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  • 1. Arruda, L. C. M.
    et al.
    Gaballa, A.
    Uhlin, Michael
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biophysics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Graft γδ TCR Sequencing Identifies Public Clonotypes Associated with Hematopoietic Stem Cell Transplantation Efficacy in Acute Myeloid Leukemia Patients and Unravels Cytomegalovirus Impact on Repertoire Distribution2019In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 202, no 6, p. 1859-1870Article in journal (Refereed)
    Abstract [en]

    Although the impact of donor graft composition on clinical outcomes after hematopoietic stem cell transplantation (HSCT) has been studied, little is known about the role of intragraft γδ TCR repertoire on clinical outcomes following HSCT. Using a high-throughput sequencing platform, we sought to analyze the TCR γ-chain (TRG) repertoire of γδ T cells within donor stem cell grafts and address its potential impact on clinical response in the corresponding patients. A total of 20 peripheral blood stem cell grafts were analyzed, and donors were classified as CMV+/- The respective acute myeloid leukemia recipients were followed for disease relapse and acute graft-versus-host disease (aGvHD) development post-HSCT. In all samples, TRG repertoire showed a reduced diversity and displayed overrepresented clones. This was more prominent in grafts from CMV+ donors, which presented a more private repertoire, lower diversity, skewed distribution, and reduced usage of the V9-JP pairing. Grafts given to nonrelapse patients presented a more public repertoire and increased presence of long sequence clonotypes. Variable-joining gene segment usage was not associated with aGvHD development, but a higher usage of V2-JP1 pairing and lower usage of V4-J2/V5-J2/V8-JP2 were observed in grafts given to nonrelapse patients. Our work identified five private overrepresented and one public CDR3 sequence (CATWDGPYYKKLF) associated with CMV infection, in addition to 12 highly frequent public sequences present exclusively in grafts given to nonrelapse patients. Our findings show that, despite CMV infection reshaping the TRG repertoire, TRG composition is not associated with aGvHD development, and several public sequences are associated with clinical remission.

  • 2. Brändström, J.
    et al.
    Vetander, M.
    Lilja, G.
    Sundqvist, A.
    Kalm, Frida
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Johansson, S.
    Nilsson, C.
    Nopp, A.
    Peanut oral immunotherapy during omalizumab protection; a clinical trial on severely peanut allergic adolescents2017In: Allergy. European Journal of Allergy and Clinical Immunology, ISSN 0105-4538, E-ISSN 1398-9995, Vol. 72, p. 65-66Article in journal (Other academic)
  • 3. Chen, G.
    et al.
    Sidhu, S. S.
    Nilvebrant, Johan
    KTH, School of Biotechnology (BIO), Protein Technology. University of Toronto, Canada.
    Synthetic antibodies in infectious disease2017In: Recombinant Antibodies for Infectious Diseases, Springer-Verlag New York, 2017, p. 79-98Chapter in book (Refereed)
    Abstract [en]

    Rapid spread of microbial resistance and recent outbreaks of viral disease have led to renewed interest in antibody-based therapies for infectious diseases. Synthetic antibody libraries displayed on phage offer unique advantages over traditional immunization-based antibody generation, including full control over library design and selection conditions. The technology has matured beyond natural antibodies and is capable of providing novel insights into infectious disease and can generate novel antibodies that cannot be produced by the natural immune system. This chapter gives an overview of recombinant antibody library technology with an emphasis on our work using a highly successful synthetic single framework Fab library. We demonstrate its utility in targeting viruses and bacterial toxins in five case studies.

  • 4.
    Edfors, Fredrik
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hober, Andreas
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Linderbäck, Klas
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Maddalo, Gianluca
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Azimi, Alireza
    Karolinska Inst, Karolinska Univ Hosp, Dept Oncol Pathol, SE-17177 Stockholm, Sweden..
    Sivertsson, Åsa
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Tegel, Hanna
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hober, Sophia
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Al-Khalili Szigyarto, Cristina
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Fagerberg, Linn
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    von Feilitzen, Kalle
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Oksvold, Per
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lindskog, Cecilia
    Uppsala Univ, Dept Immunol Genet & Pathol, SE-75185 Uppsala, Sweden..
    Forsström, Björn
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Uhlén, Mathias
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab. Biosustainabil, DK-2970 Horsholm, Denmark..
    Enhanced validation of antibodies for research applications2018In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 9, article id 4130Article in journal (Refereed)
    Abstract [en]

    There is a need for standardized validation methods for antibody specificity and selectivity. Recently, five alternative validation pillars were proposed to explore the specificity of research antibodies using methods with no need for prior knowledge about the protein target. Here, we show that these principles can be used in a streamlined manner for enhanced validation of research antibodies in Western blot applications. More than 6,000 antibodies were validated with at least one of these strategies involving orthogonal methods, genetic knockdown, recombinant expression, independent antibodies, and capture mass spectrometry analysis. The results show a path forward for efforts to validate antibodies in an application-specific manner suitable for both providers and users.

  • 5.
    Falk, Ronny
    KTH, School of Biotechnology (BIO).
    Systems enabling antibody-mediated proteomics research2006Doctoral thesis, comprehensive summary (Other scientific)
    Abstract [en]

    As many genome sequencing efforts today are completed, we are now provided with the genetic maps for several organisms, including man. With these maps at hand, the scientific focus is now shifting towards investigations of the functionality of proteins. This task is even more challenging than the genomic field since proteins, in contrast to DNA, do not allow themselves to be specifically probed or amplified by easy and generic methods. However, to achieve knowledge regarding protein function, useful information includes where, when and how much certain proteins are expressed in an organism. Such information can be obtained if protein-specific binding molecules are available as tools. One such class of target specific binders are the antibody molecules, traditionally employed in a broad variety of biotechnical applications, including protein localization studies on both cellular and sub cellular levels.

