Cell and Tissue Research

, 334:199 | Cite as

Distribution of ectonucleotidases in the rodent brain revisited

  • David Langer
  • Klaus Hammer
  • Patrycja Koszalka
  • Jürgen Schrader
  • Simon Robson
  • Herbert Zimmermann
Regular Article


Nucleotides comprise a major class of signaling molecules in the nervous system. They can be released from nerve cells, glial cells, and vascular cells where they exert their function via ionotropic (P2X) or metabotropic (P2Y) receptors. Signaling via extracellular nucleotides and also adenosine is controlled and modulated by cell-surface-located enzymes (ectonucleotidases) that hydrolyze the nucleotide to the respective nucleoside. Extracellular hydrolysis of nucleotide ligands involves a considerable number of enzymes with differing catalytic properties differentially affecting the nucleotide signaling pathway. It is therefore important to investigate which type of ectonucleotidase(s) contributes to the control of nucleotide signaling in distinct cellular and physiological settings. By using a classical enzyme histochemical approach and employing various substrates, inhibitors, and knockout animals, we provide, for the first time, a comparative analysis of the overall distribution of catalytic activities reflecting four ectonucleotidase families: ecto-5′-nucleotidase, alkaline phosphatases, ectonucleoside triphosphate diphosphohydrolases (E-NTPDases), and ectonucleotide pyrophyphatases/phosphodiesterases (E-NPPs). We place into perspective the earlier literature and provide novel evidence for a parenchymal localization of tissue non-specific alkaline phosphatase, E-NPPs, and E-NTPDases in the mouse brain. In addition, we specify the location of ectonucleotidases within the brain vasculature. Most notably, brain vessels do not express ecto-5′-nucleotidase. The preponderance of individual enzymes differs considerably between brain locations. The contribution of all types of ectonucleotidases thus needs to be considered in physiological and pharmacological studies of purinergic signaling in the brain.


Alkaline phosphatase ATP Brain Ectonucleotidase Ecto-5′-nucleotidase Mouse (C57BL/6; CD73 Knockout; 129 Sv; NTPDase1 null) 


  1. Asensio AC, Rodriguez-Ferrera CR, Castaneyra-Perdomo A, Oaknin S, Rotllán P (2007) Biochemical analysis of ecto-nucleotide pyrophosphatase phosphodiesterase activity in brain membranes indicates involvement of NPP1 isoenzyme in extracellular hydrolysis of diadenosine polyphosphates in central nervous system. Neurochem Int 50:581–590PubMedCrossRefGoogle Scholar
  2. Belcher SM, Zsarnovzky A, Crawford PA, Hemani H, Spurling L, Kirley TL (2006) Immunolocalization of ecto-nucleoside triphosphate diphosphohydrolase 3 in rat brain: implications for modulation of multiple homeostatic systems including feeding and sleep wake bahaviors. Neuroscience 137:1331–1346PubMedCrossRefGoogle Scholar
  3. Bianchi V, Spychala J (2003) Mammalian 5′-nucleotidases. J Biol Chem 278:46195–46198PubMedCrossRefGoogle Scholar
  4. Bjelobaba I, Nedeljkovic N, Subasic S, Lavrnja I, Pekovic S, Stojkov D, Rakic L, Stojiljkovic M (2006) Immunolocalization of ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) in the rat forebrain. Brain Res 1120:54–63PubMedCrossRefGoogle Scholar
  5. Bjelobaba I, Stojiljkovic M, Pekovic S, Dacic S, Lavrnja I, Stojkov D, Rakic L, Nedeljkovic N (2007) Immunohistological determination of ecto-nucleoside triphosphate diphosphohydrolase1 (NTPDase1) and 5′-nucleotidase in rat hippocampus reveals overlapping distribution. Cell Mol Neurobiol 27:731–743PubMedCrossRefGoogle Scholar
