AI Chat Paper
Note: Please note that the following content is generated by AMiner AI. SciOpen does not take any responsibility related to this content.
{{lang === 'zh_CN' ? '文章概述' : 'Summary'}}
{{lang === 'en_US' ? '中' : 'Eng'}}
Chat more with AI
Powered by AMiner. Clicking this button will take you away from SciOpen.
Home Food Science and Human Wellness Article
PDF (1.6 MB)
Cite
EndNote(RIS) BibTeX
Collect
Submit Manuscript AI Chat Paper
Show Outline
Outline
Show full outline
Hide outline
Outline
Show full outline
Hide outline
Review Article | Open Access

Application of in vitro and in vivo models in the study of food allergy

Jianjian Huanga, Changjun Liuc, Yanbo Wanga,b, Chong Wanga, Menghua Xiea, Yi Qiana, Linglin Fua,b( )
School of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou, China
Zhejiang Engineering Institute of Food Quality and Safety, Zhejiang Gongshang University, Hangzhou, China
Xiangshan County Aquatic Technology Promotion Station, Xiangshan, 315700, China

Peer review under responsibility of KeAi Communications Co., Ltd.

Show Author Information

Abstract

Food allergy is one of the most important food safety problems that has attracted increasing attention. The food allergy experimental models provide not only the accurate allergen detection and evaluation methods but also the powerful approaches for mechanism investigations. In this paper, we reviewed the common food allergy cell models including mast cell, basophil granulocyte and basophil, as well as the animal models of BALB/c mouse, C3H/HeJ mouse, and BN rat. We also introduced zebrafish, a promising model organism for investigating immunity though lacking direct applications in food allergy to date, and focused on traumatic inflammation, bacterial infection and viral infection models. In addition, we also summarized the clinical diagnostic research methods for food allergy. The elucidation of these topics will help researchers to understand the characteristics and mechanisms of various models and thus select the proper models for particular study, so as to support further investigations of food allergy.

Keywords

Clinical studyZebrafishAnimal modelFood allergyCell model

References

[1]

J. Savage, C.B. Johns, Food allergy: epidemiology and natural history, Immunol. Allergy Clin. North Am. 35 (2015) 45–59.
Crossref Google Scholar
[2]

S.L. Prescott, P. Ruby, K.J. Allen, D.E. Campbell, K.H. Sinn John, et al., A global survey of changing patterns of food allergy burden in children, World Allergy Organ. J. 6 (2013) 1–12.
Crossref Google Scholar
[3]

S.H. Sicherer, Food allergy, J. Allergy Clin. Immunol. 125 (2010) S116–S125.
Crossref Google Scholar
[4]

J.A. Boyce, A. Assa'Ad, A.W. Burks, S.M. Jones, H.A. Sampson, et al., Guidelines for the diagnosis and management of food allergy in the United States: summary of the NIAID-sponsored expert panel report, Nutrition 27 (2011) 253.
Crossref Google Scholar
[5]

R. Nakamura, Y. Uchida, M. Higuchi, I. Tsuge, A. Urisu, et al., A convenient and sensitive allergy test: IgE crosslinking-induced luciferase expression in cultured mast cells, Allergy 65 (2010) 1266–1273.
Crossref Google Scholar
[6]
Report of the FAO Technical Consultation on Food Allergies, 1995.
[7]

L.B. Schwartz, P.C. Atkins, T.R. Bradford, P. Fleekop, M. Shalit, et al., Release of tryptase together with histamine during the immediate cutaneous response to allergen, J. Allergy Clin. Immunol. 80 (1987) 850–855.
Crossref Google Scholar
[8]

L. Perezábad, M. Reche, T. Valbuena, R. López-Fandiño, E. Molina, et al., Oral food desensitization in children with IgE-Mediated cow's milk allergy: immunological changes underlying desensitization, Allergy Asthma Immunol. Res. 9 (2017) 35.
Crossref Google Scholar
[9]

B. Yang, C. Yang, P. Wang, J. Li, H. Huang, et al., Food allergen–Induced mast cell degranulation is dependent on PI3K‐Mediated reactive oxygen species production and upregulation of store‐operated calcium channel subunits, Scand. J. Immunol. 78 (2013) 35–43.
Crossref Google Scholar
[10]

