Michael H Gelb

University of Washington Seattle, Seattle, Washington, United States

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Publications (410)2401.45 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: We have previously shown that secreted phospholipases A2 (sPLA2s) from animal venoms inhibit the in vitro development of P. falciparum, the agent of malaria. In addition, the inflammatory-type human group IIA (hGIIA) sPLA2 circulates at high levels in the serum of malaria patients. However, the role of the different human sPLA2s in host defense against P. falciparum has not been investigated. We show here that 4 out of 10 human sPLA2s, namely hGX, hGIIF, hGIII and hGV, exhibit potent in vitro anti-Plasmodium properties with IC50 values of 2.9±2.4, 10.7±2.1, 16.5±9.7 and 94.2±41.9 nM, respectively. Other human sPLA2s including hGIIA are inactive. The inhibition is dependent on sPLA2 catalytic activity and primarily due to hydrolysis of plasma lipoproteins from the parasite culture. Accordingly, purified lipoproteins that have been pre-hydrolyzed by hGX, hGIIF, hGIII and hGV are more toxic to P. falciparum than native lipoproteins. However, the total enzymatic activities of human sPLA2s on purified lipoproteins or plasma did not reflect their inhibitory activities on P. falciparum. For instance, hGIIF is 9-fold more toxic than hGV but releases much less non-esterified fatty acids (NEFAs). Lipidomics analyses of released NEFAs from lipoproteins demonstrate that sPLA2s with anti-Plasmodium properties are those that release polyunsaturated fatty acids (PUFAs), with hGIIF being the most selective enzyme. NEFAs purified from lipoproteins hydrolyzed by hGIIF were more potent at inhibiting P. falciparum than those from hGV, and PUFA-enriched liposomes hydrolyzed by sPLA2s were highly toxic, demonstrating the critical role of PUFAs. The selectivity of sPLA2s towards LDL and HDL lipoproteins and their ability to directly attack parasitized erythrocytes further explain their anti-Plasmodium activity. Together, our findings indicate that 4 human sPLA2s are active against P. falciparum in vitro and pave the way to future investigations on their in vivo contribution in malaria pathophysiology. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
    Infection and immunity 03/2015; 83(6). DOI:10.1128/IAI.02474-14 · 4.16 Impact Factor
  • Clinical Chemistry 03/2015; 61(5). DOI:10.1373/clinchem.2014.236448 · 7.77 Impact Factor
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    ABSTRACT: We describe a computationally designed enzyme, formolase (FLS), which catalyzes the carboligation of three one-carbon formaldehyde molecules into one three-carbon dihydroxyacetone molecule. The existence of FLS enables the design of a new carbon fixation pathway, the formolase pathway, consisting of a small number of thermodynamically favorable chemical transformations that convert formate into a three-carbon sugar in central metabolism. The formolase pathway is predicted to use carbon more efficiently and with less backward flux than any naturally occurring one-carbon assimilation pathway. When supplemented with enzymes carrying out the other steps in the pathway, FLS converts formate into dihydroxyacetone phosphate and other central metabolites in vitro. These results demonstrate how modern protein engineering and design tools can facilitate the construction of a completely new biosynthetic pathway.
