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  • 1.
    Al-Amin, Rasel A.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Science for Life Laboratory, SciLifeLab, Science for Life Laboratory, SciLifeLab.
    Johansson, Lars
    Division of Translational Medicine & Chemical Biology, Department of Medical Biochemistry & Biophysics, Karolinska Institutet.
    Landegren, Nils
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Autoimmunity. Uppsala University, Science for Life Laboratory, SciLifeLab. Department of Medicine (Solna), Karolinska University Hospital, Karolinska Institutet.
    Löf, Liza
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Abdurakhmanov, Eldar
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Blokzijl, Andries
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Svensson, Richard
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lönn, Peter
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Dept. Of Immunology, Genetics and Pathology,.
    Söderberg, Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Kamali-Moghaddam, Masood
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Artursson, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lundbäck, Thomas
    Division of Translational Medicine & Chemical Biology, Department of Medical Biochemistry & Biophysics, Karolinska Institutet.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Target Engagement-Mediated Amplification for Monitoring Drug-Target Interactions in SituManuscript (preprint) (Other academic)
    Abstract [en]

    It is important to determine the localization of drugs or drug candidates at cellular and subcellular resolution in relevant clinical specimens. This is necessary to evaluate drug candidates from early stages of drug development to clinical evaluation of mutations potentially causing resistance to targeted therapy. We describe a technology where oligonucleotide-conjugated drug molecules are used to visualize and measure target engagement in situ via rolling-circle amplification (RCA) of circularized oligonucleotide probes (padlock probes). We established this target engagement-mediated amplification (TEMA) technique using kinase inhibitor precursor compounds, and we applied the assay to investigate target interactions by microscopy in pathology tissue sections and using flow cytometry for blood samples from patients, as well as in commercial arrays including almost half of all human proteins.  In the variant proxTEMAtechnique, in situ proximity ligation assays were performed by combining drug-DNA conjugates with antibody-DNA conjugates to specifically reveal drug binding to particular on- or off-targets in pathological tissues sections. In conclusion, the TEMA methods successfully visualize drug-target interaction by experimental and clinically approved kinase inhibitors in situ and with kinases among a large collection of arrayed proteins. 

  • 2.
    Arngården, Linda
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Löf, Liza
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Crosstalk between WNT and Hippo signaling pathways changes upon colon cancer stage and is affected by cell density and loss of or mutated E-cadherin proteinManuscript (preprint) (Other academic)
  • 3.
    Dahlin, Joakim S.
    et al.
    Karolinska Univ Hosp, Karolinska Inst, Dept Med, Stockholm, Sweden..
    Ekoff, Maria
    Karolinska Univ Hosp, Karolinska Inst, Dept Med, Stockholm, Sweden..
    Grootens, Jennine
    Karolinska Univ Hosp, Karolinska Inst, Dept Med, Stockholm, Sweden..
    Löf, Liza
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Amini, Rose-Marie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical and experimental pathology.
    Hagberg, Hans
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology.
    Ungerstedt, Johanna S.
    Karolinska Inst, Dept Med Huddinge, Stockholm, Sweden.;Karolinska Univ Hosp, Hematol Ctr, Stockholm, Sweden..
    Olsson-Strömberg, Ulla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Haematology.
    Nilsson, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Haematology. Karolinska Univ Hosp, Karolinska Inst, Dept Med, Stockholm, Sweden.
    KIT signaling is dispensable for human mast cell progenitor development2017In: Blood, ISSN 0006-4971, E-ISSN 1528-0020, Vol. 130, no 16, p. 1785-1794Article in journal (Refereed)
    Abstract [en]

    Human hematopoietic progenitors are generally assumed to require stem cell factor (SCF) and KIT signaling during differentiation for the formation of mast cells. Imatinib treatment, which inhibits KIT signaling, depletes mast cells in vivo. Furthermore, the absence of SCF or imatinib treatment prevents progenitors from developing into mast cells in vitro. However, these observations do not mean that mast cell progenitors require SCF and KIT signaling throughout differentiation. Here, we demonstrate that circulating mast cell progenitors are present in patients undergoing imatinib treatment. In addition, we show that mast cell progenitors from peripheral blood survive, mature, and proliferate without SCF and KIT signaling in vitro. Contrary to the prevailing consensus, our results show that SCF and KIT signaling are dispensable for early mast cell development.

