Differential expression of anti-glycan antibodies in schistosome-infected humans, rhesus monkeys and mice

Glycobiology

Volumn 24, Issue 7, 2014, Pages 602-608

  Luyai, Anthony E ^a,e   Heimburg Molinaro, Jamie ^a   Prasanphanich, Nina Salinger ^a   Mickum, Megan L ^a   Lasanajak, Yi ^a   Song, Xuezheng ^a   Nyame, A Kwame ^b   Wilkins, Patricia ^c   Rivera Marrero, Carlos A ^c   Smith, David F ^a   Van Die, Irma ^d   Secor, W Evan ^c   Cummings, Richard D ^a  

^a EMORY UNIVERSITY SCHOOL OF MEDICINE   (United States)

^b UNIVERSITY OF MARYLAND   (United States)

^c CENTERS FOR DISEASE CONTROL AND PREVENTION   (United States)

^d ACADEMIC MEDICAL CENTER   (Netherlands)

^e Ihrc Inc   (United States)

Author keywords

core xylose core 3 fucose; glycan microarrays; glycans; schistosomiasis; schistosomula killing

Indexed keywords

ANTIBODY; EPITOPE; FUCOSE; GLYCAN ANTIBODY; GLYCOPEPTIDE; IMMUNOGLOBULIN G; IMMUNOGLOBULIN M; LECTIN; PRAZIQUANTEL; UNCLASSIFIED DRUG; XYLOSE; HELMINTH ANTIBODY; LACTOSE; N-ACETYLGALACTOSAMINYL-1-4-N-ACETYLGLUCOSAMINE;

ANIMAL EXPERIMENT; ANIMAL MODEL; ANTIBODY BLOOD LEVEL; ANTIBODY RESPONSE; ANTIGEN BINDING; ARTICLE; CARBOHYDRATE ANALYSIS; CONTROLLED STUDY; HUMORAL IMMUNITY; MICROARRAY ANALYSIS; MOUSE; NONHUMAN; PRIORITY JOURNAL; PROTEIN EXPRESSION; RHESUS MONKEY; SCHISTOSOMA MANSONI; SCHISTOSOMIASIS; ADULT; ANALOGS AND DERIVATIVES; ANIMAL; HUMAN; IMMUNOLOGY; SCHISTOSOMIASIS MANSONI; SPECIES DIFFERENCE;

ADULT; ANIMALS; ANTIBODIES, HELMINTH; EPITOPES; HUMANS; IMMUNOGLOBULIN G; LACTOSE; MACACA MULATTA; MICE; PRAZIQUANTEL; SCHISTOSOMIASIS MANSONI; SPECIES SPECIFICITY;

EID: 84901777062     PISSN: 09596658     EISSN: 14602423     Source Type: Journal    
DOI: 10.1093/glycob/cwu029     Document Type: Article

References (88)