S. Toyoshima, E. Wakamatsu, Y. Ishida, Y. Obata, Y. Kurashima, et al., The spleen is the site where mast cells are induced in the development of food allergy, Int. Immunol. (2017) 29.
Crossref Google Scholar
[11]

K. Nagai, T. Fukushima, H. Oike, M. Kobori, High glucose increases the expression of proinflammatory cytokines and secretion of TNFα and β-hexosaminidase in human mast cells, Eur. J. Pharmacol. 687 (2012) 39.
Crossref Google Scholar
[12]

Q. Zhang, Y. Xiong, G.B. Li, Q. Tang, M. Cao, et al., Xinqin exhibits the anti-allergic effect through the JAK2/STAT5 signaling pathway, J. Ethnopharmacol. 193 (2016) 466–473.
Crossref Google Scholar
[13]

N.R. Han, P.D. Moon, K.J. Ryu, J.B. Jang, H.M. Kim, et al., β‐Eudesmol suppresses allergic reactions via inhibiting mast cell degranulation, Clin. Exp. Pharmacol. Physiol. (2016) 44.
Crossref Google Scholar
[14]

K. Obata, K. Mukai, Y. Tsujimura, K. Ishiwata, Y. Kawano, et al., Basophils are essential initiators of a novel type of chronic allergic inflammation, Blood 110 (2007) 913.
Crossref Google Scholar
[15]

H. Karasuyama, K. Obata, T. Wada, Y. Tsujimura, K. Mukai, Newly appreciated roles for basophils in allergy and protective immunity, Allergy 66 (2011) 1133–1141.
Crossref Google Scholar
[16]

K. Mukai, K. Matsuoka, C. Taya, H. Suzuki, H. Yokozeki, et al., Basophils play a critical role in the development of IgE-mediated chronic allergic inflammation independently of t cells and mast cells, Immunity 23 (2005) 191–202.
Crossref Google Scholar
[17]

D. Mittag, J. Akkerdaas, B.K. Ballmer-Weber, L. Vogel, M. Wensing, et al., Ara h 8, a Bet v 1–homologous allergen from peanut, is a major allergen in patients with combined birch pollen and peanut allergy, J. Allergy Clin. Immunol. 114 (2004) 1410–1417.
Crossref Google Scholar
[18]

M. Toda, G. Reese, G. Gadermaier, V. Schulten, I. Lauer, et al., Protein unfolding strongly modulates the allergenicity and immunogenicity of Pru p 3, the major peach allergen, J. Allergy Clin. Immunol. 128 (2011) 1–7.
Crossref Google Scholar
[19]

A.D. Crockard, M. Ennis, Basophil histamine release tests in the diagnosis of allergy and asthma, Clin. Exp. Allergy 31 (2001) 345–350.
Crossref Google Scholar
[20]

E. Passante, N. Frankish, The RBL-2H3 cell line: its provenance and suitability as a model for the mast cell, Inflamm. Res. 58 (2009) 737–745.
Crossref Google Scholar
[21]

S. Kaul, A. Hoffmann, Mediator release assay of rat basophil leukemia cells as alternative for passive cutaneous anaphylaxis testing (PCA) in laboratory animals, Altex 18 (2001) 55.
Google Scholar
[22]

M. Bodinier, C. Brossard, S. Triballeau, M. Morisset, C. Guã©Rin-Marchand, et al., Evaluation of an in vitro mast cell degranulation test in the context of food allergy to wheat, Int. Arch. Allergy Immunol. 146 (2008) 307–320.
Crossref Google Scholar
[23]

H. Hochwallner, U. Schulmeister, I. Swoboda, N. Balic, B. Geller, et al., Microarray and allergenic activity assessment of milk allergens, Clin. Exp. Allergy J. Br. Soc. Allergy Clin. Immunol. 40 (2010) 1809–1818.
Crossref Google Scholar
[24]

G.S. Ladics, J.H. van Bilsen, H.M. Brouwer, L. Vogel, S. Vieths, et al., Assessment of three human FcepsilonRI-transfected RBL cell-lines for identifying IgE induced degranulation utilizing peanut-allergic patient sera and peanut protein extract, Regul. Toxicol. Pharmacol. 51 (2008) 288–294.
Crossref Google Scholar
[25]