    Proceedings of the National Academy of Sciences 03/2015; DOI:10.1073/pnas.1500545112 · 9.81 Impact Factor
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    Molecular Genetics and Metabolism 02/2015; 114(2):S105-S106. DOI:10.1016/j.ymgme.2014.12.238 · 2.83 Impact Factor
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    ABSTRACT: Microparticles, also called microvesicles, are submicron extracellular vesicles produced by plasma membrane budding and shedding recognized as key actors in numerous physio(patho)logical processes. Since they can be released by virtually any cell lineages and are retrieved in biological fluids, microparticles appear as potent biomarkers. However, the small dimensions of microparticles and soluble factors present in body fluids can considerably impede their quantification. Here, flow cytometry with improved methodology for microparticle resolution was used to detect microparticles of human and mouse species generated from platelets, red blood cells, endothelial cells, apoptotic thymocytes and cells from the male reproductive tract. A family of soluble proteins, the secreted phospholipases A2 (sPLA2), comprises enzymes concomitantly expressed with microparticles in biological fluids and that catalyze the hydrolysis of membrane phospholipids. As sPLA2 can hydrolyze phosphatidylserine, a phospholipid frequently used to assess microparticles, and might even clear microparticles, we further considered the impact of relevant sPLA2 enzymes, sPLA2 group IIA, V and X, on microparticle quantification. We observed that if enriched in fluids, certain sPLA2 enzymes impair the quantification of microparticles depending on the species studied, the source of microparticles and the means of detection employed (surface phosphatidylserine or protein antigen detection). This study provides analytical considerations for appropriate interpretation of microparticle cytofluorometric measurements in biological samples containing sPLA2 enzymes.
    PLoS ONE 01/2015; 10(1):e0116812. DOI:10.1371/journal.pone.0116812 · 3.53 Impact Factor
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    ABSTRACT: The thymus is a primary lymphoid organ, home of maturation and selection of thymocytes for generation of functional T-cells. Multiple factors are involved throughout the different stages of the maturation process to tightly regulate T-cell production. The metabolism of arachidonic acid by cyclooxygenases, lipoxygenases and specific isomerases generates eicosanoids, lipid mediators capable of triggering cellular responses. In this study, we determined the profile of expression of the eicosanoids present in the mouse thymus at different stages of thymocyte development. As the group IVA cytosolic phospholipase A2 (cPLA2α) catalyzes the hydrolysis of phospholipids, thereby generating arachidonic acid, we further verified its contribution by including cPLA2α deficient mice to our investigations. We found that a vast array of eicosanoids is expressed in the thymus, which expression is substantially modulated through thymocyte development. The cPLA2α was dispensable in the generation of most eicosanoids in the thymus and consistently, the ablation of the cPLA2α gene in mouse thymus and the culture of thymuses from human newborns in presence of the cPLA2α inhibitor pyrrophenone did not impact thymocyte maturation. This study provides information on the eicosanoid repertoire present during thymocyte development and suggests that thymocyte maturation can occur independently of cPLA2α.
    PLoS ONE 01/2015; 10(5):e0126204. DOI:10.1371/journal.pone.0126204 · 3.53 Impact Factor
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    ABSTRACT: Matrix metalloproteinase (MMP)-2 deficiency makes humans and mice susceptible to inflammation. Here, we reveal an MMP-2-mediated mechanism that modulates the inflammatory response via secretory phospholipase A2 (sPLA2), a phospholipid hydrolase that releases fatty acids, including precursors of eicosanoids. Mmp2(-/-) (and, to a lesser extent, Mmp7(-/-) and Mmp9(-/-)) mice had between 10- and 1000-fold elevated sPLA2 activity in plasma and heart, increased eicosanoids and inflammatory markers (both in the liver and heart), and exacerbated lipopolysaccharide-induced fever, all of which were blunted by adenovirus-mediated MMP-2 overexpression and varespladib (pharmacological sPLA2 inhibitor). Moreover, Mmp2 deficiency caused sPLA2-mediated dysregulation of cardiac lipid metabolic gene expression. Compared with liver, kidney, and skeletal muscle, the heart was the single major source of the Ca(2+)-dependent, ≈20-kDa, varespladib-inhibitable sPLA2 that circulates when MMP-2 is deficient. PLA2G5, which is a major cardiac sPLA2 isoform, was proinflammatory when Mmp2 was deficient. Treatment of wild-type (Mmp2(+/+)) mice with doxycycline (to inhibit MMP-2) recapitulated the Mmp2(-/-) phenotype of increased cardiac sPLA2 activity, prostaglandin E2 levels, and inflammatory gene expression. Treatment with either indomethacin (to inhibit cyclooxygenase-dependent eicosanoid production) or varespladib (which inhibited eicosanoid production) triggered acute hypertension in Mmp2(-/-) mice, revealing their reliance on eicosanoids for blood pressure homeostasis. A heart-centric MMP-2/sPLA2 axis may modulate blood pressure homeostasis, inflammatory and metabolic gene expression, and the severity of fever. This discovery helps researchers to understand the cardiovascular and systemic effects of MMP-2 inhibitors and suggests a disease mechanism for human MMP-2 gene deficiency. © 2015 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.