1. Abdul-Ghani RA, Hassan AA. 2010. Murine schistosomiasis as a model for human schistosomiasis mansoni: Similarities and discrepancies. Parasitol Res. 107:1-8.
2. Altmann F. 2007. The role of protein glycosylation in allergy. Int Arch Allergy Immunol. 142:99-115. (Pubitemid 46095922)
3. Alvarez RA, Blixt O. 2006. Identification of ligand specificities for glycanbinding proteins using glycan arrays. Methods Enzymol. 415:292-310. (Pubitemid 44751104)
4. Aranzamendi C, Tefsen B, Jansen M, Chiumiento L, Bruschi F, Kortbeek T, Smith DF, Cummings RD, Pinelli E, Van Die I. 2011. Glycan microarray profiling of parasite infection sera identifies the LDNF glycan as a potential antigen for serodiagnosis of trichinellosis. Exp Parasitol. 129:221-226.
5. Baenziger JU, Fiete D. 1979. Structural determinants of concanavalin A specificity for oligosaccharides. J Biol Chem. 254:2400-2407.
6. Blixt O, Head S, Mondala T, Scanlan C, Huflejt ME, Alvarez R, Bryan MC, Fazio F, Calarese D, Stevens J, et al. 2004. Printed covalent glycan array for ligand profiling of diverse glycan binding proteins. Proc Natl Acad Sci USA. 101:17033-17038. (Pubitemid 39627769)
7. de Boer AR, Hokke CH, Deelder AM, Wuhrer M. 2007. General microarray technique for immobilization and screening of natural glycans. Anal Chem. 79:8107-8113. (Pubitemid 350060007)
8. de Boer AR, Hokke CH, Deelder AM, Wuhrer M. 2008. Serum antibody screening by surface plasmon resonance using a natural glycan microarray. Glycoconj J. 25:75-84.
9. Brenckle R, Kornfeld R. 1980. Structure of the oligosaccharides of mouse immunoglobulin M secreted by the MOPC 104E plasmacytoma. Arch Biochem Biophys. 201:160-173. (Pubitemid 11190268)
10. Brewer CF, Bhattacharyya L. 1986. Specificity of concanavalin A binding to asparagine-linked glycopeptides. A nuclear magnetic relaxation dispersion study. J Biol Chem. 261:7306-7310. (Pubitemid 17224729)
11. Cheever AW. 1969. Quantitative comparison of the intensity of Schistosoma manosni infections and man and experimental animals. Trans R Soc Trop Med Hyg. 63:781-795.
12. Cheever AW, Powers KG. 1969. Schistosoma mansoni infection in rhesus monkeys: Changes in egg production and egg distribution in prolonged infections in intact and splenectomized monkeys. Ann Trop Med Parasitol. 63:83-93.
13. Chitsulo L, Loverde P, Engels D. 2004. Schistosomiasis. Nat Rev Microbiol. 2:12-13. (Pubitemid 39490043)
14. Cioli D, Pica-Mattoccia L. 2003. Praziquantel. Parasitol Res. 90(Supp 1): S3-S9. (Pubitemid 36831287)
15. Clegg JA, Smithers SR. 1972. The effects of immune rhesus monkey serum on schistosomula of Schistosoma manosni during cultivation in vitro. Int J Parasitol. 2:79-98.
16. Clegg JA, Smithers SR, Terry RJ. 1971. Concomitant immunity and host antigens associated with schistosomiasis. Int J Parasitol. 1:43-49.
17. Correa-Oliveira R, Pearce EJ, Oliveira GC, Golgher DB, Katz N, Bahia LG, Carvalho OS, Gazzinelli G, Sher A. 1989. The human immune response to defined immunogens of Schistosoma mansoni: Elevated antibody levels to paramyosin in stool-negative individuals from two endemic areas in Brazil. Trans R Soc Trop Med Hyg. 83:798-804. (Pubitemid 20004495)
18. Cummings RD. 1994. Use of lectins in analysis of glycoconjugates. Methods Enzymol. 230:66-86. (Pubitemid 24080128)
19. Cummings RD, Etzler ME. 2009. Antibodies and lectins in glycan analysis. In: Varki A, Cummings RD, Esko JD, Freeze HH, Stanley P, Bertozzi CR, Hart GW, Etzler ME, editors. Essentials of Glycobiology. Boston: Cold Spring Harbor Laboratory Press.
20. Cummings RD, Nyame AK. 1999. Schistosome glycoconjugates. Biochim Biophys Acta. 1455:363-374. (Pubitemid 29436001)