    In a first serie of studies, new methodology for recombinant production and purification of antigens for generation of antibodies via immunization routes were investigated. Parallel affinity gene fusion-based expression systems were used for evaluation of different concepts for production of antigen and post-immunization antibody purification. Carefully designed protein antigens from different organisms were produced and used to raise antisera which were affinity purified on their respective antigens to obtain highly specific polyclonal antibodies (monospecific antibodies). One of the constructed expression systems includes an affinity handle, ZSPA-1, previously selected from a combinatorial protein library for its capacity to selectively bind protein A. This allows for convenient, non IgG-dependent, affinity purification of proteins on conventional protein A resins.

    A strategy where highly target specific antibody preparations could be affinity purified in a more streamlined setup is also presented. By this strategy it was possible to fractionate antibodies showing reactivity to different parts of the antigen into separate fractions. This resulted in affinity purified antibodies showing monospecific but still multi-epitope reactivity. Purified monospecific antibodies were used in different studies including Western blot immunofluorescence and recovery applications. For affinity purification of endogenous target from its native surrounding a selective elution strategy where the recombinant antigen was used to competitively elute the captured target was developed.

  • 6.
    Falk, Ronny
    et al.
    KTH, Superseded Departments, Biotechnology.
    Agaton, Charlotta
    KTH, Superseded Departments, Biotechnology.
    Kiesler, E.
    Jin, S.
    Wieslander, L.
    Visa, N.
    Hober, Sophia
    KTH, Superseded Departments, Biotechnology.
    Ståhl, Stefan
    KTH, Superseded Departments, Biotechnology.
    An improved dual-expression concept, generating high-quality antibodies for proteomics research2003In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 38, p. 231-239Article in journal (Refereed)
    Abstract [en]

    A novel, improved dual bacterial-expression system, designed for large-scale generation of high-quality polyclonal antibody preparations intended for proteomics research, is presented. The concept involves parallel expression of cDNA-encoded proteins, as a fusion with two different tags in two separate vector systems. Both systems enable convenient blotting procedures for expression screening on crude bacterial cell cultures and single-step affinity purification under denaturing conditions. One of the fusion proteins is used to elicit antibodies, and the second fusion protein is used in an immobilized form as an affinity ligand to enrich antibodies with selective reactivity to the cDNA-encoded part, common for the two fusion proteins. To evaluate the system, four cDNA clones from putative nuclear proteins from the non-biting midge Chironomus tentans were expressed. Antibodies to these cDNA-encoded proteins were generated, enriched and used in blotting and immunofluorescence procedures to determine expression patterns for the native proteins corresponding to the cDNAs. The four antibody preparations showed specific reactivity to the corresponding recombinant cDNA-encoded proteins, and three of the four antibodies gave specific staining in Western-blot analysis of nuclear cell extracts. Furthermore, two of the antibody preparations gave specific staining in immunofluorescence analysis of C. tentans cells. We conclude that the dual-vector concept presented offers a highly stringent strategy for the generation of monospecific polyclonal antibodies, which are useful in proteomics research.

  • 7.
    Falk, Ronny
    et al.
    KTH, Superseded Departments, Biotechnology.
    Gräslund, Susanne
    KTH, Superseded Departments, Biotechnology.
    Brundell, Eva
    Höög, Christer
    Ståhl, Stefan
    KTH, Superseded Departments, Biotechnology.
    A high-stringency proteomics concept aimed for generation of antibodies specific for cDNAencoded proteins2002In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 35, no 2, p. 75-82Article in journal (Refereed)
    Abstract [en]

    A novel dual bacterial expression system, designed for high-throughput generation of antibodies specific for cDNA-encoded proteins, is presented. The concept involves parallel expression of cDNA-encoded proteins, in two vector systems, as fusions with two different tags, both enabling single-step affinity purification under denaturing conditions. One of the fusion tags includes a portion with documented immunopotentiating effect to stimulate antibody production, and the generated fusion proteins are used to elicit antibodies. The second fusion protein is used in an immobilized form as an affinity ligand to enrich, from the generated antisera, antibodies with selective reactivity to the cDNA-encoded part. To evaluate the system, five cDNA clones from a mouse testis cDNA library were expressed, and antibodies to these cDNA-encoded proteins were generated, enriched and used in blotting procedures to determine expression patterns for the native proteins corresponding to the cDNAs. The five antibody preparations showed specific reactivity to the corresponding recombinant cDNA-encoded proteins, and three of the five antibodies gave specific staining in Western-blot screening of various cell types and tissue homogenates. When the same five cDNAs were processed and analysed using a single-vector method, antibodies with a more non-specific staining were generated. We thus conclude that the presented dual-vector method offers a highly stringent strategy for generation of monospecific polyclonal antibodies.

