基于不同种属细胞体外血脑屏障模型的药物渗透性比较

doi:10.5246/jcps.2023.04.021

摘要/Abstract

摘要：

血脑屏障(BBB)对维持中枢神经系统(CNS)内环境稳态发挥着重要作用。由于BBB的存在, 许多CNS药物难以进入脑组织发挥药效, 严重影响CNS疾病的治疗。基于细胞的体外BBB模型是研究CNS药物递送的重要工具。现已开发利用小鼠、大鼠、牛、猪和人的脑内皮细胞用于建立体外BBB模型。然而, 由于物种的差异, 不同种属来源的体外BBB模型在转运体、受体和紧密连接等蛋白表达上的差异是显著的。本文综述了常用的几种不同种属的体外细胞BBB模型, 为研究者选择BBB模型提供参考。

关键词: 血脑屏障, 脑内皮细胞, 药物递送, 体外模型

Abstract:

The specific structure of the blood-brain barrier (BBB) leads to the low permeability of central nervous system (CNS) drugs. Many cell-based in vitro BBB models have been developed to aid the study of CNS drug delivery. Mouse, rat, bovine, pig, and human brain endothelial cells are mainly used to build in vitro BBB models. However, interspecies differences in the expressions of transporters, receptors, and tight junction proteins are significant. In this review, several commonly used cell-based in vitro BBB models were introduced. The transendothelial electrical resistance and permeability coefficients of the representative substances, as well as specific endothelial markers, were highlighted. The review provided a clearer overview of these common models and a reference for readers when choosing BBB models.

Key words: Blood-brain barrier, Brain endothelial cell, Drug delivery, In vitro models

Supporting:

拓文静, 华若辰, 李星玥, 宋继红, 卢闻. 基于不同种属细胞体外血脑屏障模型的药物渗透性比较[J]. 中国药学(英文版), 2023, 32(4): 237-249.

Wenjing Ta, Ruochen Hua, Xingyue Li, Jihong Song, Wen Lu. In vitro blood-brain barrier models from different species: an overview on permeability associated with drug delivery[J]. Journal of Chinese Pharmaceutical Sciences, 2023, 32(4): 237-249.

图/表 2

Figure 1. Micromorphology of endothelial cells from different species. (A) MBCECs were cultured for 5 d[17]; (B) b.End3 were cultured for 6 d[18]; (C) cEND were cultured for 1 month[19]; (D–E) RBEC and rat spinal cord endothelial cells (RSCECs) were cultured for 5–7 d[20]; (F) PBECs were co-cultured with astrocytes for 7 d[21]; (G) BBCECs were cultured for 5 d[22]; (H–K) BB19, hBMEC, hCMEC/D3, and TY10 were cultured for 2 d[23]; (L) EPDCs were cultured for 14 d[24].

Table 1. Compilation of validation markers of in vitro BBB models.

