Lipid raft-dependent adhesion of Giardia intestinalis trophozoites to a cultured human enterocyte-like Caco-2/TC7 cell monolayer leads to cytoskeleton-dependent functional injuries

Cellular Microbiology

Volumn 13, Issue 11, 2011, Pages 1683-1702

  Humen, Martín A ^a,b,c   Pérez, Pablo F ^c,d   Liévin Le Moal, Vanessa ^a,b  

^a INSERM   (France)

^b UNIVERSITÉ PARIS SACLAY   (France)

^c CENTRO DE INVESTIGACIÓN Y DESARROLLO EN CRIOTECNOLOGÍA DE ALIMENTOS CIDCA   (Argentina)

^d FACULTAD DE CIENCIAS EXACTAS   (Argentina)

Author keywords

[No Author keywords available]

Indexed keywords

CLAUDIN 1; DIPEPTIDYL PEPTIDASE; F ACTIN; GLUCOSE TRANSPORTER 5; MEMBRANE LIPID; OCCLUDIN; SUCRASE ISOMALTASE;

ARTICLE; BACTERIUM ADHERENCE; BRUSH BORDER; CELL DAMAGE; CELL STRAIN CACO 2; CONTROLLED STUDY; CYTOSKELETON; ELECTRIC RESISTANCE; ENZYME ACTIVITY; GIARDIA LAMBLIA; HUMAN; HUMAN CELL; HUMAN CELL CULTURE; INTESTINE CELL; INTESTINE EPITHELIUM; LIPID RAFT; MONOLAYER CULTURE; NONHUMAN; PRIORITY JOURNAL; TIGHT JUNCTION; TRANSEPITHELIAL ELECTRIC RESISTANCE; TROPHOZOITE;

ACTINS; BIOLOGICAL TRANSPORT; CELL ADHESION; CELL LINE; ENTEROCYTES; GIARDIA LAMBLIA; HOST-PATHOGEN INTERACTIONS; HUMANS; MEMBRANE MICRODOMAINS; MEMBRANE PROTEINS; TIGHT JUNCTIONS; TROPHOZOITES;

GIARDIA; GIARDIA INTESTINALIS;

EID: 80054092933     PISSN: 14625814     EISSN: 14625822     Source Type: Journal    
DOI: 10.1111/j.1462-5822.2011.01647.x     Document Type: Article

References (98)