  • 4.
    Dubois, Louise
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Chemistry.
    Löf, Liza
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Larsson, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Chemistry.
    Hultenby, Kjell
    Department of Laboratory Medicine, Karolinska Institutet, SE-141 86 Huddinge, Sweden.
    Waldenström, Anders
    Department of Public Health and Clinical Medicine, Umeå University, SE-901 85 Umeå, Sweden.
    Kamali-Moghaddam, Masood
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Ronquist, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Chemistry.
    Ronquist, K. Göran
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Chemistry.
    Human erythrocyte-derived nanovesicles can readily be loaded with doxorubicin and act as anticancer agents2018In: Cancer Research Frontiers, ISSN 2328-5249, Vol. 4, no 1, p. 13-26Article in journal (Refereed)
    Abstract [en]

    Purpose: In future therapeutics new formulas are needed that assure lower doses, fewer side effects, targeted administration and protection of the drug from degradation. In a first step to fulfil the requirements defined above, we carried out an in vitro study by developing a new procedure to encapsulate drugs using native vesicles first from prostasomes and then from erythrocyte membranes known to be well tolerated. The new method for production of drug delivery vesicles utilized osmotic loading of detergent resistant membranes (DRMs).

    Materials and methods: DRMs of prostasomes and prepared human erythrocyte membranes were extracted and separated in a sucrose gradient at a density of 1.10 g/mL containing 1% Triton X-100. These DRMs were characterized by electron microscopy (transmission and scanning EM) and loaded with low and high molecular compounds. PC3 prostate cancer cells were treated with doxorubicin loaded DRMs in triplicate. DAPI (nuclear fluorescent stain) was included and fluorescence microscopic pictures were taken before the cells were trypsinized and counted after 48h.

    Results: The content of the well separated band was observed ultrastructurally as small spherical, double layered membrane vesicles, (DRM vesicles) which harbored hyperosmolar sucrose of the gradient. Encapsulated hyperosmolar sucrose induced a transient osmotic lysis of the DRM vesicles when suspended in isotonic buffer containing loading molecules allowing vesicular inclusion. After this proof of concept, the method was finally employed for doxorubicin loading of DRM vesicles from human erythrocytes. When incubating such vesicles with PC3 cells a complete arrest of growth was observed in sharp contrast to PC3 cells incubated with plain doxorubicin in similar conditions.

    Conclusion: The present results open up new possibilities for using DRM vesicles as drug delivery vesicles.

  • 5.
    Ebai, Tonge
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    de Oliveira, Felipe Marques Souza
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Löf, Liza
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Wik, Lotta
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Schweiger, Caroline
    Charité Comprehensive Cancer Center, University of Berlin, Berlin, Germany; Institute of Pathology, Medical University of Graz, Graz, Austria; .
    Larsson, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Biochemial structure and function.
    Keilholtz, Ulrich
    Institute of Pathology, Medical University of Graz, Graz, Austria; .
    Haybaeck, Johannes
    Charité Comprehensive Cancer Center, University of Berlin, Berlin, Germany; Department of Pathology, Otto von Guericke University Magdeburg, Magdeburg, Germany..
    Landegren, Ulf
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Kamali-Moghaddam, Masood
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Analytically Sensitive Protein Detection in Microtiter Plates by Proximity Ligation with Rolling Circle Amplification2017In: Clinical Chemistry, ISSN 0009-9147, E-ISSN 1530-8561, Vol. 63, no 9, p. 1497-1505Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Detecting proteins at low concentrations in plasma is crucial for early diagnosis. Current techniques in clinical routine, such as sandwich ELISA, provide sensitive protein detection because of a dependence on target recognition by pairs of antibodies, but detection of still lower protein concentrations is often called for. Proximity ligation assay with rolling circle amplification (PLARCA) is a modified proximity ligation assay (PLA) for analytically specific and sensitive protein detection via binding of target proteins by 3 antibodies, and signal amplification via rolling circle amplification (RCA) in microtiter wells, easily adapted to instrumentation in use in hospitals.

    METHODS: Proteins captured by immobilized antibodies were detected using a pair of oligonucleotide-conjugated antibodies. Upon target recognition, these PLA probes guided oligonucleotide ligation, followed by amplification via RCA of circular DNA strands that formed in the reaction. The RCA products were detected by horseradish peroxidase-labeled oligonucleotides to generate colorimetric reaction products with readout in an absorbance microplate reader.

    RESULTS: We compared detection of interleukin (IL)-4, IL-6, IL-8, p53, and growth differentiation factor-15 by PLARCA and conventional sandwich ELISA or immuno RCA. PLARCA detected lower concentrations of proteins and exhibited a broader dynamic range compared ELISA and iRCA using the same antibodies. IL-4 and IL-6 were detected in clinical samples at femtomolar concentrations, considerably lower than for ELISA.