D.D. Jr, G.W. Palmer, P.B. Williams, S.A. Bock, S.C. Dreskin, RBL cells expressing human Fc epsilon RI are a sensitive tool for exploring functional IgE-allergen interactions: studies with sera from peanut-sensitive patients, J. Immunol. Methods 274 (2003) 37–45.
Crossref Google Scholar
[26]

L. Vogel, D. Lüttkopf, L. Hatahet, D. Haustein, S. Vieths, Development of a functional in vitro assay as a novel tool for the standardization of allergen extracts in the human system, Allergy 60 (2005) 1021–1028.
Crossref Google Scholar
[27]

H.S. Porterfield, K.S. Murray, D.G. Schlichting, X. Chen, K.C. Hansen, et al., Effector activity of peanut allergens: a critical role for Ara h 2, Ara h 6, and their variants, Clin. Exp. Allergy 39 (2010) 1099–1108.
Crossref Google Scholar
[28]

T. Liu, S. Navarro, A.L. Lopata, Current advances of murine models for food allergy, Mol. Immunol. 70 (2016) 104–117.
Crossref Google Scholar
[29]

J.C. Kips, G.P. Anderson, J.J. Fredberg, U. Herz, M.D. Inman, et al., Murine models of asthma, Eur. Respir. J. Eur. Respir. J. 22 (2003) 374–382.
Crossref Google Scholar
[30]

C. Zhou, T. Ludmila, N. Sun, C. Wang, Q. Pu, et al., BALB/c mice can be used to evaluate allergenicity of different food protein extracts, Food Agric. Immunol. 27 (2016) 589–603.
Crossref Google Scholar
[31]

K. Thomas, S. Macintosh, G. Bannon, C. Herouetguicheney, M. Holsapple, et al., Scientific advancement of novel protein allergenicity evaluation: an overview of work from the HESI Protein Allergenicity Technical Committee (2000-2008), Food Chem. Toxicol. 47 (2009) 1041–1050.
Crossref Google Scholar
[32]

P. Rupa, S. Nakamura, Y. Mine, Genetically glycosylated ovomucoid third domain can modulate Immunoglobulin E antibody production and cytokine response in BALB/c mice, Clin. Exp. Allergy J. Br. Soc. Allergy Clin. Immunol. 37 (2007) 918–928.
Crossref Google Scholar
[33]

C.Y. Sun, J. Bai, T.Y. Hu, B.H. Cheng, L. Ma, et al., CD4+ T cell responses in Balb/c mice with food allergy induced by trinitrobenzene sulfonic acid and ovalbumin, Mol. Med. Rep. 13 (2016) 5349–5357.
Crossref Google Scholar
[34]

J. Fotschki, A.M. Szyc, J.M. Laparra, L.H. Markiewicz, B. Wrã3Blewska, Immune-modulating properties of horse milk administered to mice sensitized to cow milk, J. Dairy Sci. 99 (2016) 9395–9404.
Crossref Google Scholar
[35]

S. Wavrin, H. Bernard, J.M. Wal, K. Adelpatient, Cutaneous or respiratory exposures to peanut allergens in mice and their impacts on subsequent oral exposure, Int. Arch. Allergy Immunol. 164 (2014) 189.
Crossref Google Scholar
[36]

F. Gizzarelli, S. Corinti, B. Barletta, P. Iacovacci, B. Brunetto, et al., Evaluation of allergenicity of genetically modified soybean protein extract in a murine model of oral allergen-specific sensitization, Clin. Exp. Allergy 36 (2006) 238–248.
Crossref Google Scholar
[37]

A.C. Gomes-Santos, R.C. Fonseca, L. Lemos, D.S. Reis, T.G. Moreira, et al., Hydrolyzed whey protein prevents the development of food allergy to β-lactoglobulin in sensitized mice, Cell. Immunol. 298 (2015) 47–53.
Crossref Google Scholar
[38]

V. Morafo, K. Srivastava, C.K. Huang, G. Kleiner, S.Y. Lee, et al., Genetic susceptibility to food allergy is linked to differential TH2-TH1 responses in C3H/HeJ and BALB/c mice, J. Allergy Clin. Immunol. 111 (2003) 1122–1128.
Crossref Google Scholar
[39]