  6. Blass-Kampmann S, Kindler-Rohrborn A, Deissler H, D’Urso D, Rajewsky MF (1997) In vitro differentiation of neural progenitor cells from prenatal rat brain: common cell surface glycoprotein on three glial cell subsets. J Neurosci Res 48:95–111PubMedCrossRefGoogle Scholar
  7. Braun JS, Lehir M, Kaissling B (1994) Morphology and distribution of ecto-5′-nucleotidase-positive cells in the rat choroid plexus. J Neurocytol 23:193–200PubMedCrossRefGoogle Scholar
  8. Braun N, Sévigny J, Robson SC, Enjyoji K, Guckelberger O, Hammer K, Di Virgilio F, Zimmermann H (2000a) Assignment of ecto-nucleoside triphosphate diphosphohydrolase-1/cd39 expression to microglia and vasculature of the brain. Eur J Neurosci 12:4357–4366PubMedCrossRefGoogle Scholar
  9. Braun N, Fengler S, Ebeling C, Servos J, Zimmermann H (2000b) Sequencing, functional expression and characterization of NTPDase6, a nucleoside diphosphatase and novel member of the ecto-nucleoside triphosphate diphosphohydrolase family. Biochem J 351:639–647PubMedCrossRefGoogle Scholar
  10. Braun N, Sévigny J, Mishra S, Robson SC, Barth SW, Gerstberger R, Hammer K, Zimmermann H (2003) Expression of the ecto-ATPase NTPDase2 in the germinal zones of the developing and adult rat brain. Eur J Neurosci 17:1355–1364PubMedCrossRefGoogle Scholar
  11. Brundege JM, Diao LH, Proctor WR, Dunwiddie TV (1997) The role of cyclic AMP as a precursor of extracellular adenosine in the rat hippocampus. Neuropharmacology 36:1201–1210PubMedCrossRefGoogle Scholar
  12. Burnstock G (2007a) Physiology and pathophysiology of purinergic neurotransmission. Physiol Rev 87:659–797PubMedCrossRefGoogle Scholar
  13. Burnstock G (2007b) Purine and pyrimidine receptors. Cell Mol Life Sci 64:1471–1483PubMedCrossRefGoogle Scholar
  14. Chilingaryan A, Chilingaryan AM, Martin GG (2006) The three-dimensional detection of microvasculatory bed in the brain of white rat Rattus norvegicus by a Ca2+-ATPase method. Brain Res 1070:131–138PubMedCrossRefGoogle Scholar
  15. Coles JA, Deitmer JW (2005) Extracellular potassium and pH: homeostasis and signaling. In: Kettenmann H, Ransom BR (eds) Neuroglia. Oxford University Press, Oxford, pp 334–345Google Scholar
  16. Cunha RA (2001a) Adenosine as a neuromodulator and as a homeostatic regulator in the nervous system: different roles, different sources and different receptors. Neurochem Int 38:107–125PubMedCrossRefGoogle Scholar
  17. Cunha RA (2001b) Regulation of the ecto-nucleotidase pathway in rat hippocampal nerve terminals. Neurochem Res 26:979–991PubMedCrossRefGoogle Scholar
  18. Doengi M, Deitmer JW, Lohr C (2008) New evidence for purinergic signaling in the olfactory bulb: A2A and P2Y1 receptors mediate intracellular calcium release in astrocytes. FASEB J 22:2368–2378PubMedCrossRefGoogle Scholar
  19. Dulla CG, Dobelis P, Pearson T, Frenguelli BG, Staley KJ, Masino SA (2005) Adenosine and ATP link P-CO2 to cortical excitability via pH. Neuron 48:1011–1023PubMedCrossRefGoogle Scholar
  20. Enjyoji K, Sévigny J, Lin Y, Frenette P, Christie PD, Schulte am Esch J, Imai M, Edelberger JM, Rayburn H, Lech M, Beeler DM, Csizmadia E, Wagner DD, Robson SC, Rosenberg RD (1999) Targeted disruption of cd39/ATP diphosphohydrolase results in disordered hemostasis and thromboregulation. Nat Med 5:1010–1017PubMedCrossRefGoogle Scholar
  21. Fastbom J, Pazos A, Palacios JM (1987) The distribution of adenosine A1 receptors and 5′-nucleotidase in the brain of some commonly used experimental animals. Neuroscience 22:813–826PubMedCrossRefGoogle Scholar