    Journal of the American Heart Association 01/2015; 4(4). DOI:10.1161/JAHA.115.001868 · 2.88 Impact Factor
  • Chemical Reviews 11/2014; DOI:10.1021/cr500365f · 45.66 Impact Factor
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    ABSTRACT: Mitochondrial DNA (mtDNA) is a highly potent inflammatory trigger and is reportedly found outside the cells in blood in various pathologies. Platelets are abundant in blood where they promote hemostasis. While lacking a nucleus, platelets contain functional mitochondria. Upon activation, platelets produce extracellular vesicles known as microparticles. We hypothesized that activated platelets could also release their mitochondria. We show that activated platelets release respiratory-competent mitochondria, both within membrane-encapsulated microparticles and as free organelles. Extracellular mitochondria are found in platelet concentrates used for transfusion and are present at higher levels in those that induced acute reactions (febrile non-hemolytic reactions, skin manifestations and cardiovascular events) in transfused patients. We establish that the mitochondrion is an endogenous substrate of secreted phospholipase A2 IIA (sPLA2-IIA), a phospholipase otherwise specific for bacteria, likely reflecting the ancestral proteobacteria origin of mitochondria. The hydrolysis of the mitochondrial membrane by sPLA2-IIA yields inflammatory mediators (i.e. lysophospholipids, fatty acids and mtDNA) that promote leukocyte activation. Two-photon microscopy in live transfused-animals revealed that extracellular mitochondria interact with neutrophils in vivo, triggering neutrophil adhesion to the endothelial wall. Our findings identify extracellular mitochondria, produced by platelets, at the midpoint of a potent mechanism leading to inflammatory responses.
    Blood 07/2014; 124(14). DOI:10.1182/blood-2014-05-573543 · 9.78 Impact Factor
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    ABSTRACT: We report new substrates for quantitative enzyme activity measurements of human palmitoyl protein thioesterase (PPT1) and tripeptidyl peptidase (TPP1) in dried blood spots from newborns using tandem mass spectrometry. Deficiencies in these enzyme activities due to inborn errors of metabolism cause neuronal ceroid lipofuscinoses. The assays use synthetic compounds that were designed to mimic the natural substrates. Incubation produces nanomole quantities of enzymatic products per a blood spot that are quantified by tandem mass spectrometry using synthetic internal standards and selected reaction monitoring. The assays utilize a minimum steps for sample work up and can be run in a duplex format for the detection of neuronal ceroid lipofuscinoses or potentially multiplexed with other mass spectrometry-based assays for newborn screening of lysosomal storage disorders.
    Analytical Chemistry 07/2014; 86(15). DOI:10.1021/ac501994b · 5.83 Impact Factor
  • Michael H Gelb
    Clinical Biochemistry 06/2014; DOI:10.1016/j.clinbiochem.2014.05.015 · 2.23 Impact Factor
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    ABSTRACT: Metabolic disorders, including obesity and insulin resistance, have their basis in dysregulated lipid metabolism and low-grade inflammation. In a microarray search of unique lipase-related genes whose expressions are associated with obesity, we found that two secreted phospholipase A2s (sPLA2s), PLA2G5 and PLA2G2E, were robustly induced in adipocytes of obese mice. Analyses of Pla2g5(-/-) and Pla2g2e(-/-) mice revealed distinct roles of these sPLA2s in diet-induced obesity. PLA2G5 hydrolyzed phosphatidylcholine in fat-overladen low-density lipoprotein to release unsaturated fatty acids, which prevented palmitate-induced M1 macrophage polarization. As such, PLA2G5 tipped the immune balance toward an M2 state, thereby counteracting adipose tissue inflammation, insulin resistance, hyperlipidemia, and obesity. PLA2G2E altered minor lipoprotein phospholipids, phosphatidylserine and phosphatidylethanolamine, and moderately facilitated lipid accumulation in adipose tissue and liver. Collectively, the identification of "metabolic sPLA2s" adds this gene family to a growing list of lipolytic enzymes that act as metabolic coordinators.