    CONCLUSIONS: PLARCA offers detection of lower protein levels and increased dynamic ranges compared to ELISA. The PLARCA procedure may be adapted to routine instrumentation available in hospitals and research laboratories.

  • 6.
    Kamali-Moghaddam, Masood
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Löf, Liza
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Oliveir, Felipe
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Wik, Lotta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Wu, Di
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Yan, Junhong
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Advanced Molecular Tools for Proteomic Analyses of Microvesicles2014In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 23, p. 102-102Article in journal (Other academic)
  • 7.
    Kamali-Moghaddam, Masood
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Yan, Junhong
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Wu, Di
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Löf, Liza
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Gu, Jijuan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Molecular tools for sensitive detection of microvesicles as biomarkers2012In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 21, no S1, p. 164-164Article in journal (Other academic)
  • 8.
    Koos, Björn
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Cane, Gaëlle
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Grannas, Karin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Löf, Liza
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Arngården, Linda
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Heldin, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Clausson, Carl-Magnus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Klaesson, Axel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Hirvonen, M Karoliina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    de Oliveira, Felipe Marques Souza
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Talibov, Vladimir O
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Pham, Nhan T
    School of Biological Sciences and School of Biomedical Sciences, University of Edinburgh, UK.
    Auer, Manfred
    School of Biological Sciences and School of Biomedical Sciences, University of Edinburgh, UK.
    Danielson, U Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Haybaeck, Johannes
    Institute of Pathology, Medical University of Graz, Austria.
    Kamali-Moghaddam, Masood
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Söderberg, Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Proximity-dependent initiation of hybridization chain reaction2015In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 6, article id 7294Article in journal (Refereed)
    Abstract [en]

    Sensitive detection of protein interactions and post-translational modifications of native proteins is a challenge for research and diagnostic purposes. A method for this, which could be used in point-of-care devices and high-throughput screening, should be reliable, cost effective and robust. To achieve this, here we design a method (proxHCR) that combines the need for proximal binding with hybridization chain reaction (HCR) for signal amplification. When two oligonucleotide hairpins conjugated to antibodies bind in close proximity, they can be activated to reveal an initiator sequence. This starts a chain reaction of hybridization events between a pair of fluorophore-labelled oligonucleotide hairpins, generating a fluorescent product. In conclusion, we show the applicability of the proxHCR method for the detection of protein interactions and posttranslational modifications in microscopy and flow cytometry. As no enzymes are needed, proxHCR may be an inexpensive and robust alternative to proximity ligation assays.

  • 9. Larssen, Pia
    et al.
    Wik, Lotta
    Uppsala University, Science for Life Laboratory, SciLifeLab.
    Czarnewski, Paulo
    Eldh, Maria
    Löf, Liza
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Ronquist, Göran
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Biochemial structure and function.
    Dubois, Louise
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Biochemial structure and function.
    Freyhult, Eva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Gallant, Caroline
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Oelrich, Johan
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Larsson, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Biochemial structure and function.
    Ronquist, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Biochemial structure and function.
    Villablanca, Eduardo
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Gabrielsson, Susanne
    Kamali-Moghaddam, Masood
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Tracing Cellular Origin of Human Exosomes Using Multiplex Proximity Extension Assay2017In: Molecular & cellular proteomics (online), ISSN 1535-9476, E-ISSN 1535-9484, Vol. 16, no 3, p. 502-511Article in journal (Refereed)
    Abstract [en]

    Extracellular vesicles (EVs) are membrane-coated objects such as exosomes and microvesicles, released by many cell-types. Their presence in body fluids and the variable surface composition and content render them attractive potential biomarkers. The ability to determine their cellular origin could greatly move the field forward. We used multiplex proximity extension assays (PEA) to identify with high specificity and sensitivity the protein profiles of exosomes of different origins, including seven cell lines and two different body fluids. By comparing cells and exosomes, we successfully identified the cells originating the exosomes. Furthermore, by principal component analysis of protein patterns human milk EVs and prostasomes released from prostate acinar cells clustered with cell lines from breast and prostate tissues, respectively. Milk exosomes uniquely expressed CXCL5, MIA and KLK6, while prostasomes carried NKX31, GSTP1 and SRC, highlighting that EVs originating from different origins express distinct proteins. In conclusion, PEA provides a powerful protein screening tool in exosome research, for purposes of identifying the cell source of exosomes, or new biomarkers in diseases such as cancer and inflammation.