参考文献 78

[1]	Hajar, A.F.; Al-Majdoub, Z.M.; Brahim, A.; Narciso, C.; Amin, R.H.; Jill, B. Identification and quantification of blood-brain barrier transporters in isolated rat brain microvessels. J. Neurochem. 2018, 146, 670–685.
[2]	Abbott, N.J. Blood-brain barrier structure and function and the challenges for CNS drug delivery. J. Inherit. Metab. Dis. 2013, 36, 437–449.
[3]	Yin, F.C.; Su, W.T.; Wang, L.; Hu, Q.Z. Microfluidic strategies for the blood-brain barrier construction and assessment. TrAc Trends Anal. Chem. 2022, 155, 116689.
[4]	Erickson, M.A.; Wilson, M.L.; Banks, W.A. In vitro modeling of blood-brain barrier and interface functions in neuroimmune communication. Fluids Barriers CNS. 2020, 17, 26.
[5]	Pardridge, W.M. The isolated brain microvessel: a versatile experimental model of the blood-brain barrier. Front. Physiol. 2020, 11, 398.
[6]	Fu, B.M. Quantification of In vitro blood-brain barrier permeability. Methods Mol. Biol. 2022, 2375, 217–228.
[7]	Helms, H.C.; Abbott, N.J.; Burek, M.; Cecchelli, R.; Couraud, P.O.; Deli, M.A.; Förster, C.; Galla, H.J.; Romero, I.A.; Shusta, E.V.; Stebbins, M.J.; Vandenhaute, E.; Weksler, B.; Brodin, B. In vitro models of the blood-brain barrier: an overview of commonly used brain endothelial cell culture models and guidelines for their use. J. Cereb. Blood Flow Metab. 2016, 36, 862–890.
[8]	Kim, W.; Kim, J.; Lee, S.Y.; Kim, H.M.; Joo, K.M.; Nam, D.H. Simplified in vitro 3D co-culture-based blood-brain barrier model using transwell. Biochem. Biophys. Res. Commun. 2022, 620, 63–68.
[9]	Kaisar, M.A.; Sajja, R.K.; Prasad, S.; Abhyankar, V.V.; Liles, T.; Cucullo, L. New experimental models of the blood-brain barrier for CNS drug discovery. Expert Opin. Drug Discov. 2017, 12, 89–103.
[10]	Gomes, M.J.; Mendes, B.; Martins, S.; Sarmento, B. Cell-based in vitro models for studying blood-brain barrier (BBB) permeability. Concepts Models Drug Permeabil. Stud. 2015, 1, 169–188.
[11]	Rice, O.; Surian, A.; Chen, Y.P. Modeling the blood-brain barrier for treatment of central nervous system (CNS) diseases. J. Tissue Eng. 2022, 13, 20417314221095997.
[12]	Wolff, A.; Antfolk, M.; Brodin, B.; Tenje, M. In vitro blood-brain barrier models—an overview of established models and new microfluidic approaches. J. Pharm. Sci. 2015, 104, 2727–2746.
[13]	Santa-Maria, A.R.; Heymans, M.; Walter, F.R.; Culot, M.; Gosselet, F.; Deli, M.A.; Neuhaus, W. Transport studies using blood-brain barrier In vitro models: a critical review and guidelines. Handb. Exp. Pharmacol. 2022, 273, 187–204.
[14]	Morris, M.E.; Rodriguez-Cruz, V.; Felmlee, M.A. SLC and ABC transporters: expression, localization, and species differences at the blood-brain and the blood-cerebrospinal fluid barriers. AAPS. J. 2017, 19, 1317–1331.
[15]	Warren, M.S.; Zerangue, N.; Woodford, K.; Roberts, L.M.; Tate, E.H.; Feng, B.; Li, C.; Feuerstein, T.J.; Gibbs, J.; Smith, B.; de Morais, S.M.; Dower, W.J.; Koller, K.J. Comparative gene expression profiles of ABC transporters in brain microvessel endothelial cells and brain in five species including human. Pharmacol. Res. 2009, 59, 404–413.
[16]	Hoshi, Y.; Uchida, Y.; Tachikawa, M.; Inoue, T.; Ohtsuki, S.; Terasaki, T. Quantitative atlas of blood-brain barrier transporters, receptors, and tight junction proteins in rats and common marmoset. J. Pharm. Sci. 2013, 102, 3343–3355.
[17]	Weidenfeller, C.; Schrot, S.; Zozulya, A.; Galla, H.J. Murine brain capillary endothelial cells exhibit improved barrier properties under the influence of hydrocortisone. Brain Res. 2005, 1053, 162–174.