21. Dean DA. 1983. Schistosoma and related genera: Acquired resistance in mice. Exp Parasitol. 55:1-104. (Pubitemid 13130666)
22. Debeire P, Montreuil J, Moczar E, van Halbeek H, Vliegenthart JF. 1985. Primary structure of two major glycans of bovine fibrinogen. Eur J Biochem. 151:607-611. (Pubitemid 16215797)
23. Dell A, Morris HR, Easton RL, Panico M, Patankar M, Oehniger S, Koistinen R, Koistinen H, Seppala M, Clark GF. 1995. Structural analysis of the oligosaccharides derived from glycodelin, a human glycoprotein with potent immunosuppressive and contraceptive activities. J Biol Chem. 270: 24116-24126.
24. Demeure CE, Rihet P, Abel L, Ouattara M, Bourgois A, Dessein AJ. 1993. Resistance to Schistosoma mansoni in humans: Influence of the IgE/IgG4 balance and IgG2 in immunity to reinfection after chemotherapy. J Infect Dis. 168:1000-1008. (Pubitemid 23282577)
25. De Vries T, Palcic MP, Schoenmakers PS, Van Den Eijnden DH, Joziasse DH. 1997. Acceptor specificity of GDP-Fuc:Gal beta 1->4GlcNAc-R alpha 3-fucosyltransferase VI (FucT VI) expressed in insect cells as soluble, secreted enzyme. Glycobiology. 7:921-927. (Pubitemid 27455658)
26. van Die I, Gomord V, Kooyman FNJ, Berg TKVD, Cummings RD, Vervelde L. 1999. Core alpha1,3-fucose is a common modification of N-glycans in parasitic helminths and constitutes an important epitope for IgE from Haemonchus contortus infected sheep. FEBS Lett. 463:189-193. (Pubitemid 30001745)
27. van Diepen A, Smit CH, van Egmond L, Kabatereine NB, Pinot de Moira A, Dunne DW, Hokke CH. 2012. Differential anti-glycan antibody responses in Schistosoma mansoni-infected children and adults studied by shotgun glycan microarray. PLoS Neglect Trop Dis. 6:e1922.
28. Dissous C, Grzych JM, Capron A. 1982. Schistosoma mansoni surface antigen define by a rat monoclonal IgGa. J Immunol. 129:2232-2234. (Pubitemid 12004400)
29. Dubois M, Gilles K, Hamilton JK, Rebers PA, Smith F. 1956. A colorimetric method for the determination of sugars. Nature. 168:167.
30. Dunne DW, Butterworth AE, Fulford AJ, Kariuki HC, Langley JG, Ouma JH, Capron A, Pierce RJ, Sturrock RF. 1992. Immunity after treatment of human schistosomiasis: Association between IgE antibodies to adult worm antigens and resistance to reinfection. Eur J Immunol. 22:1483-1494.
31. Eberl M, Langermans JA, Vervenne RA, Nyame AK, Cummings RD, Thomas AW, Coulson PS, Wilson RA. 2001. Antibodies to glycans dominate the host response to schistosome larvae and eggs: Is their role protective or subversive? J Infect Dis. 183:1238-1247. (Pubitemid 32281563)
32. Ellis LA, Reason AJ, Morris HR, Dell A, Iglesias R, Ubeira FM, Appleton JA. 1994. Glycans as targets for monoclonal antibodies that protect rats against Trichinella spiralis. Glycobiology. 4:585-592. (Pubitemid 24327723)
33. Faveeuw C, Mallevaey T, Paschinger K, Wilson IB, Fontaine J, Mollicone R, Oriol R, Altmann F, Lerouge P, CapronM, et al. 2003. Schistosome N-glycans containing core alpha 3-fucose and core beta 2-xylose epitopes are strong inducers of Th2 responses in mice. Eur J Immunol. 33:1271-1281. (Pubitemid 36609145)
34. Gryseels B, Polman K, Clerinx J, Kestens L. 2006. Human schistosomiasis. Lancet. 368:1106-1118. (Pubitemid 44402679)
35. Grzych JM, Capron M, Lambert PH, Dissous C, Torres S, Capron A. 1985. An anti-idiotype vaccine against experimental schistosomiasis. Nature. 316: 74-76. (Pubitemid 15013717)
36. Grzych J-M, Dissous C, Capron M, Torres S, Lambert P-H, Capron A. 1987. Schistosoma mansoni shares a protective carbohydrate epitope with keyhole limpet hemocyanin. J Exp Med. 165:865-878. (Pubitemid 17059458)
37. Hagan P, Blumenthal UJ, Dunn D, Simpson AJ, Wilkins HA. 1991. Human IgE, IgG4 and resistance to reinfection with Schistosoma haematobium. Nature. 349:243-245. (Pubitemid 21912056)