  • 10.
    Löf, Liza
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Applications of in situ proximity ligation assays for cancer research and diagnostics2016Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    In the field of cancer research and diagnostics it is crucial to have reliable methods for detecting molecules involved in the disease. New and better methods for diagnostics, prognostics and drug delivery therefore remain a permanent aim. In this thesis applications of the in situ proximity ligation assay (in situ PLA) were developed for diagnostics and research. Two new methods were developed, one more cost effective proximity assay without the use of enzymes and one method for loading pharmaceuticals in lipid rafts made from detergent resistant membranes (DRMs) to be used as a drug delivery platform.

    In Paper I the aim was to develop a flow cytometric detection method of the fusion protein BCR-ABL that is the hallmark of chronic myeloid leukemia (CML). By using in situ PLA the malignant cells carrying the fusion protein could be detected in patients in a convenient workflow.

    Paper II describes an application of multiplex in situ PLA, where extracellular vesicles (EVs) are detected and identified using flow cytometry. Up to five different antigens are targeted on the EVs, reflected in three different colors during detection in the flow cytometer. By using antibodies targeting proteins specific for prostasomes a population of prostasomes could be identified in human blood plasma.

    In Paper III a new method is described for using lipid raft for drug delivery. In this method, lipid rafts, derived from prostasomes or erythrocytes, are loaded with pharmaceuticals. The vehicles were loaded with doxorubicin, added to cells and counted. Cells that received the vehicle with doxorubicin stopped proliferating and died, while controls that received the lipid raft vehicle without doxorubicin were not affected, suggesting that the vehicles are effectively loaded with the drug and that they are safe. This lipid raft vehicle could provide a safe drug delivery system.     

    Paper IV investigates the crosstalk between the two major signal pathways Hippo and Wnt, and how these are affected in gastric cancer. When looking at different colon cancer tumor stages, we found that the cellular localization of TAZ/β-catenin interactions were different. We also found that protein complexes involved in the crosstalk increased in sparsely growing cells compared to more densely growing cells. On the basis of these results the protein E-cadherin, involved in maintenance of the epithelial integrity, was investigated and was found to have a probable role in regulating the crosstalk between Hippo and Wnt.    

    A new method for localized protein detection is described in paper V. Here a proximity assay, based on the hybridization chain reaction (HCR), was developed. This assay, proxHCR, is more cost effective than in situ PLA because no enzymes are required. ProxHCR successfully detects protein interactions and can be used together with both fluorescence microscopy and flow cytometry. 

     

    List of papers
    1. Crosstalk between WNT and Hippo signaling pathways changes upon colon cancer stage and is affected by cell density and loss of or mutated E-cadherin protein
    Open this publication in new window or tab >>Crosstalk between WNT and Hippo signaling pathways changes upon colon cancer stage and is affected by cell density and loss of or mutated E-cadherin protein
    (English)Manuscript (preprint) (Other academic)
    National Category
    Medical and Health Sciences
    Research subject
    Molecular Medicine
    Identifiers
    urn:nbn:se:uu:diva-300107 (URN)
    Available from: 2016-08-02 Created: 2016-08-02 Last updated: 2016-08-26Bibliographically approved
    2. Detergent resistant membranes from erythrocytes can form vesicles and be loaded for delivery of pharmaceutics
    Open this publication in new window or tab >>Detergent resistant membranes from erythrocytes can form vesicles and be loaded for delivery of pharmaceutics
    Show others...
    (English)Manuscript (preprint) (Other academic)
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-300090 (URN)
    Available from: 2016-08-02 Created: 2016-08-02 Last updated: 2016-08-26
    3. Proximity-dependent initiation of hybridization chain reaction
    Open this publication in new window or tab >>Proximity-dependent initiation of hybridization chain reaction
    Show others...
    2015 (English)In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 6, article id 7294Article in journal (Refereed) Published
    Abstract [en]

    Sensitive detection of protein interactions and post-translational modifications of native proteins is a challenge for research and diagnostic purposes. A method for this, which could be used in point-of-care devices and high-throughput screening, should be reliable, cost effective and robust. To achieve this, here we design a method (proxHCR) that combines the need for proximal binding with hybridization chain reaction (HCR) for signal amplification. When two oligonucleotide hairpins conjugated to antibodies bind in close proximity, they can be activated to reveal an initiator sequence. This starts a chain reaction of hybridization events between a pair of fluorophore-labelled oligonucleotide hairpins, generating a fluorescent product. In conclusion, we show the applicability of the proxHCR method for the detection of protein interactions and posttranslational modifications in microscopy and flow cytometry. As no enzymes are needed, proxHCR may be an inexpensive and robust alternative to proximity ligation assays.