  • 8.
    Ge, Changrong
    et al.
    Karolinska Inst, Stockholm, Sweden..
    Xu, Bingze
    Karolinska Inst, Stockholm, Sweden..
    Liang, Bibo
    Karolinska Inst, Stockholm, Sweden.;Southern Med Univ, Guangzhou, Guangdong, Peoples R China..
    Lonnblom, Erik
    Karolinska Inst, Stockholm, Sweden..
    Lundstrom, Susanna L.
    Karolinska Inst, Stockholm, Sweden..
    Zubarev, Roman A.
    Karolinska Inst, Stockholm, Sweden..
    Ayoglu, Burcu
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Nilsson, Peter
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Skogh, Thomas
    Linkoping Univ, Linkoping, Sweden..
    Kastbom, Alf
    Linkoping Univ, Linkoping, Sweden..
    Malmstrom, Vivianne
    Karolinska Inst, Stockholm, Sweden.;Karolinska Univ Hosp, Stockholm, Sweden..
    Klareskog, Lars
    Karolinska Inst, Stockholm, Sweden.;Karolinska Univ Hosp, Stockholm, Sweden..
    Toes, Rene E. M.
    Leiden Univ, Med Ctr, Leiden, Netherlands..
    Rispens, Theo
    Univ Amsterdam, Amsterdam, Netherlands..
    Dobritzsch, Doreen
    Uppsala Univ, Uppsala, Sweden..
    Holmdahl, Rikard
    Karolinska Inst, Stockholm, Sweden.;Southern Med Univ, Guangzhou, Guangdong, Peoples R China..
    Structural Basis of Cross-Reactivity of Anti-Citrullinated Protein Antibodies2019In: Arthritis & Rheumatology, ISSN 2326-5191, E-ISSN 2326-5205, Vol. 71, no 2, p. 210-221Article in journal (Refereed)
    Abstract [en]

    Objective Anti-citrullinated protein antibodies (ACPAs) develop many years before the clinical onset of rheumatoid arthritis (RA). This study was undertaken to address the molecular basis of the specificity and cross-reactivity of ACPAs from patients with RA. Methods Antibodies isolated from RA patients were expressed as monoclonal chimeric antibodies with mouse Fc. These antibodies were characterized for glycosylation using mass spectrometry, and their cross-reactivity was assessed using Biacore and Luminex immunoassays. The crystal structures of the antigen-binding fragment (Fab) of the monoclonal ACPA E4 in complex with 3 different citrullinated peptides were determined using x-ray crystallography. The prevalence of autoantibodies reactive against 3 of the citrullinated peptides that also interacted with E4 was investigated by Luminex immunoassay in 2 Swedish cohorts of RA patients. Results Analysis of the crystal structures of a monoclonal ACPA from human RA serum in complex with citrullinated peptides revealed key residues of several complementarity-determining regions that recognized the citrulline as well as the neighboring peptide backbone, but with limited contact with the side chains of the peptides. The same citrullinated peptides were recognized by high titers of serum autoantibodies in 2 large cohorts of RA patients. Conclusion These data show, for the first time, how ACPAs derived from human RA serum recognize citrulline. The specific citrulline recognition and backbone-mediated interactions provide a structural explanation for the promiscuous recognition of citrullinated peptides by RA-specific ACPAs.

  • 9.
    Gräslund, Susanne
    et al.
    KTH, Superseded Departments, Biotechnology.
    Eklund, Malin
    KTH, Superseded Departments, Biotechnology.
    Falk, Ronny
    KTH, Superseded Departments, Biotechnology.
    Uhlén, Mathias
    KTH, Superseded Departments, Biotechnology.
    Nygen, Per-Åke
    KTH, Superseded Departments, Biotechnology.
    Ståhl, Stefan
    KTH, Superseded Departments, Biotechnology.
    A novel affinity gene fusion system allowing protein A-based recovery of non-immunoglobulin gene products2002In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Journal of biotechnology, Vol. 99, no 1, p. 41-50Article in journal (Refereed)
    Abstract [en]

    An expression vector system has been developed, taking advantage of a novel, Staphylococcus aureus protein A (SPA)-binding affinity tag ZSPA-1, enabling straightforward affinity blotting procedures and efficient recovery by affinity purification of expressed gene products on readily available reagents and chromatography media. The 58 amino acid SPA-binding affinity tag ZSPA-1, was previously selected from a library constructed by combinatorial mutagenesis of a protein domain from SPA. An Escherichia coli expression vector for intracellular T7 promoter (PT7) driven production was constructed with an N-terminal dual affinity tag, consisting of a hexahistidyl (His6) tag in frame with the ZSPA-1 tag, thus allowing alternative affinity recovery methods. To evaluate the system, five cDNA clones from a mouse testis cDNA library were expressed, and two alternative blotting procedures were developed for convenient screening of expression efficiencies. The five produced fusion proteins were recovered on both immobilized metal-ion affinity chromatography (IMAC) columns and on Protein A-based chromatography media, to allow comparative studies. It was found that the Protein A-based recovery resulted in the highest degree of purity, and furthermore, gene products that were produced as inclusion bodies could after denaturation be efficiently affinity purified on Protein A-Sepharose in the presence of 0.5 M guanidine hydrochloride. The convenience and robustness of the presented expression system should make it highly suitable for various high-throughput protein expression efforts.

  • 10.
    Guldevall, Karolin
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Single Cell Investigations of the Functional Heterogeneity Within Immune Cell Populations: a Microchip-based Study2014Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Immune cell populations are constantly divided into smaller and smaller subsets defined by newly emerging cellular markers. However, there is a growing awareness of the functional heterogeneities in between cells even within small populations, in addition to the heterogeneity over time. One may ask whether a population is correctly defined only by cellular markers or if the functionality should be regarded as well? Many of today’s techniques only measure at the population level, giving an average estimate of the behavior of that pool of cells, but failing to detect rare possibly important events. Thus, high-throughput experimental approaches to analyze single cells over time are required to address cellular heterogeneity.