R.J. Dearman, H. Caddick, S. Stone, J.G. Kenna, D.A. Basketter, et al., Immunogenic properties of rapidly digested food proteins following gavage exposure of mice: a comparison of ovalbumin with a potato acid phosphatase preparation, Food Chem. Toxicol. 40 (2002) 625–633.
Crossref Google Scholar
[40]

X.M. Li, B.H. Schofield, C.K. Huang, G.I. Kleiner, H.A. Sampson, A murine model of IgE-mediated cow's milk hypersensitivity, J. Allergy Clin. Immunol. 103 (1999) 206.
Crossref Google Scholar
[41]

R.J. Dearman, I. Kimber, Animal models of protein allergenicity: potential benefits, pitfalls and challenges, Clin. Exp. Allergy 39 (2009) 458.
Crossref Google Scholar
[42]

A. Poltorak, X. He, I. Smirnova, M.Y. Liu, H.C. Van, et al., Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene, Science 282 (1998) 2085.
Crossref Google Scholar
[43]

M.E. Bashir, S. Louie, H.N. Shi, C. Nagler-Anderson, Toll-like receptor 4 signaling by intestinal microbes influences susceptibility to food allergy, J. Immunol. 172 (2004) 6978.
Crossref Google Scholar
[44]

F. Capobianco, C. Butteroni, B. Barletta, S. Corinti, C. Afferni, et al., Oral sensitization with shrimp tropomyosin induces in mice allergen-specific IgE, T cell response and systemic anaphylactic reactions, Int. Immunol. 20 (2008) 1077.
Crossref Google Scholar
[45]

M.C. Berin, Y. Zheng, M. Domaradzki, X.M. Li, H.A. Sampson, Role of TLR4 in allergic sensitization to food proteins in mice, Allergy 61 (2006) 64–71.
Crossref Google Scholar
[46]

D. Dunkin, M.C. Berin, L. Mayer, Allergic sensitization can Be induced via multiple physiologic routes in an adjuvant-dependent manner, J. Allergy Clin. Immunol. 128 (2011) 1251.
Crossref Google Scholar
[47]

B.C. van Esch, J.H. van Bilsen, P.V. Jeurink, J. Garssen, A.H. Penninks, et al., Interlaboratory evaluation of a cow's milk allergy mouse model to assess the allergenicity of hydrolysed cow's milk based infant formulas, Toxicol. Lett. 220 (2013) 95–102.
Crossref Google Scholar
[48]

A. Monica, B. Thomas, W. Odd-Gunnar, B. Johanna, T. Terje, et al., Investigations of immunogenic, allergenic and adjuvant properties of Cry1Ab protein after intragastric exposure in a food allergy model in mice, BMC Immunol. 17 (2016) 10.
Crossref Google Scholar
[49]

C.A. Lino, A.K. Batista, M.A. Soares, A.E. de Freitas, L.C. Gomes, et al., Bronchiolitis obliterans: clinical and radiological profile of children followed-up in a reference outpatient clinic, Rev. Paul. Pediatr. 31 (2013) 10–16.
Crossref Google Scholar
[50]

L. Knippels, M.M. Penninks, G. Houben, Humoral and cellular immune responses in different rat strains on oral exposure to ovalbumin, Food Chem. Toxicol. Int. J. Publ. Br. Ind. Biol. Res. Assoc. 37 (1999) 881–888.
Crossref Google Scholar
[51]

H. Akiyama, R. Teshima, J.I. Sakushima, H. Okunuki, Y. Goda, et al., Examination of oral sensitization with ovalbumin in Brown Norway rats and three strains of mice, Immunol. Lett. 78 (2001) 1–5.
Crossref Google Scholar
[52]

M. Abril-Gil, A. Garcia-Just, F.J. Pã©Rez-Cano, Ã Franch, M. Castell, Development and characterization of an effective food allergy model in Brown Norway rats, PLoS One 10 (2015) e0125314.
Crossref Google Scholar
[53]

R. Chen, X. Tang, B. Fan, J. Liu, X. Jia, et al., IgG expression upon oral sensitization in association with maternal exposure to Ovalbumin, PLoS One 11 (2016) e0148251.
Crossref Google Scholar
[54]