1. Anderson, J.M., and Van Itallie, C.M. (2009) Physiology and function of the tight junction. Cold Spring Harb Perspect Biol 1: a002584.
2. Ankarklev, J., Jerlstrom-Hultqvist, J., Ringqvist, E., Troell, K., and Svard, S.G. (2010) Behind the smile: cell biology and disease mechanisms of Giardia species. Nat Rev Microbiol 8: 413-422.
3. Beau, I., Cotte-Laffitte, J., Amsellem, R., and Servin, A.L. (2007) A protein kinase A-dependent mechanism by which rotavirus affects the distribution and mRNA level of the functional tight junction-associated protein, occludin, in human differentiated intestinal Caco-2 cells. J Virol 81: 8579-8586.
4. Belosevic, M., Faubert, G.M., and MacLean, J.D. (1989) Disaccharidase activity in the small intestine of gerbils (Meriones unguiculatus) during primary and challenge infections with Giardia lamblia. Gut 30: 1213-1219.
5. Berkes, J., Viswanathan, V.K., Savkovic, S.D., and Hecht, G. (2003) Intestinal epithelial responses to enteric pathogens: effects on the tight junction barrier, ion transport, and inflammation. Gut 52: 439-451.
6. Bernander, R., Palm, J.E., and Svard, S.G. (2001) Genome ploidy in different stages of the Giardia lamblia life cycle. Cell Microbiol 3: 55-62.
7. Boyle, E.C., and Finlay, B.B. (2003) Bacterial pathogenesis: exploiting cellular adherence. Curr Opin Cell Biol 15: 633-639.
8. Buret, A.G. (2007) Mechanisms of epithelial dysfunction in giardiasis. Gut 56: 316-317.
9. Buret, A.G. (2008) Pathophysiology of enteric infections with Giardia duodenalis. Parasite 15: 261-265.
10. Buret, A., Gall, D.G., and Olson, M.E. (1990) Effects of murine giardiasis on growth, intestinal morphology, and disaccharidase activity. J Parasitol 76: 403-409.
11. Buret, A., Gall, D.G., and Olson, M.E. (1991) Growth, activities of enzymes in the small intestine, and ultrastructure of microvillous border in gerbils infected with Giardia duodenalis. Parasitol Res 77: 109-114.
12. Buret, A., Hardin, J.A., Olson, M.E., and Gall, D.G. (1992) Pathophysiology of small intestinal malabsorption in gerbils infected with Giardia lamblia. Gastroenterology 103: 506-513.
13. Buret, A.G., Mitchell, K., Muench, D.G., and Scott, K.G. (2002) Giardia lamblia disrupts tight junctional ZO-1 and increases permeability in non-transformed human small intestinal epithelial monolayers: effects of epidermal growth factor. Parasitology 125: 11-19.
14. Cevallos, A., Carnaby, S., James, M., and Farthing, J.G. (1995) Small intestinal injury in a neonatal rat model of giardiasis is strain dependent. Gastroenterology 109: 766-773.
15. Chantret, I., Rodolosse, A., Barbat, A., Dussaulx, E., Brot-Laroche, E., Zweibaum, A., and Rousset, M. (1994) Differential expression of sucrase-isomaltase in clones isolated from early and late passages of the cell line Caco-2: evidence for glucose-dependent negative regulation. J Cell Sci 107: 213-225.
16. Chavez, B., Knaippe, F., Gonzalez-Mariscal, L., and Martinez-Palomo, A. (1986) Giardia lamblia: electrophysiology and ultrastructure of cytopathology in cultured epithelial cells. Exp Parasitol 61: 379-389.
17. Chen, N., Upcroft, J.A., and Upcroft, P. (1995) A Giardia duodenalis gene encoding a protein with multiple repeats of a toxin homologue. Parasitology 111: 423-431.
18. Chin, A.C., Teoh, D.A., Scott, K.G., Meddings, J.B., Macnaughton, W.K., and Buret, A.G. (2002) Strain-dependent induction of enterocyte apoptosis by Giardia lamblia disrupts epithelial barrier function in a caspase-3-dependent manner. Infect Immun 70: 3673-3680.
19. Dagci, H., Ustun, S., Taner, M.S., Ersoz, G., Karacasu, F., and Budak, S. (2002) Protozoon infections and intestinal permeability. Acta Trop 81: 1-5.
20. Daniels, C.W., and Belosevic, M. (1992) Disaccharidase activity in the small intestine of susceptible and resistant mice after primary and challenge infections with Giardia muris. Am J Trop Med Hyg 46: 382-390.