    Progress in the fields of microfabrication, microscopy and computing have paved the way for increasingly efficient tools for studies on the single cell level, and a variety of devices have been described by others. However, few of them are suitable for long-term imaging of dynamic events such as cell-cell interactions or migration. In addition, for efficient recording of many individual events it is desirable to scale down the cells’ interaction volume; not only to shorten the time to interaction, but also to increase the number of individual events in a given area; thereby pushing a screening approach.

    To address these questions, a complete microwell array system for imaging of immune cell responses with single-cell resolution was designed. The platform consists of a range of silicon-glass microchips with arrays of miniature wells for incubation of cells and a custom made holder that fits conventional microscopes. The device has been designed to allow cells to be kept viable for several days in the wells, to be easy to use and to allow high-resolution imaging. Five different designs were fabricated; all with a specific type of assay in mind, and were evaluated regarding biocompatibility and functionality. Here, the design aimed for screening applications is the main focus. In this approach a large amount, tens of thousands, of small wells are imaged two to three times: first directly post-seeding of effector and target cells to register the well’s content, and second after some time has passed to allow for cell-cell interactions. The final read-out is the number of killed target cells in each well, making an automatic cell counting protocol necessary in order to analyze the massive amount of data generated.

    We here show that our silicon microwell platform allows long-term studies with the possibility of both time-lapse and high-resolution imaging of a variety of immune cell behavior. Using both time-lapse imaging and the screening approach we confirmed and investigated immune cell heterogeneity within NK cell populations in regards to both cytotoxicity and migrational behavior. In addition, two different types of cytolytic behavior in NK cells, termed fast and slow killing, were described and evaluated in regards to dynamic parameters; like conjugation and attachment time. We could also quantify the type of cytolytic response in relation to serial killing NK cells, and saw that serial killing NK cells more often induced fast target cell death. Further investigations using the screening approach have shown that serial killing NK cells also differ from other NK cells in their morphology, being both larger and with a more elongated shape. So far the platform has been used to gain better understanding of some aspects of NK cell biology, but there is still much left to explore. With the addition of an automatic counting program, the large numbers of wells that can be simultaneously imaged will provide new statistical information and enable higher throughput.

    Altogether, our family of techniques enables novel types of cellular imaging assays allowing data collection at a level of resolution not previously obtained – this was shown to be important for performing basic cell biological studies, but may also prove valuable in the proposed future medical applications such as adoptive cell therapy and stem cell transplantation.

  • 11.
    Guldevall, Karolin
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Gustafsson, Karin
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Forslund, Elin
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Frisk, Thomas
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Manneberg, Otto
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Olofsson, Per E.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Tauriainen, Johanna
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Stikvoort, Arwen
    Karolinska Institute.
    Vanherberghen, Bruno
    KTH, School of Engineering Sciences (SCI), Applied Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Mattsson, Jonas
    Karolinska Institute.
    Kärre, Klas
    Karolinska Institute.
    Uhlin, Michael
    Karolinska Institute.
    Önfelt, Bjorn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Microchip screening platform for assessment of cytotoxic effector cellsManuscript (preprint) (Other academic)
    Abstract [en]

    Here we report a screening platform for assessment of the cytotoxic potential of individual natural killer (NK) or T cells within larger populations. Human primary NK cells or human Epstein-Barr virus (EBV)- specific T cells were distributed across a silicon-glass microchip containing 32 400 individual microwells loaded with target cells. Through fluorescence screening and automated image analysis the numbers of effector and live or dead target cells in each well could be assessed at different time-points after initial mixing. Cytotoxicity was also studied by time-lapse live-cell imaging in microwells quantifying the killing potential of individual NK cells. Although most resting NK cells (≈75%) were non-cytotoxic to the leukemia cell line K562, some NK cells were able to kill several (≥3) target cells within the 12 hours long experiment. We demonstrate that this assay can be used to enumerate and characterize cytotoxic cells, something that could find clinical applications, e.g. in the selection of donors for stem cell transplantation or generation of highly specific and cytotoxic cells for adoptive immunotherapy.

  • 12. Hertting, Olof
    et al.
    Lüthje, Petra
    Sullivan, Devin
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Aspenström, Pontus
    Brauner, Annelie
    Vitamin D-deficient mice have more invasive urinary tract infection2017In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 12, no 7, article id e0180810Article in journal (Refereed)
    Abstract [en]

    Vitamin D deficiency is a common health problem with consequences not limited to bone and calcium hemostasis. Low levels have also been linked to tuberculosis and other respiratory infections as well as autoimmune diseases. We have previously shown that supplementation with vitamin D can induce the antimicrobial peptide cathelicidin during ex vivo infection of human urinary bladder. In rodents, however, cathelicidin expression is not linked to vitamin D and therefore this vitamin D-related effect fighting bacterial invasion is not relevant. To determine if vitamin D had further protective mechanisms during urinary tract infections, we therefore used a mouse model. In vitamin D-deficient mice, we detected more intracellular bacterial communities in the urinary bladder, higher degree of bacterial spread to the upper urinary tract and a skewed cytokine response. Furthermore, we show that the vitamin D receptor was upregulated in the urinary bladder and translocated into the cell nucleus after E. coli infection. This study supports a more general role for vitamin D as a local immune response mediator in the urinary tract.