38. Harn DA, Mitsuyama M, David JR. 1984. Schistosoma manosoni anti-egg monoclonal antibodies protect against cercarial challenge in vivo. J Exp Med. 159:1371-1387. (Pubitemid 14106455)
39. Heimburg-Molinaro J, Song X, Smith DF, Cummings RD. 2011. Preparation and analysis of glycan microarrays. Curr Prot Protein Sci. Chapter 12.10:1-33.
40. Hokke CH, Yazdanbakhsh M. 2005. Schistosome glycans and innate immunity. Parasite Immunol. 27:257-264. (Pubitemid 41344617)
41. Hotez PJ, Bethony JM, Diemert DJ, Pearson M, Loukas A. 2010. Developing vaccines to combat hookworm infection and intestinal schistosomiasis. Nat Rev Microbiol. 8:814-826.
42. Hotez PJ, Ferris MT. 2006. The antipoverty vaccines. Vaccine. 24:5787-5799. (Pubitemid 43947393)
43. Kabatereine NB, Vennervald BJ, Ouma JH, Kemijumbi J, Butterworth AE, Dunne DW, Fulford AJ. 1999. Adult resistance to schistosomiasis mansoni: Age-dependence of reinfection remains constant in communities with diverse exposure patterns. Parasitology. 118 (Pt 1):101-105. (Pubitemid 29043568)
44. Karanja DM, Hightower AW, Colley DG, Mwinzi PN, Galil K, Andove J, Secor WE. 2002. Resistance to reinfection with Schistosoma mansoni in occupationally exposed adults and effect of HIV-1 co-infection on susceptibility to schistosomiasis: A longitudinal study. Lancet. 360:592-596. (Pubitemid 35232192)
45. Kaulfuerst-Soboll H, Rips S, Koiwa H, Kajiura H, Fujiyama K, von Schaewen A. 2011. Reduced immunogenicity of Arabidopsis hybrid glycosylation1 (hgl1) N-glycans due to altered accessibility of xylose and core fucose epitopes. J Biol Chem. 286:22955-22964.
46. Kawar ZS, Van Die I, Cummings RD. 2002. Molecular cloning and enzymatic characterization of a UDP-GalNAc:GlcNAc(beta)-R beta1,4-N-acetylgalac tosaminyltransferase from Caenorhabditis elegans. J Biol Chem. 277: 34924-34932.
47. Khoo K-h, Chatteriee D, Caulfield JP, Morris HR, Dell A. 1997. Structural mapping of the glycans from the egg glycoproteins of Schistosoma mansoni and Schistosoma japonicum: Identification of novel core structures and terminal sequences. Glycobiology. 7:663-677. (Pubitemid 27308494)
48. Khoo KH, Huang HH, Lee KM. 2001. Characteristic structural features of schistosome cercarial N-glycans: Expression of Lewis X and core xylosylation. Glycobiology. 11:149-163.
49. King CH, Dangerfield-Cha M. 2008. The unacknowledged impact of chronic schistosomiasis. Chronic Illn. 4:65-79. (Pubitemid 351341878)
50. Ko AI, Dräger UC, Harn DA. 1990. A Schistosoma mansoni epitope recognized by a protective monoclonal antibody is identical to the stage-specific embryonic antigen 1. Proc Natl Acad Sci USA. 87:4159-4163.
51. Kochibe N, Furukawa K. 1980. Purification and properties of a novel fucosespecific hemagglutinin of Aleuria aurantia. Biochemistry. 19:2841-2846.
52. Kupferschmidt K. 2013. Coming at a snail's pace. Science. 339:502-503.
53. Lamberton PH, Hogan SC, Kabatereine NB, Fenwick A, Webster JP. 2010. In vitro praziquantel test capable of detecting reduced in vivo efficacy in Schistosoma mansoni human infections. Am J Trop Med Hyg. 83: 1340-1347.
54. Lazdins JK, Stein MJ, David JR, Sher A. 1982. Schistosoma mansoni: Rapid isolation and purification of schistosomula of different developmental stages by centrifugation on discontinuous density gradients of Percoll. Exp Parasitol. 53:39-44. (Pubitemid 12148205)
55. Mandalasi M, Dorabawila N, Smith DF, Heimburg-molinaro J, Nyame AK, Cummings RD. 2013. Development and characterization of a specific IgG monoclonal antibody toward the Lewis x antigen using splenocytes of Schistosoma mansoni infected mice. Glycobiology. 23:877-892.