[18]	Omidi, Y.; Campbell, L.; Barar, J.; Connell, D.; Akhtar, S.; Gumbleton, M. Evaluation of the immortalised mouse brain capillary endothelial cell line, b.End3, as an in vitro blood-brain barrier model for drug uptake and transport studies. Brain Res. 2003, 990, 95–112.
[19]	Burek, M.; Salvador, E.; Förster, C.Y. Generation of an immortalized murine brain microvascular endothelial cell line as an in vitro blood brain barrier model. J. Vis. Exp. 2012, e4022.
[20]	Watson, M.; Paterson, J.; Thom, G.; Ginman, U.; Lundquist, S.; Webster, C. Modelling the endothelial blood-CNS barriers: a method for the production of robust in vitro models of the rat blood-brain barrier and blood-spinal cord barrier. BMC Neurosci. 2013, 14, 59.
[21]	Hoheisel, D.; Nitz, T.; Franke, H.; Wegener, J.; Hakvoort, A.; Tilling, T.; Galla, H.J. Hydrocortisone reinforces the blood-brain barrier properties in a serum free cell culture system. Biochem. Biophys. Res. Commun. 1998, 244, 312–316.
[22]	Dehouck, M.P.; Méresse, S.; Delorme, P.; Fruchart, J.C.; Cecchelli, R. An easier, reproducible, and mass-production method to study the blood-brain barrier in vitro. J. Neurochem. 1990, 54, 1798–801.
[23]	Eigenmann, D.E.; Xue, G.D.; Kim, K.S.; Moses, A.V.; Hamburger, M.; Oufir, M. Comparative study of four immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, and optimization of culture conditions, for an in vitro blood-brain barrier model for drug permeability studies. Fluids Barriers CNS. 2013, 10, 33.
[24]	Boyer-Di Ponio, J.; El-Ayoubi, F.; Glacial, F.; Ganeshamoorthy, K.; Driancourt, C.; Godet, M.; Perrière, N.; Guillevic, O.; Couraud, P.O.; Uzan, G. Instruction of circulating endothelial progenitors in vitro towards specialized blood-brain barrier and arterial phenotypes. PLoS One. 2014, 9, e84179.
[25]	Thomsen, M.S.; Birkelund, S.; Burkhart, A.; Stensballe, A.; Moos, T. Synthesis and deposition of basement membrane proteins by primary brain capillary endothelial cells in a murine model of the blood-brain barrier. J. Neurochem. 2017, 140, 741–754.
[26]	Wuest, D.M.; Lee, K.H. Optimization of endothelial cell growth in a murine in vitro blood-brain barrier model. Biotechnol. J. 2012, 7, 409–417.
[27]	Coisne, C.; Dehouck, L.; Faveeuw, C.; Delplace, Y.; Miller, F.; Landry, C.; Morissette, C.; Fenart, L.; Cecchelli, R.; Tremblay, P.; Dehouck, B. Mouse syngenic in vitro blood–brain barrier model: a new tool to examine inflammatory events in cerebral endothelium. Lab. Investig. 2005, 85, 734–746.
[28]	Wagner, E.F.; Risau, W. Oncogenes in the study of endothelial cell growth and differentiation. Semin. Cancer Biol. 1994, 5, 137–145.
[29]	Paolinelli, R.; Corada, M.; Ferrarini, L.; Devraj, K.; Artus, C.; Czupalla, C.J.; Rudini, N.; Maddaluno, L.; Papa, E.; Engelhardt, B.; Couraud, P.O.; Liebner, S.; Dejana, E. Wnt activation of immortalized brain endothelial cells as a tool for generating a standardized model of the blood brain barrier in vitro. PLoS One. 2013, 8, e70233.
[30]	Wang, J.D.; Khafagy, E.S.; Khanafer, K.; Takayama, S.; ElSayed, M.E.H. Organization of endothelial cells, pericytes, and astrocytes into a 3D microfluidic in vitro model of the blood-brain barrier. Mol. Pharm. 2016, 13, 895–906.
[31]	Silwedel, C.; Förster, C. Differential susceptibility of cerebral and cerebellar murine brain microvascular endothelial cells to loss of barrier properties in response to inflammatory stimuli. J. Neuroimmunol. 2006, 179, 37–45.