  • 13. Hilderman, Marie
    et al.
    Qureshi, Abdul R
    Al-Abed, Yousef
    Abtahi, Farhad
    KTH, School of Technology and Health (STH), Medical Engineering, Medical sensors, signals and systems.
    Lindecrantz, Kaj
    Anderstam, Björn
    Bruchfeld, Annette
    Cholinergic anti-inflammatory pathway activity in dialysis patients: a role for neuroimmunomodulation?2015In: Clinical Kidney Journal, ISSN 2048-8505, E-ISSN 2048-8513, Vol. 8, no 5Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: The cholinergic anti-inflammatory pathway (CAP) modulates inflammatory responses through the vagus nerve and the α-7-nicotinic acetylcholine receptor (α7nAChR) on macrophages and immune cells. Sympathetic/parasympathetic imbalance and chronic inflammation are both linked to poor outcome in dialysis patients. The aim of this study was to investigate CAP activity in these patients.

    METHODS: Twenty dialysis patients, 12 hemodialysis (HD) and 8 peritoneal dialysis (PD) patients (12 male, 8 female; age range 47-83 years) and 8 controls (5 male, 3 female; age range 31-52 years) were analyzed for C-reactive protein (CRP), tumor necrosis factor (TNF), interleukin-1b (IL-1b), IL-6 and IL-10 at baseline. The cytokines were then assessed after whole blood stimulation ex vivo with lipopolysaccharide (LPS) (10 and 100 ng/mL) and again in the presence of 45 and 90 μmol/L GTS-21, a cholinergic α7nAChR agonist.

    RESULTS: CRP, TNF, IL-1 and IL-6 were significantly higher, whereas IL-10 was significantly lower at baseline in patients compared with controls. After LPS stimulation, TNF increased significantly more in patients than in controls but decreased to similar levels in both groups after addition of GTS-21. IL-6 attenuation was comparable with TNF and the IL-1b pattern was similar but remained significantly higher in patients. Interestingly, IL-10 increased after GTS-21 in a dose-dependent manner, but only in patients. Results in HD and PD patients did not differ.

    CONCLUSIONS: The response of immune cells after LPS exposure and cholinergic stimulation suggests a functional CAP in dialysis patients. It may thus be possible to target the α7nAChR control of cytokine release as an anti-inflammatory strategy and thereby improve outcome in these patients.

  • 14. Hilderman, Marie
    et al.
    Qureshi, Abdul Rashid
    Abtahi, Farhad
    Witt, Nils
    Jägren, Christina
    Olbers, Joakim
    Delle, Martin
    Lindecrantz, Kaj
    Bruchfeld, Annette
    The cholinergic anti-inflammatory pathway in resistant hypertension treated with renal denervation2019In: Molecular medicine (Cambridge, Mass. Print), ISSN 1076-1551, E-ISSN 1528-3658, Vol. 25, no 1Article in journal (Refereed)
    Abstract [en]

    Renal denervation (RDN) reduces sympathetic tone and may alter the sympathetic-parasympathetic balance. The autonomic nervous system is partly a regulator of innate immunity via the cholinergic anti-inflammatory pathway (CAP) which inhibits inflammation via the vagus nerve. Placental Growth Factor (PlGF) influences a neuro-immunological pathway in the spleen which may contribute to hypertension. The aim of this study was to investigate if modulation of renal sympathetic nerve activity affects CAP in terms of cytokine release as well as levels of PlGF.

  • 15. Honarvar, Hadis
    et al.
    Westerlund, Kristina
    KTH, School of Biotechnology (BIO), Protein Technology.
    Altai, Mohamed
    Sandstrom, Mattias
    Orlova, Anna
    Tolmachev, Vladimir
    Eriksson Karlström, Amelie
    KTH, School of Biotechnology (BIO), Protein Technology.
    Feasibility of Affibody Molecule-Based PNA-Mediated Radionuclide Pretargeting of Malignant Tumors2016In: Theranostics, ISSN 1838-7640, E-ISSN 1838-7640, Vol. 6, no 1, p. 93-103Article in journal (Refereed)
    Abstract [en]

    Affibody molecules are small (7 kDa), non-immunoglobulin scaffold proteins with a potential as targeting agents for radionuclide imaging of cancer. However, high renal re-absorption of Affibody molecules prevents their use for radionuclide therapy with residualizing radiometals. We hypothesized that the use of Affibody-based peptide nucleic acid (PNA)-mediated pretargeting would enable higher accumulation of radiometals in tumors than in kidneys. To test this hypothesis, we designed an Affibody-PNA chimera Z(HER2:342)-SR-HP1 containing a 15-mer HP1 PNA recognition tag and a complementary HP2 hybridization probe permitting labeling with both I-125 and In-111. In-111-Z(HER2:342)-SR-HP1 bound specifically to HER2-expressing BT474 and SKOV-3 cancer cells in vitro, with a K-D of 6+/-2 pM for binding to SKOV-3 cells. Specific high affinity binding of the radiolabeled complementary PNA probe In-111-/I-125-HP2 to Z(HER2:342)-SR-HP1 pre-treated cells was demonstrated. In-111-Z(HER2:342)-SR-HP1 demonstrated specific accumulation in SKOV-3 xenografts in BALB/C nu/nu mice and rapid clearance from blood. Pre-saturation of SKOV-3 with non-labeled anti-HER2 Affibody or the use of HER2-negative Ramos xenografts resulted in significantly lower tumor uptake of In-111-Z(HER2:342)-SR-HP1. The complementary PNA probe In-111/I-125-HP2 accumulated in SKOV-3 xenografts when Z(HER2:342)-SR-HP1 was injected 4 h earlier. The tumor accumulation of In-111/I-125-HP2 was negligible without Z(HER2:342)-SR-HP1 pre-injection. The uptake of In-111-HP2 in SKOV-3 xenografts was 19+/-2 % ID/g at 1 h after injection. The uptake in blood and kidneys was approximately 50- and 2-fold lower, respectively. In conclusion, we have shown that the use of Affibody-based PNA-mediated pretargeting enables specific delivery of radiometals to tumors and provides higher radiometal concentration in tumors than in kidneys.