21. Daniels, C.W., and Belosevic, M. (1995) Disaccharidase activity in male and female C57BL/6 mice infected with Giardia muris. Parasitol Res 81: 143-147.
22. Das, S., Traynor-Kaplan, A., Reiner, D.S., Meng, T.C., and Gillin, F.D. (1991) A surface antigen of Giardia lamblia with a glycosylphosphatidylinositol anchor. J Biol Chem 266: 21318-21325.
23. Das, S., Traynor-Kaplan, A., Kachintorn, U., Aley, S.B., and Gillin, F.D. (1994) GP49, an invariant GPI-anchored antigen of Giardia lamblia. Braz J Med Biol Res 27: 463-469.
24. Fanning, A.S., Jameson, B.J., Jesaitis, L.A., and Anderson, J.M. (1998) The tight junction protein ZO-1 establishes a link between the transmembrane protein occludin and the actin cytoskeleton. J Biol Chem 273: 29745-29753.
25. Farshori, P., and Kachar, B. (1999) Redistribution and phosphorylation of occludin during opening and resealing of tight junctions in cultured epithelial cells. J Membr Biol 170: 147-156.
26. Favennec, L., Magne, D., and Gobert, J.G. (1991) Cytopathogenic effect of Giardia intestinalis, in vitro. Parasitol Today 7: 141.
27. Fogh, J., Orfeo, T., Tiso, J., and Sharkey, F.E. (1979) Establishment of human colon carcinoma lines in nude mice. Exp Cell Biol 47: 136-144.
28. Furuse, M., Hirase, T., Itoh, M., Nagafuchi, A., Yonemura, S., and Tsukita, S. (1993) Occludin: a novel integral membrane protein localizing at tight junctions. J Cell Biol 123: 1777-1788.
29. Furuse, M., Itoh, M., Hirase, T., Nagafuchi, A., Yonemura, S., Tsukita, S., and Tsukita, S. (1994) Direct association of occludin with ZO-1 and its possible involvement in the localization of occludin at tight junctions. J Cell Biol 127: 1617-1626.
30. Furuse, M., Fujita, K., Hiiragi, T., Fujimoto, K., and Tsukita, S. (1998) Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin. J Cell Biol 141: 1539-1550.
31. Gonzalez-Mariscal, L., Betanzos, A., and Avila-Flores, A. (2000) MAGUK proteins: structure and role in the tight junction. Semin Cell Dev Biol 11: 315-324.
32. van der Goot, F.G., and Harder, T. (2001) Raft membrane domains: from a liquid-ordered membrane phase to a site of pathogen attack. Semin Immunol 13: 89-97.
33. Gorowara, S., Ganguly, N.K., Mahajan, R.C., and Walia, B.N. (1992) Study on the mechanism of Giardia lamblia induced diarrhoea in mice. Biochim Biophys Acta 1138: 122-126.
34. Guignot, J., and Servin, A.L. (2008) Maintenance of the Salmonella-containing vacuole in the juxtanuclear area: a role for intermediate filaments. Microb Pathog 45: 415-422.
35. Guignot, J., Chaplais, C., Coconnier-Polter, M.H., and Servin, A.L. (2007) The secreted autotransporter toxin, Sat, functions as a virulence factor in Afa/Dr diffusely adhering Escherichia coli by promoting lesions in tight junction of polarized epithelial cells. Cell Microbiol 9: 204-221.
36. Guttman, J.A., and Finlay, B.B. (2008a) Tight junctions as targets of infectious agents. Biochim Biophys Acta 1788: 832-841.
37. Guttman, J.A., and Finlay, B.B. (2008b) Subcellular alterations that lead to diarrhea during bacterial pathogenesis. Trends Microbiol 16: 535-542.
38. Hardin, J.A., Buret, A.G., Olson, M.E., Kimm, M.H., and Gall, D.G. (1997) Mast cell hyperplasia and increased macromolecular uptake in an animal model of giardiasis. J Parasitol 83: 908-912.
39. Hare, D.F., Jarroll, E.L., and Lindmark, D.G. (1989) Giardia lamblia: characterization of proteinase activity in trophozoites. Exp Parasitol 68: 168-175.
40. Hiltpold, A., Frey, M., Hulsmeier, A., and Kohler, P. (2000) Glycosylation and palmitoylation are common modifications of Giardia variant surface proteins. Mol Biochem Parasitol 109: 61-65.
41. Humen, M.A., De Antoni, G.L., Benyacoub, J., Costas, M.E., Cardozo, M.I., Kozubsky, L., etal. (2005) Lactobacillus johnsonii La1 antagonizes Giardia intestinalis in vivo. Infect Immun 73: 1265-1269.
