conclusion, the tools developed in this thesis, from highly sensitive detection of EV subtype to efficient display of biotherapeutics cargo on EV surfaces, holds great future potential and applicability in numerous biomedical applications of EVs.
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| 7. |
- Last, Torben, et al.
(författare)
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Micro Swirl-Nozzle Enables Portable Delivery ofLarge-Molecule Biopharmaceuticals to the Lung
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Large-molecule pharmaceuticals offer new treatment options for severe lung disease. However, delivering these drugs to the lung is challenging due to the elevated shear rates during the aerosolization process. So far, this has prevented an application in portable inhalers, holding back the portable use of biopharmaceuticals for drug delivery. We demonstrate that a micro swirl nozzle can aerosolize fragile biopharmaceuticals in an aqueous solution. Shear rate simulations of the nozzle unit indicate orders of magnitude in shear rate reduction compared with conventional aqueous aerosolization units. Catalase protein can survive the aerosolization process at up to 50 bar without significant degradation. The protein further remains enzymatically active after the spray event.Using an in-vitro model, we present the delivery of more complex and fragile mRNA structures (Nanoluc mRNA) at high concentrations when encapsulated in solid lipid nanoparticles (LNPs) or Extracellular vesicles (EVs). These vesicles maintain their capability to pass the cell wall in in-vitro cell cultures, leading to an expression of the encapsulated protein structure within the celll. Micro swirl nozzles can enable the portable delivery of large molecule pharmaceuticals and bring new treatment options to patients who have so far had to rely on stationary devices.
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| 8. |
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