  • 16.
    Johansson, Henrik J.
    et al.
    Karolinska Inst, Sci Life Lab, Dept Oncol Pathol, S-17121 Stockholm, Sweden..
    Vallhov, Helen
    Karolinska Inst, Dept Clin Sci & Educ, Sodersjukhuset, SE-11883 Stockholm, Sweden.;Soder Sjukhuset, Unit Sachs Children & Youth Hosp, SE-11883 Stockholm, Sweden..
    Holm, Tina
    Karolinska Inst, Translat Immunol Unit, Dept Med Solna, S-17176 Stockholm, Sweden.;Univ Hosp, S-17176 Stockholm, Sweden..
    Gehrmann, Ulf
    Karolinska Inst, Translat Immunol Unit, Dept Med Solna, S-17176 Stockholm, Sweden.;Univ Hosp, S-17176 Stockholm, Sweden..
    Andersson, Anna
    Karolinska Inst, Translat Immunol Unit, Dept Med Solna, S-17176 Stockholm, Sweden.;Univ Hosp, S-17176 Stockholm, Sweden..
    Johansson, Catharina
    Karolinska Inst, Dept Clin Sci & Educ, Sodersjukhuset, SE-11883 Stockholm, Sweden.;Soder Sjukhuset, Unit Sachs Children & Youth Hosp, SE-11883 Stockholm, Sweden..
    Blom, Hans
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Carroni, Marta
    Sci Life Lab, Cryo EM Natl Facil, S-17177 Stockholm, Sweden..
    Lehtio, Janne
    Karolinska Inst, Sci Life Lab, Dept Oncol Pathol, S-17121 Stockholm, Sweden..
    Scheynius, Annika
    Karolinska Inst, Dept Clin Sci & Educ, Sodersjukhuset, SE-11883 Stockholm, Sweden.;Soder Sjukhuset, Unit Sachs Children & Youth Hosp, SE-11883 Stockholm, Sweden.;Sci Life Lab, Clin Genom, S-17177 Stockholm, Sweden..
    Extracellular nanovesicles released from the commensal yeast Malassezia sympodialis are enriched in allergens and interact with cells in human skin2018In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, article id 9182Article in journal (Refereed)
    Abstract [en]

    Malassezia sympodialis is a dominant commensal fungi in the human skin mycobiome but is also associated with common skin disorders including atopic eczema (AE). M. sympodialis releases extracellular vesicles, designated MalaEx, which are carriers of small RNAs and allergens, and they can induce inflammatory cytokine responses. Here we explored how MalaEx are involved in hostmicrobe interactions by comparing protein content of MalaEx with that of the parental yeast cells, and by investigating interactions of MalaEx with cells in the skin. Cryo-electron tomography revealed a heterogeneous population of MalaEx. iTRAQ based quantitative proteomics identified in total 2439 proteins in all replicates of which 110 were enriched in MalaEx compared to the yeast cells. Among the MalaEx enriched proteins were two of the M. sympodialis allergens, Mala s 1 and s 7. Functional experiments indicated an active binding and internalization of MalaEx into human keratinocytes and monocytes, and MalaEx were found in close proximity of the nuclei using super-resolution fluorescence 3D-SIM imaging. Our results provides new insights into host-microbe interactions, supporting that MalaEx may have a role in the sensitization and maintenance of inflammation in AE by containing enriched amounts of allergens and with their ability to interact with skin cells.

  • 17. Kalderén, Christina
    et al.
    Stadler, Charlotte
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Forsgren, Margareta
    Kvastad, Linda
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Johansson, Elin
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Sydow-Bäckman, Mona
    Gelius, Stefan Svensson
    CCL2 mediates anti-fibrotic effects in human fibroblasts independently of CCR22014In: International Immunopharmacology, ISSN 1567-5769, E-ISSN 1878-1705, Vol. 20, no 1, p. 66-73Article in journal (Refereed)
    Abstract [en]

    CCL2 is known for its major role as a chemoattractant of monocytes for immunological surveillance and to site of inflammation. CCL2 acts mainly through the G-protein-coupled receptor CCR2 but has also been described to mediate its effects independently of this receptor in vitro and in vivo. Emerging pieces of evidence indicate that the CCL2/CCR2 axis is involved in fibrotic diseases, such as increased plasma levels of CCL2 and the presence of CCL2-hyperresponsive fibroblasts explanted from patients with systemic sclerosis and idiopathic pulmonary fibrosis. One of the profibrotic key mediators is the myofibroblast characterized by overexpression of alpha-smooth muscle actin and collagen I. However, the correlation between the CCL2/CCR2 axis and the activation of fibroblasts is not yet fully understood. We have screened human fibroblasts of various origins, human pulmonary fibroblasts (HPF), human fetal lung fibroblasts (HFL-1) and primary preadipocytes (SPF-1) in regard to CCL2 stimulated fibrotic responses. Surprisingly we found that CCL2 mediates anti-fibrotic effects independently of CCR2 in human fibroblasts of different origins.