56. Marques ET, Jr, Ichikawa Y, Strand M, August JT, Hart GW, Schnaar RL. 2001. Fucosyltransferases in Schistosoma mansoni development. Glycobiology. 11:249-259. (Pubitemid 32472000)
57. McHugh SM, Coulson PS, Wilson RA. 1987. Pathologically induced alterations in the dimensions of the hepatic portal vasculature of mice infected with Schistosoma mansoni. Parasitology. 94 (Pt 1):69-80. (Pubitemid 17013468)
58. Meevissen MHJ, Balog CIA, Koeleman CAM, Doenhoff MJ, Schramm G, Haas H, Deelder AM, Wuhrer M, Hokke CH. 2011. Targeted glycoproteomic analysis reveals that kappa-5 is a major, uniquely glycosylated component of Schistosoma mansoni egg antigens. Mol Cell Proteomics. 10: M110.005710.
59. Naus CW, van Remoortere A, Ouma JH, Kimani G, Dunne DW, Kamerling JP, Deelder AM, Hokke CH. 2003. Specific antibody responses to three schistosome-related carbohydrate structures in recently exposed immigrants and established residents in an area of Schistosoma mansoni endemicity. Infect Immun. 71:5676-5681. (Pubitemid 37205620)
60. Nyame AK, Kawar ZS, Cummings RD. 2004. Antigenic glycans in parasitic infections: Implications for vaccines and diagnostics. Arch Biochem Biophys. 426:182-200. (Pubitemid 38680616)
61. Nyame AK, Leppanen AM, Bogitsh BJ, Cummings RD. 2000. Antibody responses to the fucosylated LacdiNAc glycan antigen in Schistosoma mansoni-infected mice and expression of the glycan among schistosomes. Exp Parasitol. 96:202-212. (Pubitemid 32195398)
62. Nyame AK, Leppanen AM, DeBose-Boyd R, Cummings RD. 1999. Mice infected with Schistosoma mansoni generate antibodies to LacdiNAc (GalNAc beta 1->4GlcNAc) determinants. Glycobiology. 9:1029-1035. (Pubitemid 29473960)
63. Nyame AK, Lewis FA, Doughty BL, Correa-Oliveira R, Cummings RD. 2003. Immunity to schistosomiasis: Glycans are potential antigenic targets for immune intervention. Exp Parasitol. 104:1-13. (Pubitemid 41123180)
64. Nyame AK, Pilcher JB, Tsang VC, Cummings RD. 1996. Schistosoma mansoni infection in humans and primates induces cytolytic antibodies to surface Le(x) determinants on myeloid cells. Exp Parasitol. 82:191-200.
65. Ohsuga H, Su SN, Takahashi N, Yang SY, Nakagawa H, Shimada I, Arata Y, Lee YC. 1996. The carbohydrate moiety of the bermuda grass antigen BG60. New oligosaccharides of plant origin. J Biol Chem. 271:26653-26658. (Pubitemid 26359069)
66. Pearce EJ, MacDonald AS. 2002. The immunobiology of schistosomiasis. Nat Rev Immunol. 2:499-511. (Pubitemid 37328741)
67. Prasanphanich NS, Luyai A, Song X, Heimburg-Molinaro J, Mandalasi M, Mickum M, Smith DF, Nyame AK, Cummings RD. 2014. Immunization with recombinantly expressed glycan antigens from Schistosoma mansoni induces glycan-specific antibodies against the parasite. Glycobiology. 24:619-637.
68. Prasanphanich NS, Mickum ML, Heimburg-Molinaro J, Cummings RD. 2013. Glycoconjugates in host-helminth interactions. Front Immunol. 4 (240):1-22.
69. van Remoortere A, Bank CM, Nyame AK, Cummings RD, Deelder AM, van Die I. 2003. Schistosoma mansoni-infected mice produce antibodies that crossreact with plant, insect, and mammalian glycoproteins and recognize the truncated biantennary N-glycan Man3GlcNAc2-R. Glycobiology. 13:217-225. (Pubitemid 36511757)
70. van Remoortere A, van Dam GJ, Hokke CH, van den Eijnden DH, van Die I, Deelder AM. 2001. Profiles of immunoglobulin M (IgM) and IgG antibodies against defined carbohydrate epitopes in sera of Schistosoma-infected individuals determined by surface plasmon resonance. Infect Immun. 69:2396-2401. (Pubitemid 32239709)
71. Rihet P, Demeure CE, Bourgois A, Prata A, Dessein AJ. 1991. Evidence for an association between human resistance to Schistosoma mansoni and high anti-larval IgE levels. Eur J Immunol. 21:2679-2686.