  22. Fonta C, Negyessy L, Renaud L, Barone P (2004) Areal and subcellular localization of the ubiquitous alkaline phosphatase in the primate cerebral cortex: evidence for a role in neurotransmission. Cereb Cortex 14:595–609PubMedCrossRefGoogle Scholar
  23. Fonta C, Negyessy L, Renaud L, Barone P (2005) Postnatal development of alkaline phosphatase activity correlates with the maturation of neurotransmission in the cerebral cortex. J Comp Neurol 486:179–196PubMedCrossRefGoogle Scholar
  24. Friede RL (1966) A quantitative mapping of alkaline phosphatase in the brain of the rhesus monkey. J Neurochem 13:197–203PubMedCrossRefGoogle Scholar
  25. Fuss B, Baba H, Phan T, Tuohy VK, Macklin WB (1997) Phosphodiesterase I, a novel adhesion molecule and/or cytokine involved in oligodendrocyte function. J Neurosci 17:9095–9103PubMedGoogle Scholar
  26. Goding JW, Grobben B, Slegers H (2003) Physiological and pathophysiological functions of the ecto-nucleotide pyrophosphatase/phosphodiesterase family. Biochim Biophys Acta Mol Basis Dis 1638:1–19Google Scholar
  27. Gordon JS, Torack RM (1967) Inhibition of cerebral adenosinetriphosphatase activity by various aldehyde fixatives. J Neurochem 14:1155–1160PubMedCrossRefGoogle Scholar
  28. Harahap AR, Goding JW (1988) Distribution of murine plasma cell antigen PC-1 in non-lymphoid tissues. J Immunol 141:2317–2320PubMedGoogle Scholar
  29. Heine P, Braun N, Zimmermann H (1999) Functional characterization of rat ecto-ATPase and ecto-ATP diphosphohydrolase after heterologous expression in CHO cells. Eur J Biochem 262:102–107PubMedCrossRefGoogle Scholar
  30. Ibrahim MZM, Khreis Y, Koshayan DS (1974) The histochemical identification of microglia. J Neurol Sci 22:211–233PubMedCrossRefGoogle Scholar
  31. King AE, Ackley MA, Cass CE, Young JD, Baldwin SA (2006) Nucleoside transporters: from scavengers to novel therapeutic targets. Trends Pharmacol Sci 27:416–425PubMedCrossRefGoogle Scholar
  32. Kittel A (1994) Distribution of Ca-ATPases in the medial habenula in mouse. Scanning Microsc 8:337–343PubMedGoogle Scholar
  33. Kittel A, Siklós L, Thuróczy G, Somosy Z (1996) Qualitative enzyme histochemistry and microanalysis reveals changes in ultrastructural distribution of calcium and calcium-activated ATPases after microwave irradiation of the medial habenula. Acta Neuropathol (Berl) 92:362–368CrossRefGoogle Scholar
  34. Koszalka P, Ozuyaman B, Huo YQ, Zernecke A, Flogel U, Braun N, Buchheiser A, Decking UKM, Smith ML, Sévigny J, Gear A, Weber AA, Molojavyi A, Ding ZP, Weber C, Ley K, Zimmermann H, Gödecke A, Schrader J (2004) Targeted disruption of cd73/ecto-5′-nucleotidase alters thromboregulation and augments vascular inflammatory response. Circ Res 95:814–821PubMedCrossRefGoogle Scholar
  35. Kozlenkov A, LeDu MH, Cuniasse P, Ny T, Hoylaerts MF, Millán JL (2004) Residues determining the binding specificity of uncompetitive inhibitors to tissue-nonspecific alkaline phosphatase. J Bone Miner Res 19:1862–1872PubMedCrossRefGoogle Scholar
  36. Kukulski F, Lévesque SA, Lavoie ÉG, Lecka J, Bigonnesse F, Knowles AF, Robson SC, Kirley TL, Sévigny J (2005) Comparative hydrolysis of P2 receptor agonists by NTPDase 1, 2, 3 and 8. Purinergic Signalling 1:193–204PubMedCrossRefGoogle Scholar
  37. Langer D, Ikehara Y, Takebayashi H, Hawkes R, Zimmermann H (2007) The ectonucleotidases alkaline phosphatase and nucleoside triphosphate diphosphohydrolas 2 are associated with subsets of progenitor cell populations in the mouse embryonic, postnatal and adult neurogenic zones. Neuroscience 150:863–879PubMedCrossRefGoogle Scholar