  • 18.
    Montanez, Maria I.
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Coating Technology.
    Ruiz-Sanchez, Antonio J.
    Perez-Inestrosa, Ezequiel
    A perspective of nanotechnology in hypersensitivity reactions including drug allergy2010In: Current Opinion in Allergy and Clinical Immunology, ISSN 1528-4050, E-ISSN 1473-6322, Vol. 10, no 4, p. 297-302Article, review/survey (Refereed)
    Abstract [en]

    Purpose of review We provide an overview of the application of the concepts of nanoscience and nanotechnology as a novel scientific approach to the area of nanomedicine related to the domain of the immune system. Particular emphasis will be paid to studies on drug allergy reactions. Recent findings Several well defined chemical structures arranged in the dimension of the nanoscale are currently being studied for biomedical purposes. By interacting with the immune system, some of these show promising applications as vaccines, diagnostic tools and activators/effectors of the immune response. Even a brief listing of some key applications of nanostructured materials shows how broad and intense this area of nanomedicine is. Summary As a result of the development of nanoscience and nanotechnology applied to medicine, new approaches can be envisioned for problems related to the modulation of the immune response, as well as in immunodiagnosis, and to design new tools to solve related medical challenges. Nanoparticles offer unique advantages with which to exploit new properties and for materials to play a major role in new diagnostic techniques and therapies. Fullerene-C-60 and multivalent functionalized gold nanoparticles of various sizes have led to new tools and opened up new ways to study and interact with the immune system. Some of the most versatile nanostructures are dendrimers. In their interaction with the immune system they can naturally occurring macromolecules, taking advantage of the fact that dendrimers can be synthesized into nanosized structures. Their multivalence can be successfully exploited in vaccines and diagnostic tests for allergic reactions.

  • 19. Norström, Melissa M.
    et al.
    Radestad, Emelie
    Sundberg, Berit
    Mattsson, Jonas
    Henningsohn, Lars
    Levitsky, Victor
    Uhlin, Michael
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. Karolinska Institutet, Sweden.
    Progression of benign prostatic hyperplasia is associated with pro-inflammatory mediators and chronic activation of prostate-infiltrating lymphocytes2016In: OncoTarget, ISSN 1949-2553, E-ISSN 1949-2553, Vol. 7, no 17, p. 23581-23593Article in journal (Refereed)
    Abstract [en]

    Benign prostatic hyperplasia (BPH) is a common chronic non-malignant condition whose prevalence substantially increases with age. Immune cell infiltration and pro-inflammatory mediators have been implicated in the pathogenesis. Here, we characterized 21 extracellular markers on prostate-infiltrating lymphocytes (PILs) and analyzed expression of 26 soluble proteins in prostate tissue obtained from BPH patients (n = 31). These data were correlated with clinical parameters and compared with peripheral blood mononuclear cells (PBMCs) (n = 10). Increased frequencies of T cells expressing co-inhibitory receptors LAG-3, PD-1, TIM-3 or CTLA-4, and co-stimulatory receptors CD28, OX40 or 4-1BB were observed in BPH tissue compared to PBMCs. These findings are consistent with chronic activation and possible functional exhaustion of PILs that may be further augmented by several identified pro-inflammatory factors, such as IL-8 and MCP-1, promoting inflammation and chemotaxis of immune cells to the prostate. Prostate size and plasma prostate-specific antigen levels positively correlated with IL-8 and MCP-1 concentrations, and frequencies of T cells expressing CTLA-4 and TIM-3. It remains to be established whether the link between inflammation and BPH progression supported by our findings reflects a progressive failure of the immune system leading to decreased immune surveillance and development of prostate cancer.

  • 20.
    Parsa, Roham
    KTH, School of Biotechnology (BIO).
    Macrophage activation phenotypes in type 1 diabetes pathogenesis and therapy: Master thesis2009Independent thesis Advanced level (degree of Master (One Year)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Macrophages are an important key effector cell in the immune system which can practically be found in every tissue. Macrophages have for a long time been considered a population of cells only responsible for pro-inflammatory responses and anti-microbial activities. But over the past decade, many have come to realize the amazing plasticity of macrophages in response to different stimulations. The anti-microbial and pro-inflammatory macrophage is known as classically activated macrophages but newly discovered phenotypes have been revealed named wound-healing macrophages and regulatory macrophages. Through systematic screening we have identified an inducible macrophage activation state which has both wound-healing and regulatory capabilities activated by the novel cytokine combination IL-4/IL-10 with or without TGF-β.

  • 21.
    Prager, Isabel
    et al.
    Department for Immunology, Leibniz Research Centre for Working Environment and Human Factors at TU Dortmund, Dortmund, Germany.
    Liesche, Clarissa
    Division of Theoretical Bioinformatics, German Cancer Research Center and BioQuant Center, Heidelberg, Germany.
    van Ooijen, Hanna
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biophysics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Urlaub, Doris
    Department for Immunology, Leibniz Research Centre for Working Environment and Human Factors at TU Dortmund, Dortmund, Germany.
    Verron, Quentin
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biophysics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Sandström, Niklas
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biophysics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Fasbender, Frank
    Department for Immunology, Leibniz Research Centre for Working Environment and Human Factors at TU Dortmund, Dortmund, Germany.
    Claus, Maren
    Department for Immunology, Leibniz Research Centre for Working Environment and Human Factors at TU Dortmund, Dortmund, Germany.
    Eils, Roland
    Division of Theoretical Bioinformatics, German Cancer Research Center and BioQuant Center, Heidelberg, Germany.
    Beaudouin, Joël
    Division of Theoretical Bioinformatics, German Cancer Research Center and BioQuant Center, Heidelberg, Germany.
    Önfelt, Björn
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Watzl, Carsten
    Department for Immunology, Leibniz Research Centre for Working Environment and Human Factors at TU Dortmund, Dortmund, Germany.
    NK cells switch from granzyme B to death receptor–mediated cytotoxicity during serial killing2019In: Journal of Experimental Medicine, ISSN 0022-1007, E-ISSN 1540-9538, Vol. 7, no jem.20181454Article in journal (Refereed)
    Abstract [en]