[32]	Molino, Y, Jabès, F, Lacassagne, E, Gaudin, N, Khrestchatisky, M. Setting-up an in vitro model of rat blood-brain barrier (BBB): a focus on BBB impermeability and receptor-mediated transport. J. Vis. Exp. 2014, e51278.
[33]	Yu, F.; Kumar, N.D.S.; Foo, L.C.; Ng, S.H.; Hunziker, W.; Choudhury, D. A pump-free tricellular blood-brain barrier on-a-chip model to understand barrier property and evaluate drug response. Biotechnol. Bioeng. 2020, 117, 1127–1136.
[34]	Nakagawa, S.; Deli, M.A.; Kawaguchi, H.; Shimizudani, T.; Shimono, T.; Kittel, Á.; Tanaka, K.; Niwa, M. A new blood-brain barrier model using primary rat brain endothelial cells, pericytes and astrocytes. Neurochem. Int. 2009, 54, 253–263.
[35]	Xue, Q.; Liu, Y.; Qi, H.Y.; Ma, Q.; Xu, L.; Chen, W.H.; Chen, G.; Xu, X.Y. A novel brain neurovascular unit model with neurons, astrocytes and microvascular endothelial cells of rat. Int. J. Biol. Sci. 2013, 9, 174–189.
[36]	Kiss, L.; Bocsik, A.; Walter, F.R.; Ross, J.; Brown, D.; Mendenhall, B.A.; Crews, S.R.; Lowry, J.; Coronado, V.; Thompson, D.E.; Sipos, P.; Szabó-Révész, P.; Deli, M.A.; Petrikovics, I. From the cover: In vitro and In vivo blood-brain barrier penetration studies with the novel cyanide antidote candidate dimethyl trisulfide in mice. Toxicol. Sci. 2017, 160, 398–407.
[37]	Brightman, M.W.; Reese, T.S. Junctions between intimately apposed cell membranes in the vertebrate brain. J. Cell Biol. 1969, 40, 648–677.
[38]	Roux, F.; Durieu-Trautmann, O.; Chaverot, N.; Claire, M.; Mailly, P.; Bourre, J.; Strosberg, A.; Couraud, P. Regulation of gamma‐glutamyl transpeptidase and alkaline phosphatase activities in immortalized rat brain microvessel endothelial cells. J. Cell Physiol. 1994, 159, 101–113.
[39]	Veszelka, S.; Tóth, A.; Walter, F.R.; Tóth, A.E.; Gróf, I.; Mészáros, M.; Bocsik, A.; Hellinger, É.; Vastag, M.; Rákhely, G.; Deli, M.A. Comparison of a rat primary cell-based blood-brain barrier model with epithelial and brain endothelial cell lines: gene expression and drug transport. Front. Mol. Neurosci. 2018, 11, 166.
[40]	Bowman, P.D.; Ennis, S.R.; Rarey, K.E.; Betz, A.L.; Goldstein, G.W. Brain microvessel endothelial cells in tissue culture: a model for study of blood-brain barrier permeability. Ann. Neurol. 1983, 14, 396–402.
[41]	Geier, E.G.; Chen, E.C.; Webb, A.; Papp, A.C.; Yee, S.W.; Sadee, W.; Giacomini, K.M. Profiling solute carrier transporters in the human blood-brain barrier. Clin. Pharmacol. Ther. 2013, 94, 636–639.
[42]	Kuo, C.F.; Majd, S. An improved in vitro blood-brain barrier model for applications in therapeutics’ delivery to brain. Annu. Int. Conf. IEEE Eng. Med. Biol. Soc. EMBC. 2020, 3331–3334.
[43]	Christian, H.H.; Sønderby, W.H.; Uhd, N.C.; Birger, B. Paracellular tightness and claudin-5 expression is increased in the BCEC/astrocyte blood-brain barrier model by increasing media buffer capacity during growth. AAPS J. 2010, 12, 759–70.
[44]	Stanness, K.A.; Guatteo, E.; Janigro, D. A dynamic model of the blood-brain barrier in vitro. Neurotoxicology. 1996, 17, 481–496.
[45]	Culot, M.; Lundquist, S.; Vanuxeem, D.; Nion, S.; Landry, C.; Delplace, Y.; Dehouck, M.P.; Berezowski, V.; Fenart, L.; Cecchelli, R. An in vitro blood-brain barrier model for high throughput (HTS) toxicological screening. Toxicol. In Vitro. 2008, 22, 799–811.
[46]	Helms, H.; Hersom, M.; Kuhlmann, L.B.; Badolo, L.; Nielsen, C.U.; Brodin, B. An electrically tight in vitro blood-brain barrier model displays net brain-to-blood efflux of substrates for the ABC transporters, P-gp, Bcrp and Mrp-1. AAPS. J. 2014, 16, 1046–1055.
[47]	Walters, E.M.; Agca, Y.; Ganjam, V.; Evans, T. Animal models got you puzzled?: think pig. Ann. NY Acad. Sci. 2011, 1245, 63–64.