  38. Lee KS, Schubert P, Reddington M, Kreutzberg GW (1986) The distribution of adenosine A1 receptors and 5′-nucleotidase in the hippocampal formation of several mammalian species. J Comp Neurol 246:427–434PubMedCrossRefGoogle Scholar
  39. Lojda Z, Gossrau R, Schiebler TH (1979) Enzyme histochemistry. A laboratory manual. Springer, New YorkGoogle Scholar
  40. Millán JL (2006) Structurte, substrate specificity and functional relatedness to other members of a large superfamily of enzymes. Purinergic Signalling 2:335–341PubMedCrossRefGoogle Scholar
  41. Miras-Portugal MT, Gualix J, Mateo J, Díaz-Hernández M, Gómez-Villafuertes R, Castro E, Pintor J (1999) Diadenosine polyphosphates, extracellular function and catabolism. Prog Brain Res 120:397–409PubMedCrossRefGoogle Scholar
  42. Mori S, Nagano M (1985) Ultracytochemical demonstration of alkaline phosphatase activity in astrocytes and subependymal cells in the rat brain. Arch Hist Jpn 48:511–517Google Scholar
  43. Murabe Y, Sano Y (1981) Thiaminepyrophosphatase activity in the plasma membrane of microglia. Histochemistry 71:45–52PubMedCrossRefGoogle Scholar
  44. Murabe Y, Sano Y (1982) Morphological studies on microglia. V. Microglial cells in the cerebral cortex of the rat, with special reference to their possible involvement in synaptic function. Cell Tissue Res 223:493–506PubMedCrossRefGoogle Scholar
  45. Nagy AK (1997) Ecto-ATPases of the nervous system. In: Plesner L, Kirley TL, Knowles AF (eds) Ecto-ATPases: recent progress in structure and function. Plenum, New York, pp 1–13Google Scholar
  46. Nagy AK, Shuster TA, Delgado-Escueta AV (1986) Ecto-ATPase of mammalian synaptosomes: identification and enzymic characterization. J Neurochem 47:976–986PubMedGoogle Scholar
  47. Narisawa S, Hasegawa H, Watanabe K, Millán JL (1994) Stage-specific expression of alkaline phosphatase during neural development of the mouse. Dev Dyn 201:227–235PubMedGoogle Scholar
  48. Narisawa S, Hoylaerts MF, Doctor KS, Fukuda MN, Alpers DH, Millán JL (2007) A novel phosphatase upregulated in Akp3 knockout mice. Am J Physiol Gastrointest Liver Physiol 293:G1068–G1077PubMedCrossRefGoogle Scholar
  49. Narita M, Goji J, Nakamura H, Sano K (1994) Molecular cloning, expression, and localization of a brain- specific phosphodiesterase I/nucleotide pyrophosphatase (PD-I alpha) from rat brain. J Biol Chem 269:28235–28242PubMedGoogle Scholar
  50. Nishihara Y, Hayashi Y, Fujii T, Adachi T, Stigbrand T, Hirano K (1994) The alkaline phosphatase in human plexus chorioideus. Biochim Biophys Acta 1209:274–278PubMedGoogle Scholar
  51. Novikoff AB, Drucker J, Shin WY, Goldfischer S (1961) Further studies of the apparent adenosinetriphosphatase activity of cell membranes in formol-calcium-fixed tissues. J Histochem Cytochem 9:434–451PubMedGoogle Scholar
  52. Ogilvie A (1992) Extracellular functions for ApnA. In: McLennan AG (ed) Ap4A and other dinucleoside polyphosphates. CRC, Boca Raton, pp 229–273Google Scholar
  53. Ohkubo S, Kimura J, Matsuoka I (2000) Ecto-alkaline phosphatase in NG108-15 cells: a key enzyme mediating P1 antagonist-sensitive ATP response. Br J Pharmacol 131:1667–1672PubMedCrossRefGoogle Scholar
  54. Robson SC, Candinas D, Siegel JB, Kopp C, Millan M, Hancock WW, Bach FH (1996) Potential mechanism of abnormal thromboregulation in xenograft rejection: loss of ecto-ATPases upon endothelial cell activation. Transplant Proc 1996 28:536Google Scholar