42. Jenkins, M.C., O'Brien, C.N., Murphy, C., Schwarz, R., Miska, K., Rosenthal, B., and Trout, J.M. (2009) Antibodies to the ventral disc protein delta-giardin prevent in vitro binding of Giardia lamblia trophozoites. J Parasitol 95: 895-899.
43. Jimenez, J.C., Fontaine, J., Grzych, J.M., Dei-Cas, E., and Capron, M. (2004) Systemic and mucosal responses to oral administration of excretory and secretory antigens from Giardia intestinalis. Clin Diagn Lab Immunol 11: 152-160.
44. Johnston, J.A., Ward, C.L., and Kopito, R.R. (1998) Aggresomes: a cellular response to misfolded proteins. J Cell Biol 143: 1883-1898.
45. Jung, H.C., Eckmann, L., Yang, S.K., Panja, A., Fierer, J., Morzycka-Wroblewska, E., and Kagnoff, M.F. (1995) A distinct array of proinflammatory cytokines is expressed in human colon epithelial cells in response to bacterial invasion. J Clin Invest 95: 55-65.
46. Kamda, J.D., and Singer, S.M. (2009) Phosphoinositide 3-kinase-dependent inhibition of dendritic cell interleukin-12 production by Giardia lamblia. Infect Immun 77: 685-693.
47. Katelaris, P.H., Naeem, A., and Farthing, M.J. (1995) Attachment of Giardia lamblia trophozoites to a cultured human intestinal cell line. Gut 37: 512-518.
48. Kaur, H., Ghosh, S., Samra, H., Vinayak, V.K., and Ganguly, N.K. (2001) Identification and characterization of an excretory-secretory product from Giardia lamblia. Parasitology 123: 347-356.
49. Keister, D.B. (1983) Axenic culture of Giardia lamblia in TYI-S-33 medium supplemented with bile. Trans R Soc Trop Med Hyg 77: 487-488.
50. Lambert, D., O'Neill, C.A., and Padfield, P.J. (2007) Methyl-beta-cyclodextrin increases permeability of Caco-2 cell monolayers by displacing specific claudins from cholesterol rich domains associated with tight junctions. Cell Physiol Biochem 20: 495-506.
51. Laughlin, R.C., McGugan, G.C., Powell, R.R., Welter, B.H., and Temesvari, L.A. (2004) Involvement of raft-like plasma membrane domains of Entamoeba histolytica in pinocytosis and adhesion. Infect Immun 72: 5349-5357.
52. Lencer, W.I. (2001) Microbial invasion across the intestinal epithelial barrier. Pediatr Res 49: 4-5.
53. Lievin-Le Moal, V., and Servin, A.L. (2006) The front line of enteric host defense against unwelcome intrusion of harmful microorganisms: mucins, antimicrobial peptides, and microbiota. Clin Microbiol Rev 19: 315-337.
54. Lujan, H.D. (2006) Giardia and giardiasis. Medicina (B Aires) 66: 70-74.
55. Minnaard, J., Humen, M., and Perez, P.F. (2001) Effect of Bacillus cereus exocellular factors on human intestinal epithelial cells. J Food Prot 64: 1535-1541.
56. Mittal, K., Welter, B.H., and Temesvari, L.A. (2008) Entamoeba histolytica: lipid rafts are involved in adhesion of trophozoites to host extracellular matrix components. Exp Parasitol 120: 127-134.
57. Monajemzadeh, S.M., and Monajemzadeh, M. (2008) Comparison of iron and hematological indices in Giardia lamblia infection before and after treatment in 102 children in Ahwaz, Iran. Med Sci Monit 14: CR19-CR23.
58. Müller, N., and von Allmen, N. (2005) Recent insights into the mucosal reactions associated with Giardia lamblia infections. Int J Parasitol 35: 1339-1347.
59. Muza-Moons, M.M., Schneeberger, E.E., and Hecht, G.A. (2004) Enteropathogenic Escherichia coli infection leads to appearance of aberrant tight junctions strands in the lateral membrane of intestinal epithelial cells. Cell Microbiol 6: 783-793.
60. Nash, T.E., Lujan, H.T., Mowatt, M.R., and Conrad, J.T. (2001) Variant-specific surface protein switching in Giardia lamblia. Infect Immun 69: 1922-1923.
61. Nozawa, N., Yamauchi, Y., Ohtsuka, K., Kawaguchi, Y., and Nishiyama, Y. (2004) Formation of aggresome-like structures in herpes simplex virus type 2-infected cells and a potential role in virus assembly. Exp Cell Res 299: 486-497.
62. Oberhuber, G., Kastner, N., and Stolte, M. (1997) Giardiasis: a histologic analysis of 567 cases. Scand J Gastroenterol 32: 48-51.