72. Rollinson D, Knopp S, Levitz S, Stothard JR, Tchuente LA, Garba A, Mohammed KA, Schur N, Person B, Colley DG, et al. 2013. Time to set the agenda for schistosomiasis elimination. Acta Trop. 128:423-440.
73. Scinicariello F, Engleman CN, Jayashankar L, McClure HM, Attanasio R. 2004. Rhesus macaque antibody molecules: Sequences and heterogeneity of alpha and gamma constant regions. Immunology. 111:66-74. (Pubitemid 38091051)
74. Smithers SR, Terry RJ. 1967. Resistance to experimental infection with Schistosoma mansoni in rhesus monkeys induced by the transfer of adult worms. Trans R Soc Trop Med Hyg. 61:517-533.
75. Srivatsan J, Smith DF, Cummings RD. 1992a. The human blood fluke Schistosoma mansoni synthesizes glycoproteins containing the Lewis X antigen. The Journal of Biological Chemistry. 267:20196-20203.
76. Srivatsan J, Smith DF, Cummings RD. 1992b. Schistosoma mansoni synthesizes novel biantennary Asn-linked oligosaccharides containing terminal B-linked N-acetylgalactosamine. Glycobiology. 2:445-452.
77. van Stijn CMW, van den Broek M, Vervelde L, Alvarez Ra, Cummings RD, Tefsen B, van Die I. 2010. Vaccination-induced IgG response to Galalpha1-3GalNAc glycan epitopes in lambs protected against Haemonchus contortus challenge infection. Int J Parasitol. 40:215-222.
78. Stothard JR, Mugisha L, Standley CJ. 2012. Stopping schistosomes from 'monkeying-around' in chimpanzees. Trends Parasitol. 28:320-326.
79. Tendler M, Pinto RM, Cortes M, Gebara G. 1985. Schistosoma mansoni: Comparative evaluation of different routes of experimental infection. Rev Inst Med Trop Sao Paulo. 27:111-114. (Pubitemid 15005640)
80. The World Health Organization. 2010. Working to Overcome the Global Impact of Neglected Tropical Diseases: First WHO Report on Neglected Tropical Diseases. p. 1-184. Available: http://www.who.int/neglected-diseases/2010report/ en/.
81. Van den Eijnden DH, Neeleman AP, Van der Knaap WP, Bakker H, Agterberg M, Van Die I. 1995. Novel glycosylation routes for glycoproteins: The lacdiNAc pathway. Biochem Soc Trans. 23:175-179.
82. Van den Nieuwenhof IM, Koistinen H, Easton RL, Koistinen R, Kamarainen M, Morris HR, Van Die I, Seppala M, Dell A, Van den Eijnden DH. 2000. Recombinant glycodelin carrying the same type of glycan structures as contraceptive glycodelin-A can be produced in human kidney 293 cells but not in Chinese hamster ovary cells. Eur J Biochem. 267:4753-4762. (Pubitemid 30619466)
83. Van Roon AM, Van de Vijver KK, Jacobs W, Van Marck EA, Van Dam GJ, Hokke CH, Deelder AM. 2004. Discrimination between the anti-monomeric and the anti-multimeric Lewis X response in murine schistosomiasis. Microbes Infect. 6:1125-1132. (Pubitemid 39370081)
84. Wang W, Wang L, Liang Y-S. 2012. Susceptibility or resistance of praziquantel in human schistosomiasis: A review. Parasitol Res. 111:1871-1877.
85. Warren KS, Peters PA. 1967. Comparison of penetration and maturation of Schistosoma mansoni in the hamster, mouse, guinea pig, rabbit, and rat. Am J Trop Med Hygiene. 16:718-722.
86. Wilson RA, Langermans JA, van Dam GJ, Vervenne RA, Hall SL, Borges WC, Dillon GP, Thomas AW, Coulson PS. 2008. Elimination of Schistosoma mansoni adult worms by rhesus macaques: basis for a therapeutic vaccine? PLoS Negl Trop Dis. 2:e290.
87. Wuhrer M, Balog CI, Koeleman CA, Deelder AM, Hokke CH. 2005. New features of site-specific horseradish peroxidase (HRP) glycosylation uncovered by nano-LC-MS with repeated ion-isolation/fragmentation cycles. Biochim Biophys Acta. 1723:229-239. (Pubitemid 40744691)
88. Zhou Y-B, Liang S, Jiang Q-W. 2012. Factors impacting on progress towards elimination of transmission of schistosomiasis japonica in China. Parasites Vectors. 5:275.