[48]	Patabendige, A.; Skinner, R.A.; Morgan, L.; Joan Abbott, N. A detailed method for preparation of a functional and flexible blood-brain barrier model using porcine brain endothelial cells. Brain Res. 2013, 1521, 16–30.
[49]	Malina, K.C.K.; Cooper, I.; Teichberg, V.I. Closing the gap between the in-vivo and in-vitro blood-brain barrier tightness. Brain Res. 2009, 1284, 12–21.
[50]	Thomsen, L.B.; Burkhart, A.; Moos, T. A triple culture model of the blood-brain barrier using porcine brain endothelial cells, astrocytes and pericytes. PLoS One. 2015, 10, e0134765.
[51]	Freese, C.; Hanada, S.; Fallier-Becker, P.; Kirkpatrick, C.J.; Unger, R.E. Identification of neuronal and angiogenic growth factors in an in vitro blood-brain barrier model system: relevance in barrier integrity and tight junction formation and complexity. Microvasc. Res. 2017, 111, 1–11.
[52]	Weksler, B.; Romero, I.A.; Couraud, P.O. The hCMEC/D3 cell line as a model of the human blood brain barrier. Fluids Barriers CNS. 2013, 10, 16.
[53]	Ohtsuki, S.; Ikeda, C.; Uchida, Y.; Sakamoto, Y.; Miller, F.; Glacial, F.; Decleves, X.; Scherrmann, J.M.; Couraud, P.O.; Kubo, Y.; Tachikawa, M.; Terasaki, T. Quantitative targeted absolute proteomic analysis of transporters, receptors and junction proteins for validation of human cerebral microvascular endothelial cell line hCMEC/D3 as a human blood-brain barrier model. Mol. Pharm. 2013, 10, 289–296.
[54]	Zakharova, M.; Tibbe, M.; Koch, L.; Le The, H.; Leferink, A.; van den Berg, A.; van der Meer, A.; Broersen, K.; Segerink, L. Transwell-integrated 2 µm thick transparent polydimethylsiloxane membranes with controlled pore sizes and distribution to model the blood-brain barrier. Adv. Mater. Technol. 2021, 6, 2100138.
[55]	Birthe, G.; Kerstin, R.; Andreas, N.; Sandra, N.; Jessica, K.; Ernst, B.I.; Wolfgang, L. A face-to-face comparison of claudin-5 transduced human brain endothelial (hCMEC/D3) cells with porcine brain endothelial cells as blood-brain barrier models for drug transport studies. Fluids Barriers CNS. 2020, 17, 53.
[56]	Lippmann, E.S.; Azarin, S.M.; Kay, J.E.; Nessler, R.A.; Wilson, H.K.; Al-Ahmad, A.; Palecek, S.P.; Shusta, E.V. Derivation of blood-brain barrier endothelial cells from human pluripotent stem cells. Nat. Biotechnol. 2012, 30, 783–791.
[57]	Boyer-Di Ponio, J.; El-Ayoubi, F.; Glacial, F.; Ganeshamoorthy, K.; Driancourt, C.; Godet, M.; Perrière, N.; Guillevic, O.; Couraud, P.O.; Uzan, G. Instruction of circulating endothelial progenitors in vitro towards specialized blood-brain barrier and arterial phenotypes. PLoS One. 2014, 9, e84179.
[58]	Cecchelli, R.; Aday, S.; Sevin, E.; Almeida, C.; Culot, M.; Dehouck, L.; Coisne, C.; Engelhardt, B.; Dehouck, M.P.; Ferreira, L. A stable and reproducible human blood-brain barrier model derived from hematopoietic stem cells. PLoS One. 2014, 9, e99733.
[59]	Steiner, O.; Coisne, C.; Engelhardt, B.; Lyck, R. Comparison of immortalized bEnd5 and primary mouse brain microvascular endothelial cells as in vitro blood-brain barrier models for the study of T cell extravasation. J. Cereb. Blood Flow Metab. 2011, 31, 315–327.
[60]	Booth, R.; Kim, H. Permeability analysis of neuroactive drugs through a dynamic microfluidic In vitro blood-brain barrier model. Ann Biomed Eng. 2014, 42, 2379–2391.
[61]	Yang, S.; Jin, H.; Zhao, Z.G. Paracellular tightness and the functional expression of efflux transporters P-gp and BCRP in bEnd3 cells. Neurol. Res. 2018, 40, 644–649.