    NK cells eliminate virus-infected and tumor cells by releasing cytotoxic granules containing granzyme B (GrzB) or by engaging death receptors that initiate caspase cascades. The orchestrated interplay between both cell death pathways remains poorly defined. Here we simultaneously measure the activities of GrzB and caspase-8 in tumor cells upon contact with human NK cells. We observed that NK cells switch from inducing a fast GrzB-mediated cell death in their first killing events to a slow death receptor–mediated killing during subsequent tumor cell encounters. Target cell contact reduced intracellular GrzB and perforin and increased surface-CD95L in NK cells over time, showing how the switch in cytotoxicity pathways is controlled. Without perforin, NK cells were unable to perform GrzB-mediated serial killing and only killed once via death receptors. In contrast, the absence of CD95 on tumor targets did not impair GrzB-mediated serial killing. This demonstrates that GrzB and death receptor–mediated cytotoxicity are differentially regulated during NK cell serial killing.

  • 22.
    Steen, Johanna
    et al.
    Karolinska Inst, Karolinska Univ Hosp, Stockholm, Sweden..
    Forsström, Björn
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology.
    Sahlström, Peter
    KTH, School of Industrial Engineering and Management (ITM).
    Odowd, Victoria
    UCB Pharma, Slough, Berks, England..
    Israelsson, Lena
    Karolinska Inst, Karolinska Univ Hosp, Stockholm, Sweden..
    Krishnamurthy, Akilan
    Karolinska Inst, Karolinska Univ Hosp, Stockholm, Sweden..
    Badreh, Sara
    Karolinska Inst, Karolinska Univ Hosp, Stockholm, Sweden..
    Alm, Linda Mathsson
    Thermo Fisher Sci, Uppsala, Sweden.;Uppsala Univ, Uppsala, Sweden..
    Compson, Joanne
    UCB Pharma, Slough, Berks, England..
    Ramskold, Daniel
    Karolinska Inst, Karolinska Univ Hosp, Stockholm, Sweden..
    Ndlovu, Welcome
    UCB Pharma, Slough, Berks, England..
    Rapecki, Stephen
    UCB Pharma, Slough, Berks, England..
    Hansson, Monika
    Karolinska Inst, Karolinska Univ Hosp, Stockholm, Sweden..
    Titcombe, Philip J.
    Karolinska Inst, Karolinska Univ Hosp, Stockholm, Sweden.;Univ Minnesota, Med Sch, Minneapolis, MN 55455 USA..
    Bang, Holger
    Orgentec Diagnost, Mainz, Germany..
    Mueller, Daniel L.
    Univ Minnesota, Med Sch, Minneapolis, MN 55455 USA..
    Catrina, Anca I.
    Karolinska Inst, Karolinska Univ Hosp, Stockholm, Sweden..
    Gronwall, Caroline
    Karolinska Inst, Karolinska Univ Hosp, Stockholm, Sweden..
    Skriner, Karl
    Charite, Berlin, Germany..
    Nilsson, Peter
    Lightwood, Daniel
    UCB Pharma, Slough, Berks, England..
    Klareskog, Lars
    Karolinska Inst, Karolinska Univ Hosp, Stockholm, Sweden..
    Malmstrom, Vivianne
    Karolinska Inst, Karolinska Univ Hosp, Stockholm, Sweden..
    Recognition of Amino Acid Motifs, Rather Than Specific Proteins, by Human Plasma Cell-Derived Monoclonal Antibodies to Posttranslationally Modified Proteins in Rheumatoid Arthritis2019In: Arthritis & Rheumatology, ISSN 2326-5191, E-ISSN 2326-5205, Vol. 71, no 2, p. 196-209Article in journal (Refereed)
    Abstract [en]

    Objective Antibodies against posttranslationally modified proteins are a hallmark of rheumatoid arthritis (RA), but the emergence and pathogenicity of these autoantibodies are still incompletely understood. The aim of this study was to analyze the antigen specificities and mutation patterns of monoclonal antibodies (mAb) derived from RA synovial plasma cells and address the question of antigen cross-reactivity. Methods IgG-secreting cells were isolated from RA synovial fluid, and the variable regions of the immunoglobulins were sequenced (n = 182) and expressed in full-length mAb (n = 93) and also as germline-reverted versions. The patterns of reactivity with 53,019 citrullinated peptides and 49,211 carbamylated peptides and the potential of the mAb to promote osteoclastogenesis were investigated. Results Four unrelated anti-citrullinated protein autoantibodies (ACPAs), of which one was clonally expanded, were identified and found to be highly somatically mutated in the synovial fluid of a patient with RA. The ACPAs recognized >3,000 unique peptides modified by either citrullination or carbamylation. This highly multireactive autoantibody feature was replicated for Ig sequences derived from B cells from the peripheral blood of other RA patients. The plasma cell-derived mAb were found to target distinct amino acid motifs and partially overlapping protein targets. They also conveyed different effector functions as revealed in an osteoclast activation assay. Conclusion These findings suggest that the high level of cross-reactivity among RA autoreactive B cells is the result of different antigen encounters, possibly at different sites and at different time points. This is consistent with the notion that RA is initiated in one context, such as in the mucosal organs, and thereafter targets other sites, such as the joints.

1 - 22 of 22
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