63. Obert, G., Peiffer, I., and Servin, A.L. (2000) Rotavirus-induced structural and functional alterations in tight junctions of polarized intestinal Caco-2 cell monolayers. J Virol 74: 4645-4651.
64. O'Hara, J.R., and Buret, A.G. (2008) Mechanisms of intestinal tight junctional disruption during infection. Front Biosci 13: 7008-7021.
65. Palm, D., Weiland, M., McArthur, A.G., Winiecka-Krusnell, J., Cipriano, M.J., Birkeland, S.R., etal. (2005) Developmental changes in the adhesive disk during Giardia differentiation. Mol Biochem Parasitol 141: 199-207.
66. Papanastasiou, P., McConville, M.J., Ralton, J., and Kohler, P. (1997) The variant-specific surface protein of Giardia, VSP4A1, is a glycosylated and palmitoylated protein. Biochem J 322: 49-56.
67. Parenti, D.M. (1989) Characterization of a thiol proteinase in Giardia lamblia. J Infect Dis 160: 1076-1080.
68. Pegado, M.G., and de Souza, W. (1994) Role of surface components in the process of interaction of Giardia duodenalis with epithelial cells in vitro. Parasitol Res 80: 320-326.
69. Peiffer, I., Blanc-Potard, A.B., Bernet-Camard, M.F., Guignot, J., Barbat, A., and Servin, A.L. (2000a) Afa/Dr diffusely adhering Escherichia coli C1845 infection promotes selective injuries in the junctional domain of polarized human intestinal Caco-2/TC7 cells. Infect Immun 68: 3431-3442.
70. Peiffer, I., Guignot, J., Barbat, A., Carnoy, C., Moseley, S.L., Nowicki, B.J., etal. (2000b) Structural and functional lesions in brush border of human polarized intestinal Caco-2/TC7 cells infected by members of the Afa/Dr diffusely adhering family of Escherichia coli. Infect Immun 68: 5979-5990.
71. Peiffer, I., Bernet-Camard, M.F., Rousset, M., and Servin, A.L. (2001) Impairments in enzyme activity and biosynthesis of brush border-associated hydrolases in human intestinal Caco-2/TC7 cells infected by members of the Afa/Dr family of diffusely adhering Escherichia coli. Cell Microbiol 3: 341-357.
72. Perez, P.F., Minnaard, J., Rouvet, M., Knabenhans, C., Brassart, D., De Antoni, G.L., and Schiffrin, E.J. (2001) Inhibition of Giardia intestinalis by extracellular factors from lactobacilli: an in vitro study. Appl Environ Microbiol 67: 5037-5042.
73. Peterson, M.D., and Mooseker, M.S. (1992) Characterization of the enterocyte-like brush border cytoskeleton of the C2BBe clones of the human intestinal cell line, Caco-2. J Cell Sci 102: 581-600.
74. Peterson, M.D., and Mooseker, M.S. (1993) An in vitro model for the analysis of intestinal brush border assembly. I. Ultrastructural analysis of cell contact-induced brush border assembly in Caco-2BBe cells. J Cell Sci 105: 445-460.
75. Peterson, M.D., Bement, W.M., and Mooseker, M.S. (1993) An in vitro model for the analysis of intestinal brush border assembly. II. Changes in expression and localization of brush border proteins during cell contact-induced brush border assembly in Caco-2BBe cells. J Cell Sci 105: 461-472.
76. Pinto, M., Robine-Leon, S., Appay, M.D., Kedinger, M., Triadou, N., Dussaulx, E., etal. (1983) Enterocyte-like differentiation and polarization of the human colon carcinoma cell line Caco-2 in culture. Biol Cell 47: 323-330.
77. Ringqvist, E., Palm, J.E., Skarin, H., Hehl, A.B., Weiland, M., Davids, B.J., etal. (2008) Release of metabolic enzymes by Giardia in response to interaction with intestinal epithelial cells. Mol Biochem Parasitol 159: 85-91.
78. Ringqvist, E., Avesson, L., Soderbom, F., and Svard, S.G. (2011) Transcriptional changes in Giardia during host-parasite interactions. Int J Parasitol 41: 277-285.
79. Rodriguez-Fuentes, G.B., Cedillo-Rivera, R., Fonseca-Linan, R., Arguello-Garcia, R., Munoz, O., Ortega-Pierres, G., and Yepez-Mulia, L. (2006) Giardia duodenalis: analysis of secreted proteases upon trophozoite-epithelial cell interaction in vitro. Mem Inst Oswaldo Cruz 101: 693-696.