    National Category
    Medical and Health Sciences Chemical Sciences
    Identifiers
    urn:nbn:se:uu:diva-255596 (URN)10.1038/ncomms8294 (DOI)000357171100008 ()26065580 (PubMedID)
    Funder
    EU, FP7, Seventh Framework Programme, 278568, 316929, 259796Swedish Foundation for Strategic Research Swedish Research Council
    Available from: 2015-06-17 Created: 2015-06-17 Last updated: 2018-01-25Bibliographically approved
    4. Flow Cytometric Measurement of BCR-ABL Fusion Protein Positive Cells in Blood from Patients with Chronic Myeloid Leukemia
    Open this publication in new window or tab >>Flow Cytometric Measurement of BCR-ABL Fusion Protein Positive Cells in Blood from Patients with Chronic Myeloid Leukemia
    Show others...
    (English)Manuscript (preprint) (Other academic)
    National Category
    Medical and Health Sciences Clinical Laboratory Medicine
    Research subject
    Molecular Medicine; Pathology
    Identifiers
    urn:nbn:se:uu:diva-300087 (URN)
    Available from: 2016-08-02 Created: 2016-08-02 Last updated: 2019-04-02
    5. Detecting extracellular vesicles using a multicolor in situ proximity ligation assay with flow cytometric readout
    Open this publication in new window or tab >>Detecting extracellular vesicles using a multicolor in situ proximity ligation assay with flow cytometric readout
    (English)Manuscript (preprint) (Other academic)
    National Category
    Medical and Health Sciences
    Research subject
    Molecular Medicine
    Identifiers
    urn:nbn:se:uu:diva-300089 (URN)
    Available from: 2016-08-02 Created: 2016-08-02 Last updated: 2016-08-26
  • 11.
    Löf, Liza
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Arngården, Linda
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Olsson-Strömberg, Ulla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Haematology.
    Siart, Benjamin
    Jansson, Mattias
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medicinsk genetik och genomik. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Dahlin, Joakim S
    Thörn, Ingrid
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical and experimental pathology.
    Christiansson, Lisa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical Immunology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Hermansson, Monica
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medicinsk genetik och genomik. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Larsson, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Biochemial structure and function.
    Ahlstrand, Erik
    Wålinder, Göran
    Söderberg, Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Rosenquist, Richard
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology.
    Landegren, Ulf
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Kamali-Moghaddam, Masood
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Flow Cytometric Measurement of Blood Cells with BCR-ABL1 Fusion Protein in Chronic Myeloid Leukemia2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, p. 1-9, article id 623Article in journal (Refereed)
    Abstract [en]

    Chronic myeloid leukemia (CML) is characterized in the majority of cases by a t(9;22)(q34;q11) translocation, also called the Philadelphia chromosome, giving rise to the BCR-ABL1 fusion protein. Current treatment with tyrosine kinase inhibitors is directed against the constitutively active ABL1 domain of the fusion protein, and minimal residual disease (MRD) after therapy is monitored by real-time quantitative PCR (RQ-PCR) of the fusion transcript. Here, we describe a novel approach to detect and enumerate cells positive for the BCR-ABL1 fusion protein by combining the in situ proximity ligation assay with flow cytometry as readout (PLA-flow). By targeting of the BCR and ABL1 parts of the fusion protein with one antibody each, and creating strong fluorescent signals through rolling circle amplification, PLA-flow allowed sensitive detection of cells positive for the BCR-ABL1 fusion at frequencies as low as one in 10,000. Importantly, the flow cytometric results correlated strongly to those of RQ-PCR, both in diagnostic testing and for MRD measurements over time. In summary, we believe this flow cytometry-based method can serve as an attractive approach for routine measurement of cells harboring BCR-ABL1 fusions, also allowing simultaneously assessment of other cell surface markers as well as sensitive longitudinal follow-up.

  • 12.
    Löf, Liza
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Olsson-Strömberg, Ulla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Haematology.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Kamali-Moghaddam, Masood
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    FLOW CYTOMETRY-BASED ASSAY FOR DETECTION OF BCR-ABL FUSION PROTEIN IN BLOOD CELLS FROM CML PATIENTS2015In: Haematologica, ISSN 0390-6078, E-ISSN 1592-8721, Vol. 100, p. 694-695Article in journal (Other academic)
  • 13.
    Löf, Liza
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Olsson-Strömberg, Ulla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Haematology.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Kamali-Moghaddam, Masood
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Pla Flow; A Flow Cytometry-Based Assay For Detection Of Bcr-Abl Fusion Protein In Blood Cells From Cml Patients2016In: Haematologica, ISSN 0390-6078, E-ISSN 1592-8721, Vol. 101, p. 457-458Article in journal (Other academic)
1 - 13 of 13
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