    Cell Metabolism 06/2014; DOI:10.1016/j.cmet.2014.05.002 · 16.75 Impact Factor
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    ABSTRACT: Tandem mass spectrometry for the multiplex and quantitative analysis of enzyme activities in dried blood spots on newborn screening cards has emerged as a powerful technique for early assessment of lysosomal storage diseases. Here we report the design and process-scale synthesis of substrates for the enzymes alpha-L-iduronidase, iduronate-2-sulfatase, and N-acetylgalactosamine-4-sulfatase that are used for newborn screening of mucopolysaccharidosis types I, II and VI. The products contain a bis-amide unit that is hypothesized to readily protonate in the gas phase, which improves detection sensitivity by tandem mass spectrometry. The products contain a benzoyl group, which provides a useful site for inexpensive deuteration, thus facilitating the preparation of internal standards for the accurate quantification of enzymatic products. Finally, the reagents are designed with ease of synthesis in mind, thus permitting scale up preparation to support worldwide newborn screening of lysosomal storage diseases. The new reagents provide the most sensitive assay for the 3 lysosomal enzymes reported to date as shown by their performance in reactions using dried blood spots as the enzyme source. Also, the ratio of assay signal to that measured in the absence of blood (background) is superior to all previously reported mucopolysaccharidosis types I, II an VI assays.
    Analytical Chemistry 04/2014; 86(9). DOI:10.1021/ac5004135 · 5.83 Impact Factor
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    ABSTRACT: The role of Group X secreted phospholipase A2 (GX-sPLA2) during influenza infection has not been previously investigated. We examined the role of GX-sPLA2 during H1N1 pandemic influenza infection in a GX-sPLA2 gene targeted mouse (GX−/−) model and found that survival after infection was significantly greater in GX−/− mice than in GX+/+ mice. Downstream products of GX-sPLA2 activity, PGD2, PGE2, LTB4, cysteinyl leukotrienes and Lipoxin A4 were significantly lower in GX−/− mice BAL fluid. Lung microarray analysis identified an earlier and more robust induction of T and B cell associated genes in GX−/− mice. Based on the central role of sPLA2 enzymes as key initiators of inflammatory processes, we propose that activation of GX-sPLA2 during H1N1pdm infection is an early step of pulmonary inflammation and its inhibition increases adaptive immunity and improves survival. Our findings suggest that GX-sPLA2 may be a potential therapeutic target during influenza.
    Virology 04/2014; s 454–455:78–92. DOI:10.1016/j.virol.2014.01.030 · 3.28 Impact Factor
  • Molecular Genetics and Metabolism 02/2014; 111(2):S22–S23. DOI:10.1016/j.ymgme.2013.12.033 · 2.83 Impact Factor
  • Molecular Genetics and Metabolism 02/2014; 111(2):S23. DOI:10.1016/j.ymgme.2013.12.034 · 2.83 Impact Factor
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    ABSTRACT: Sandhoff disease is a rare progressive neurodegenerative genetic disorder with a high incidence among certain isolated communities and ethnic groups around the world. Previous reports have shown a high occurrence of Sandhoff disease in northern Saskatchewan. Newborn screening cards from northern Saskatchewan were retrospectively screened in order to investigate the incidence and determine the carrier frequency of Sandhoff disease in these communities. PCR-based screening was conducted for the c.115delG (p.(Val39fs)) variant in the HEXB gene that was previously found in 4 Sandhoff disease patients from this area. The carrier frequency for this allele was estimated to be ~1:27. MS/MS-based screening of hexosaminidase activity along with genetic sequencing allowed for the identification of additional variants based on low total hexosaminidase activity and high % hexosaminidase A activity relative to c.115delG carriers. In total 4 pathogenic variants were discovered in the population (c.115delG, c.619A>G, c.1601G>T, and c.1652G>A) of which two are previously unreported (c.1601G>T and c.1652G>A). The combined carrier frequency of these alleles in the study area was estimated at ~1:15. Based on the number of cases of Sandhoff disease from this area we estimate the incidence to be ~1:390 corresponding to a child being born with the disease every 1-2years on average. The results from our study were then compared with variants in the HEXB gene from the genomes available from the 1000 Genomes project. A total of 19 HEXB variants were found in the 1092 genomes of which 5 are suspected of having a deleterious effect on hexosaminidase activity. The estimated carrier frequency of Sandhoff disease in Saskatchewan at 1:15 is more than 3 times higher than the carrier frequency in the global sample provided by the 1000 Genomes project at 1:57.