[62]	Ugolini, G.S.; Occhetta, P.; Saccani, A.; Re, F.; Krol, S.; Rasponi, M.; Redaelli, A. Design and validation of a microfluidic device for blood-brain barrier monitoring and transport studies. J. Micromech. Microeng. 2018, 28.
[63]	Daneman, R.; Zhou, L.; Kebede, A.A.; Barres, B.A. Pericytes are required for blood–brain barrier integrity during embryogenesis. Nature. 2010, 468, 562–566.
[64]	Neuhaus, W.; Gaiser, F.; Mahringer, A.; Franz, J.; Riethmüller, C.; Förster, C. The pivotal role of astrocytes in an in vitro stroke model of the blood-brain barrier. Front. Cell Neurosci. 2014, 8, 352.
[65]	Hawkins, B.T.; Grego, S.; Sellgren, K.L. Three-dimensional culture conditions differentially affect astrocyte modulation of brain endothelial barrier function in response to transforming growth factor β1. Brain Res. 2015, 1608, 167–176.
[66]	Matsumoto, J.; Dohgu, S.; Takata, F.; Iwao, T.; Kimura, I.; Tomohiro, M.; Aono, K.; Kataoka, Y.; Yamauchi, A. Serum amyloid A-induced blood-brain barrier dysfunction associated with decreased claudin-5 expression in rat brain endothelial cells and its inhibition by high-density lipoprotein in vitro. Neurosci. Lett. 2020, 738, 135352.
[67]	Abbott, N.; Dolman, D.; Drndarski, S.; Fredriksson, S.M. An improved in vitro blood-brain barrier model: rat brain endothelial cells co-cultured with astrocytes. Methods Mol. Biol. 2012, 814, 415–430.
[68]	Fauquette, W.; Amourette, C.; Dehouck, M.P.; Diserbo, M. Radiation-induced blood-brain barrier damages: an in vitro study. Brain Res. 2012, 1433, 114–126.
[69]	Christian, H.H.; Rasmus, M.; Sønderby, W.H.; Uhd, N.C.; Birger, B. In vitro evidence for the brain glutamate efflux hypothesis: brain endothelial cells cocultured with astrocytes display a polarized brain-to-blood transport of glutamate. Glia. 2012, 60, 882–893.
[70]	Cohen-Kashi-Malina, K.; Cooper, I.; Teichberg, V.I. Mechanisms of glutamate efflux at the blood-brain barrier: involvement of glial cells. J. Cereb. Blood Flow Metab. 2012, 32, 177–189.
[71]	Cantrill, C.A.; Skinner, R.A.; Rothwell, N.J.; Penny, J.I. An immortalised astrocyte cell line maintains the in vivo phenotype of a primary porcine in vitro blood-brain barrier model. Brain Res. 2012, 1479, 17–30.
[72]	Patabendige, A.; Skinner, R.A.; Abbott, N.J. Establishment of a simplified in vitro porcine blood-brain barrier model with high transendothelial electrical resistance. Brain Res. 2013, 1521, 1–15.
[73]	Hatherell, K.; Couraud, P.O.; Romero, I.A.; Weksler, B.; Pilkington, G.J. Development of a three-dimensional, all-human in vitro model of the blood-brain barrier using mono-, co-, and tri-cultivation Transwell models. J. Neurosci. Methods. 2011, 199, 223–229.
[74]	Cucullo, L.; Marchi, N.; Hossain, M.; Janigro, D. A dynamic in vitro BBB model for the study of immune cell trafficking into the central nervous system. J. Cereb. Blood Flow Metab. 2011, 31, 767–777.
[75]	Cucullo, L.; Hossain, M.; Puvenna, V.; Marchi, N.; Janigro, D. The role of shear stress in Blood-Brain Barrier endothelial physiology. BMC Neurosci. 2011, 12, 40.
[76]	Ogunshola, O.O. In vitro modeling of the blood-brain barrier: simplicity versus complexity. Curr. Pharm. Des. 2011, 17, 2755–2761.
[77]	Thomsen, M.S.; Humle, N.; Hede, E.; Moos, T.; Burkhart, A.; Thomsen, L.B. The blood-brain barrier studied in vitro across species. PLoS One. 2021, 16, e0236770.
[78]	Nielsen, S, Siupka, P, Georgian, A, Preston, JE, Tóth, AE, Yusof, SR, Abbott, NJ, Nielsen, MS. Improved method for the establishment of an In vitro blood-brain barrier model based on porcine brain endothelial cells. J. Vis. Exp. 2017, 56277.