K.L. Bøgh, V. Barkholt, C.B. Madsen, Characterization of the immunogenicity and allergenicity of two cow's milk hydrolysates – a study in brown norway rats, Scand. J. Immunol. 81 (2015) 274–283.
Crossref Google Scholar
[55]

N.D. Meeker, N.S. Trede, Immunology and zebrafish: spawning new models of human disease, Dev. Comp. Immunol. 32 (2008) 745–757.
Crossref Google Scholar
[56]
B. Novoa, A. Figueras, Zebrafish: Model for the Study of Inflammation and the Innate Immune Response to Infectious Diseases, Springer, New York, 2012, pp. 253–275.
Crossref
[57]

Y.J. Li, B. Hu, Establishment of multi-site infection model in zebrafish larvae for studying Staphylococcus aureus infectious disease, J. Genet. Genom. 39 (2012) 521–534.
Crossref Google Scholar
[58]

X. Xu, L. Zhang, S. Weng, Z. Huang, J. Lu, et al., A zebrafish (Danio rerio) model of infectious spleen and kidney necrosis virus (ISKNV) infection, Virology 376 (2008) 1–12.
Crossref Google Scholar
[59]
P.E. Phelan, M.E. Pressley, P.E. Witten, M.T. Mellon, S. Blake, et al., Characterization of Viral Infection With Snakehead Rhabdovirus in Zebrafish, 2005.
[60]

L. Wang, L. Wang, H.X. Zhang, J.H. Zhang, W.H. Chen, et al., In vitro effects of recombinant zebrafish IFN on spring viremia of carp virus and infectious hematopoietic necrosis virus, J. Interferon Cytokine Res. Off. J. Int. Soc. Interferon Cytokine Res. 26 (2006) 256.
Crossref Google Scholar
[61]

M.W. Lu, Y.M. Chao, T.C. Guo, N. Santi, O. Evensen, et al., The interferon response is involved in nervous necrosis virus acute and persistent infection in zebrafish infection model, Mol. Immunol. 45 (2008) 1146–1152.
Crossref Google Scholar
[62]

J.L. Turnbull, H.N. Adams, D.A. Gorard, Review article: the diagnosis and management of food allergy and food intolerances, Aliment. Pharmacol. Ther. 41 (2015) 3–25.
Crossref Google Scholar
[63]

K. Soares-Weiser, Y. Takwoingi, S.S. Panesar, A. Muraro, T. Werfel, et al., The diagnosis of food allergy: a systematic review and meta‐analysis, Allergy 69 (2014) 76–86.
Crossref Google Scholar
[64]

C. Caffarelli, A. Dondi, C.P. Dascola, G. Ricci, Skin prick test to foods in childhood atopic eczema: pros and cons, Ital. J. Pediatr. 39 (2013), 48-48.
Crossref Google Scholar
[65]
U. Darsow, M. Gjomarkaj, S. W??Hrl, S. Durham, T. Haahtela, et al., The skin prick test??? European standards, Clin. Transl. Allergy 3 (1) (2013) (2013-02-01) 3: 3.
Crossref
[66]

H.A. Sampson, Update on food allergy, J. Allergy Clin. Immunol. 70 (2016) 1511–1520.
Crossref Google Scholar
[67]

M.P. Borres, N. Maruyama, S. Sato, M. Ebisawa, Recent advances in component resolved diagnosis in food allergy, Allergol. Int. 65 (2016) 378–387.
Crossref Google Scholar
[68]

L.S. Ford, K.A. Bloom, A.H. Nowakwęgrzyn, W.G. Shreffler, M. Masilamani, et al., Basophil reactivity, wheal size and immunoglobulin levels distinguish degree of cow's milk tolerance, J. Allergy Clin. Immunol. 131 (2013) 180–U261.
Crossref Google Scholar
[69]

J.C. Caubet, A. Nowakwęgrzyn, E. Moshier, J. Godbold, J. Wang, et al., Utility of casein-specific IgE levels in predicting reactivity to baked milk, J. Allergy Clin. Immunol. 131 (222-224) (2013) e224.
Crossref Google Scholar
[70]

G.J. Sturm, A. Heinemann, C. Schuster, M. Wiednig, A. Groseljstrele, et al., Influence of total IgE levels on the severity of sting reactions in Hymenoptera venom allergy, Allergy 62 (2010) 884–889.
Crossref Google Scholar
[71]