  55. Robson SC, Sévigny J, Zimmermann H (2006) The E-NTPDase family of ectonucleotidases: structure function relationships and pathophysiological significance. Purinergic Signalling 2:409–430PubMedCrossRefGoogle Scholar
  56. Salmi M, Jalkanen S (2005) Cell-surface enzymes in control of leukocyte trafficking. Nat Rev Immunol 5:760–771PubMedCrossRefGoogle Scholar
  57. Sano S, Matsuda Y, Nakagawa H (1988) Thiamine pyrophosphatase (nucleoside diphosphatase) in the Golgi apparatus is distinct from microsomal nucleoside diphosphatase. J Biochem 103:678–681PubMedGoogle Scholar
  58. Sato K, Malchinkhuu E, Muraki T, Ishikawa K, Hayashi K, Tosaka M, Mochiduki A, Inoue K, Tomura H, Mogi C, Nochi H, Tamoto K, Okajima F (2005) Identification of autotaxin as a neurite retraction- inducing factor of PC12 cells in cerebrospinal fluid and its possible sources. J Neurochem 92:904–914PubMedCrossRefGoogle Scholar
  59. Savaskan NE, Rocha L, Kotter MR, Baer A, Lubec G, vanMeeteren LA, Kishi Y, Aoki J, Moolenaar WH, Nitsch R, Brauer AU (2007) Autotaxin (NPP-2) in the brain: cell type-specific expression and regulation during development and after neurotrauma. Cell Mol Life Sci 64:230–243PubMedCrossRefGoogle Scholar
  60. Schoen SW, Graybiel AM (1993) Species-specific patterns of glycoprotein expression in the developing rodent caudoputamen—association of 5′-nucleotidase activity with dopamine islands and striosomes in rat, but with extrastriosomal matrix in mouse. J Comp Neurol 333:578–596PubMedCrossRefGoogle Scholar
  61. Schoen SW, Kreutzberg GW (1997) 5′-Nucleotidase enzyme cyctochemistry as a tool for revealing activated glial cells and malleable synapses in CNS development and regeneration. Brain Res Brain Res Protoc 1:33–43PubMedCrossRefGoogle Scholar
  62. Schoen SW, Graeber MB, Tóth L, Kreutzberg GW (1988) 5′-Nucleotidase in postnatal ontogeny of rat cerebellum: a marker for migrating nerve cells. Dev Brain Res 39:125–136CrossRefGoogle Scholar
  63. Scott TG (1967) The distribution of 5′-nucleotidase in the brain of the mouse. J Comp Neurol 129:97–114CrossRefGoogle Scholar
  64. Shukla V, Zimmermann H, Wang LP, Kettenmann H, Raab S, Hammer K, Sévigny J, Robson SC, Braun N (2005) Functional expression of the ecto-ATPase NTPDase2 and of nucleotide receptors by neuronal progenitor cells in the adult murine hippocampus. J Neurosci Res 80:600–610PubMedCrossRefGoogle Scholar
  65. Sjöstrand J (1966) Changes in nucleoside phosphatase activity in the hypoglossal nucleus during nerve regeneration. Acta Physiol Scand 67:219–228PubMedCrossRefGoogle Scholar
  66. Sommer JR, Hasselbach W (1967) The effect of glutaraldehyde and formaldehyde on the calcium pump of the sarcoplasmic reticulum. J Cell Biol 34:902–905PubMedCrossRefGoogle Scholar
  67. Stefan C, Jansen S, Bollen M (2005) NPP-type ectophosphodiesterases: unity in diversity. Trends Biochem Sci 30:542–550PubMedCrossRefGoogle Scholar
  68. Sugimura K, Mizutani A (1979) Histochemical and cytochemical studies of alkaline phosphatase activity in the synapses of rat brain. Histochemistry 61:123–129PubMedCrossRefGoogle Scholar
  69. Vollmayer P, Clair T, Goding JW, Sano K, Servos J, Zimmermann H (2003) Hydrolysis of diadenosine polyphosphates by nucleotide pyrophosphatases/phosphodiesterases. Eur J Biochem 270:2971–2978PubMedCrossRefGoogle Scholar
  70. Vorbrodt AW, Wisniewski HM (1982) Plasmalemma-bound nucleoside diphosphatase as a cytochemical marker of central nervous system (CNS) mesodermal cells. J Histochem Cytochem 30:418–424PubMedGoogle Scholar