    Molecular Genetics and Metabolism 01/2014; DOI:10.1016/j.ymgme.2014.01.002 · 2.83 Impact Factor
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    ABSTRACT: Treatments are being developed for metachromatic leukodystrophy (MLD), suggesting the need for eventual newborn screening. Previous studies have shown that sulfatide molecular species are increased in the urine of MLD patients compared to samples from non-MLD individuals, but there is no data using dried blood spots (DBS), the most common sample available for newborn screening laboratories. We used ultra-high performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS) to quantify sulfatides in DBS and dried urine spots from 14 MLD patients and 50 non-MLD individuals. Several sulfatide molecular species were increased in dried urine samples from all MLD samples compared to non-MLD samples. Sulfatides, especially low molecular species, were increased in DBS from MLD patients, but the sulfatide levels were relatively low. There was good separation in sulfatide levels between MLD and non-MLD samples when dried urine spots were used, but not with DBS, because DBS from non-MLD individuals have measurable levels of sulfatides. Sulfatide accumulation studies in urine, but not in DBS, emerges as the method of choice if newborn screening is to be proposed for MLD.
    Clinica chimica acta; international journal of clinical chemistry 12/2013; 433. DOI:10.1016/j.cca.2013.12.016 · 2.76 Impact Factor
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    ABSTRACT: A phenotypic screen of a compound library for antiparasitic activity on Trypanosoma brucei, the causative agent of human African trypanosomiasis, led to the identification of substituted 2-(3-aminophenyl)oxazolopyridines as a starting point for hit-to-lead medicinal chemistry. A total of 110 analogs were prepared, which led to the identification of 87, a substituted 2-(3-aminophenyl)imidazopyridine. This compound showed antiparasitic activity of parasites in vitro with an EC50 of 2 nM and displayed reasonable drug-like properties when tested in a number of in vitro assays. The compound was orally bioavailable and displayed good plasma and brain exposure in mice. Compound 87 cured mice infected with Trypanosoma brucei when dosed orally down to 2.5 mg/kg. Given its potent anti-parasitic properties and its ease of synthesis, compound 87 represents a new lead for the development of drugs to treat human African trypanosomiasis.