80. Roxstrom-Lindquist, K., Ringqvist, E., Palm, D., and Svard, S. (2005) Giardia lamblia-induced changes in gene expression in differentiated Caco-2 human intestinal epithelial cells. Infect Immun 73: 8204-8208.
81. Sackey, M.E., Weigel, M.M., and Armijos, R.X. (2003) Predictors and nutritional consequences of intestinal parasitic infections in rural Ecuadorian children. J Trop Pediatr 49: 17-23.
82. Sakaguchi, T., Kohler, H., Gu, X., McCormick, B.A., and Reinecker, H.C. (2002) Shigella flexneri regulates tight junction-associated proteins in human intestinal epithelial cells. Cell Microbiol 4: 367-381.
83. Saric, M., Vahrmann, A., Niebur, D., Kluempers, V., Hehl, A.B., and Scholze, H. (2009) Dual acylation accounts for the localization of {alpha}19-giardin in the ventral flagellum pair of Giardia lamblia. Eukaryot Cell 8: 1567-1574.
84. Schneeberger, E.E., and Lynch, R.D. (2004) The tight junction: a multifunctional complex. Am J Physiol Cell Physiol 286: C1213-C1228.
85. Scott, K.G., Logan, M.R., Klammer, G.M., Teoh, D.A., and Buret, A.G. (2000) Jejunal brush border microvillous alterations in Giardia muris-infected mice: role of T lymphocytes and interleukin-6. Infect Immun 68: 3412-3418.
86. Scott, K.G., Meddings, J.B., Kirk, D.R., Lees-Miller, S.P., and Buret, A.G. (2002) Intestinal infection with Giardia spp. reduces epithelial barrier function in a myosin light chain kinase-dependent fashion. Gastroenterology 123: 1179-1190.
87. Shant, J., Ghosh, S., Bhattacharyya, S., Ganguly, N.K., and Majumdar, S. (2004) The alteration in signal transduction parameters induced by the excretory-secretory product from Giardia lamblia. Parasitology 129: 421-430.
88. Simonovic, I., Rosenberg, J., Koutsouris, A., and Hecht, G. (2000) Enteropathogenic Escherichia coli dephosphorylates and dissociates occludin from intestinal epithelial tight junctions. Cell Microbiol 2: 305-315.
89. Singh, K.D., Bhasin, D.K., Rana, S.V., Vaiphei, K., Katyal, R., Vinayak, V.K., and Singh, K. (2000) Effect of Giardia lamblia on duodenal disaccharidase levels in humans. Trop Gastroenterol 21: 174-176.
90. Skarin, H., Ringqvist, E., Hellman, U., and Svard, S.G. (2011) Elongation factor 1-alpha is released into the culture medium during growth of Giardia intestinalis trophozoites. Exp Parasitol 127: 804-810.
91. Sousa, M.C., Goncalves, C.A., Bairos, V.A., and Poiares-Da-Silva, J. (2001) Adherence of Giardia lamblia trophozoites to Int-407 human intestinal cells. Clin Diagn Lab Immunol 8: 258-265.
92. Teoh, D.A., Kamieniecki, D., Pang, G., and Buret, A.G. (2000) Giardia lamblia rearranges F-actin and alpha-actinin in human colonic and duodenal monolayers and reduces transepithelial electrical resistance. J Parasitol 86: 800-806.
93. Tepass, U. (2003) Claudin complexities at the apical junctional complex. Nat Cell Biol 5: 595-597.
94. Touz, M.C., Conrad, J.T., and Nash, T.E. (2005) A novel palmitoyl acyl transferase controls surface protein palmitoylation and cytotoxicity in Giardia lamblia. Mol Microbiol 58: 999-1011.
95. Troeger, H., Epple, H.J., Schneider, T., Wahnschaffe, U., Ullrich, R., Burchard, G.D., etal. (2007) Effect of chronic Giardia lamblia infection on epithelial transport and barrier function in human duodenum. Gut 56: 328-335.
96. Wei, C.J., Tian, X.F., Adam, R.D., and Lu, S.Q. (2010) Giardia lamblia: intracellular localization of alpha8-giardin. Exp Parasitol 126: 489-496.
97. Weiland, M.E., McArthur, A.G., Morrison, H.G., Sogin, M.L., and Svard, S.G. (2005) Annexin-like alpha giardins: a new cytoskeletal gene family in Giardia lamblia. Int J Parasitol 35: 617-626.
98. Zweibaum, A., Laburthe, M., Grasset, E., and Louvard, D. (1991) Use of cultured cell lines in studies of intestinal cell differentiation and function. In Handbook of Physiology, the Gastrointestinal System, Intestinal Absorption and Secretion. Schultz, S.J., and Frizell, R.A. (eds). Bethesda: American Physiological Society, pp. 223-255.