  71. Vorbrodt AW, Lossinsky AS, Wisniewski HM (1986) Localization of alkaline phosphatase activity in endothelia of developing and mature mouse blood-brain barrier. Dev Neurosci 8:1–13PubMedCrossRefGoogle Scholar
  72. Wachstein M, Meisel E (1957) Histochemistry of hepatic phosphatases at a physiologic pH with special reference to the demonstration of bile canaliculi. Am J Clin Pathol 27:13–23PubMedGoogle Scholar
  73. Wang TF, Guidotti G (1996) CD39 is an ecto-(Ca2+,Mg2+)-apyrase. J Biol Chem 271:9898–9901PubMedCrossRefGoogle Scholar
  74. Wang TF, Guidotti G (1998) Widespread expression of ecto-apyrase (CD39) in the central nervous system. Brain Res 790:318–322PubMedCrossRefGoogle Scholar
  75. Wink MR, Braganhol E, Tamajusuku ASK, Lenz G, Zerbini LF, Libermann TA, Sévigny J, Battastini AMO, Robson SC (2006) Nucleoside triphosphate diphosphohydrolase-2 (NTPdase2/ CD39L1) is the dominant ectonucleotidase expressed by rat astrocytes. Neuroscience 138:421–432PubMedCrossRefGoogle Scholar
  76. Yegutkin GG (2008) Nucleotide- and nucleoside-converting ectoenzymes: important modulators of purinergic signalling cascade. Biochim Biophys Acta 1783:673–694PubMedCrossRefGoogle Scholar
  77. Zernecke A, Bidzhekov K, Ozuyaman B, Fraemohs L, Liehn EA, LuscherFirzlaff JM, Luscher B, Schrader J, Weber C (2006) CD73/Ecto-5′-nucleotidase protects against vascular inflammation and neointima formation. Circulation 113:2120–2127PubMedCrossRefGoogle Scholar
  78. Ziganshin AU, Hoyle CHV, Burnstock G (1994) Ecto-enzymes and metabolism of extracellular ATP. Drug Dev Res 32:134–146CrossRefGoogle Scholar
  79. Zimmermann H (1992) 5′-Nucleotidase—molecular structure and functional aspects. Biochem J 285:345–365PubMedGoogle Scholar
  80. Zimmermann H (1996a) Biochemistry, localization and functional roles of ecto-nucleotidases in the nervous system. Prog Neurobiol 49:589–618PubMedCrossRefGoogle Scholar
  81. Zimmermann H (1996b) Extracellular purine metabolism. Drug Dev Res 39:337–352CrossRefGoogle Scholar
  82. Zimmermann H (2000) Extracellular metabolism of ATP and other nucleotides. Naunyn-Schmiedeberg’s Arch Pharmacol 362:299–309CrossRefGoogle Scholar
  83. Zimmermann H (2006) Ecto-nucleotidases in the nervous system. Novartis Found Symp 275:113–128CrossRefGoogle Scholar
  84. Zimmermann H, Braun N (1999) Ecto-nucleotidases: molecular structures, catalytic properties, and functional roles in the nervous system. Prog Brain Res 120:371–385PubMedCrossRefGoogle Scholar
  85. Zimmermann H, Vogel M, Laube U (1993) Hippocampal localization of 5′-nucleotidase as revealed by immunocytochemistry. Neuroscience 55:105–112PubMedCrossRefGoogle Scholar
  86. Zimmermann H, Mishra SK, Shukla V, Langer D, Gampe K, Grimm I, Delic J, Braun N (2007) Ecto-nucleotidases, molecular properties and functional impact. A R Acad Nac Farm 73:537–566Google Scholar
  87. Zisapel N, Haklai R (1980) Localization of an alkaline phosphatase and other synaptic vesicle proteins. Neuroscience 5:2297–2303PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • David Langer
    • 1
  • Klaus Hammer
    • 1
  • Patrycja Koszalka
    • 2
  • Jürgen Schrader
    • 2
  • Simon Robson
    • 3
  • Herbert Zimmermann
    • 1
  1. 1.Institute of Cell Biology and Neuroscience, BiocenterGoethe University Frankfurt am MainFrankfurtGermany
  2. 2.Department of Cardiovascular PhysiologyHeinrich Heine University DuesseldorfDuesseldorfGermany
  3. 3.Department of Medicine, Beth Israel Deaconess Medical CenterHarvard Medical SchoolBostonUSA

Personalised recommendations