    Journal of Medicinal Chemistry 12/2013; 57(3). DOI:10.1021/jm401178t · 5.48 Impact Factor
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    ABSTRACT: Perturbation of calcium signaling that occurs during cell injury and disease, promotes cell death. In mouse lung fibroblasts A23187 triggered mitochondrial permeability transition pore (MPTP) formation, LDH release and necrotic cell death that were blocked by cyclosporin A (CsA) and EGTA. LDH release temporally correlated with arachidonic acid release but did not involve cytosolic phospholipase A2α (cPLA2α) or calcium independent PLA2. Surprisingly, release of arachidonic acid and LDH from cPLA2α-deficient fibroblasts was inhibited by the cPLA2α inhibitor pyrrophenone, and another serine hydrolase inhibitor KT195, by preventing mitochondrial calcium uptake. Inhibitors of calmodulin-dependent protein kinase II, a mitochondrial Ca2+ uniporter (MCU) regulator, also prevented MPTP formation and arachidonic acid release induced by A23187 and by H2O2. Pyrrophenone blocked MCU-mediated mitochondrial calcium uptake in permeabilized fibroblasts but not in isolated mitochondria. Unlike pyrrophenone, the diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) and CsA blocked cell death and arachidonic acid release not by preventing mitochondrial calcium uptake but by inhibiting MPTP formation. In fibroblasts stimulated with thapsigargin, which induces MPTP formation by a direct effect on mitochondria, LDH and arachidonic acid release were blocked by CsA and OAG but not by pyrrophenone or EGTA. Therefore serine hydrolase inhibitors prevent necrotic cell death by blocking mitochondrial calcium uptake but not the enzyme releasing fatty acids that occurs by a novel pathway during MPTP formation. This work reveals the potential for development of small molecule cell permeable serine hydrolase inhibitors that block MCU-mediated mitochondrial calcium overload, MPTP formation and necrotic cell death.
    Journal of Biological Chemistry 12/2013; 289(3). DOI:10.1074/jbc.M113.497651 · 4.60 Impact Factor

Publication Stats

21k Citations
2,401.45 Total Impact Points

Institutions

  • 1970–2015
    • University of Washington Seattle
      • • Department of Chemistry
      • • Department of Biochemistry
      • • Department of Biological Structure
      Seattle, Washington, United States
  • 2013
    • Tokyo Metropolitan Institute of Medical Science
      Edo, Tōkyō, Japan
  • 2000–2013
    • Showa University
      • Division of Health Chemistry
      Shinagawa, Tōkyō, Japan
    • Memorial Sloan-Kettering Cancer Center
      New York, New York, United States
  • 2012
    • University of Sfax
      Şafāqis, Şafāqis, Tunisia
  • 2011
    • University of Nice-Sophia Antipolis
      • Institut de Pharmacologie Moléculaire et Cellulaire (IPMC/UMR6097 CNRS-UNS)
      Valbonne, Provence-Alpes-Cote d'Azur, France
    • University of Toronto
      Toronto, Ontario, Canada
    • University of Kentucky
      • Graduate Center for Nutritional Sciences
      Lexington, Kentucky, United States
  • 2009
    • State University of New York Downstate Medical Center
      Brooklyn, New York, United States
  • 1990–2007
    • Howard Hughes Medical Institute
      Ashburn, Virginia, United States
  • 2005
    • City University of Seattle
      Seattle, Washington, United States
  • 1991–2005
    • Yale University
      • • Department of Chemistry
      • • Department of Molecular Biophysics and Biochemistry
      New Haven, Connecticut, United States
  • 2004
    • Cornell University
      • Department of Microbiology and Immunology
      Итак, New York, United States
  • 2003
    • Goethe-Universität Frankfurt am Main
      Frankfurt, Hesse, Germany
    • University of Milan
      • Department of Pharmacological Sciences
      Milano, Lombardy, Italy
    • University Hospital Frankfurt
      Frankfurt, Hesse, Germany
  • 2002
    • University of Alberta
      • Department of Chemistry
      Edmonton, Alberta, Canada
  • 2001
    • German Cancer Research Center
      Heidelburg, Baden-Württemberg, Germany
    • University of California, San Francisco
      • Department of Pharmaceutical Chemistry
      San Francisco, California, United States
  • 1997–1999
    • French National Centre for Scientific Research
      • Institute of Molecular and Cellular Pharmacology
      Lutetia Parisorum, Île-de-France, France
  • 1989–1998
    • University of Delaware
      • Department of Chemistry and Biochemistry
      Ньюарк, Delaware, United States
  • 1995
    • Osaka City University
      • Department of Biochemistry
      Ōsaka, Ōsaka, Japan
  • 1993–1995
    • Swiss Institute for Art Research
      Zürich, Zurich, Switzerland
  • 1991–1992
    • Uppsala University
      Uppsala, Uppsala, Sweden