K. Horimukai, K. Hayashi, Y. Tsumura, I. Nomura, M. Narita, et al., Total serum IgE level influences oral food challenge tests for IgE‐mediated food allergies, Allergy 70 (2015) 334–337.
Crossref Google Scholar
[72]

A. Muraro, T. Werfel, K. Hoffmannsommergruber, G. Roberts, K. Beyer, et al., EAACI food allergy and anaphylaxis guidelines: diagnosis and management of food allergy, Allergy 69 (2014) 1008–1025.
Crossref Google Scholar
[73]

A. Nowak-Wegrzyn, Adverse reactions to food committee of american academy of allergy, asthma & immunology. work group report : oral food challenge testing, J. Allergy Clin. Immunol. (2009) 123.
Crossref Google Scholar
[74]

X. Hong, K. Hao, C. Laddacosta, K.D. Hansen, H.J. Tsai, et al., Genome-wide association study identifies peanut allergy-specific loci and evidence of epigenetic mediation in US children, Nat. Commun. 6 (2015) 6304.
Crossref Google Scholar
[75]

X. Liu, X. Hong, H.J. Tsai, K.K. Mestan, M. Shi, et al., Genome-wide association study of maternal genetic effects and parent-of-origin effects on food allergy, Medicine 97 (9) (2018) e0043.
Crossref Google Scholar
[76]

S.H. Sicherer, R.A. Wood, Advances in diagnosing peanut allergy, J. Allergy Clin. Immunol. Pract. 1 (2013) 1–13.
Crossref Google Scholar
[77]

D. Mittag, S. Vieths, L. Vogel, W.M. Becker, H.P. Rihs, et al., Soybean allergy in patients allergic to birch pollen: clinical investigation and molecular characterization of allergens, J. Allergy Clin. Immunol. 113 (2004) 148–154.
Crossref Google Scholar
[78]

A.H. Benhamou, J.C. Caubet, P.A. Eigenmann, A. Nowak-Wegrzyn, C.P. Marcos, et al., State of the art and new horizons in the diagnosis and management of egg allergy, Allergy 65 (2010) 283–289.
Crossref Google Scholar
[79]

A. Nowak-Wegrzyn, A. Fiocchi, Rare, medium, or well done? The effect of heating and food matrix on food protein allergenicity, Curr. Opin. Allergy Clin. Immunol. 9 (2009) 234–237.
Crossref Google Scholar
[80]

S.L. Taylor, Molluscan shellfish allergy, Adv. Food Nutr. Res. 54 (2008) 139.
Crossref Google Scholar
[81]

J.E. Morgan, C.E. O'Neil, C.B. Daul, S.B. Lehrer, Species-specific shrimp allergens: RAST and RAST-inhibition studies, J. Allergy Clin. Immunol. 83 (1989) 1112–1117.
Crossref Google Scholar
[82]

L. Masthoff, L. Mattsson, L. Zuidmeerjongejan, J. Lidholm, K. Andersson, et al., Sensitization to cor a 9 and Cor a 14 is highly specific for a severe hazelnut allergy in Dutch children and adults, Clin. Transl. Allergy 132 (2013) 393–399.
Crossref Google Scholar
[83]

H. Matsuo, J. Dahlström, A. Tanaka, K. Kohno, H. Takahashi, et al., Sensitivity and specificity of recombinant omega-5 gliadin-specific IgE measurement for the diagnosis of wheat-dependent exercise-induced anaphylaxis, Allergy 63 (2010) 233–236.
Crossref Google Scholar
Food Science and Human Wellness
Volume 7 Issue 4,
December 2018
Pages 235-243
DOI: 10.1016/j.fshw.2018.10.002
Cite this article:
Huang J, Liu C, Wang Y, et al. Application of in vitro and in vivo models in the study of food allergy. Food Science and Human Wellness, 2018, 7(4): 235-243. https://doi.org/10.1016/j.fshw.2018.10.002

475

Views

15

Downloads

36

Crossref

N/A

Web of Science

40

Scopus

0

CSCD

Altmetrics
Received: 11 July 2018
Revised: 12 October 2018
Accepted: 15 October 2018
Published: 24 October 2018
© 2018 “Society information”.

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Return