Cruzipain, a major Trypanosoma cruzi cystein protease in the host-parasite interplay

Inmunologia

Volumn 25, Issue 4, 2006, Pages 225-238

  Gea, Susana ^a   Guinazu N ^a   Pellegrini, A ^a   Carrera Silva, E A ^a   Giordanengo, L ^a   Cano, R ^a   Aoki, M P ^a  

^a UNIVERSIDAD NACIONAL DE CÓRDOBA   (Argentina)

Author keywords

Autoimmunity; Cardiomyopathy; Chagas disease; Cruzipain; Pathogenesis; Trypanosoma cruzi

Indexed keywords

CRUZIPAIN; CYSTEINE; CYSTEINE PROTEINASE INHIBITOR; KETONE DERIVATIVE; PROTOZOAL VACCINE; SULFONE DERIVATIVE;

ANTIBODY TITER; CELL INTERACTION; CELL INVASION; CELLULAR IMMUNITY; CHAGAS DISEASE; DISEASE COURSE; DRUG SYNTHESIS; DRUG TARGETING; EPITOPE MAPPING; HOST PARASITE INTERACTION; HUMAN; IMMUNE RESPONSE; NONHUMAN; PATHOGENESIS; PHAGOCYTE; PROTEIN EXPRESSION; PROTEIN FAMILY; REVIEW; TRYPANOSOMA CRUZI; TRYPANOSOMIASIS;

EID: 33947596067     PISSN: 02139626     EISSN: None     Source Type: Journal    
DOI: None     Document Type: Review

References (96)

1. WE Committee. Control of Chagas disease: report of a WHO expert commitee 2002:1-109.
2. Tarleton R. Parasite persistence in the aetiology of Chagas disease. Int J Parasitol 2001;31:550-554.
3. Kierszenbaum F. Views on the autoimmunity hypothesis for Chagas disease pathogenesis. FEMS Immunol Med Microbiol 2003;37:1-11.
4. IL Cunha-Neto E, Bilate AMB, Goncalves Fonseca S, Kalil J. Autoimmunity in Chagas' Disease. Infections and Autoimmunity 2004:451-467.
5. Leon J, Engman D. The significance of autoimmunity in the pathogenesis of Chagas heart disease. Front Biosci 2003;8:e315-322.
6. Girones N, Cuervo H, Fresno M. Trypanosoma cruzi-induced molecular mimicry and Chagas' disease. Curr Top Microbiol Immunol 2005;296:89-123.
7. Girones N, Fresno M. Etiology of Chagas disease myocarditis: autoimmunity, parasite persistence, or both? Trends Parasitol 2003;19:19-22.
8. Eakin AE, Mills AA, Harth G, McKerrow JH, Craik CS. The sequence, organization, and expression of the major cysteine protease (cruzain) from Trypanosoma cruzi. J Biol Chem 1992;267:7411-7420.
9. Campetella O, Henriksson J, Aslund L, Frasch A, Pettersson U, Cazzulo J. The major cysteine proteinase (cruzipain) from Trypanosoma cruzi is encoded by multiple polymorphic tandemly organized genes located on different chromosomes. Mol Biochem Parasitol 1992;50:225-234.
10. Mottram J, Brooks D, Coombs G. Roles of cysteine proteinases of trypanosomes and Leishmania in host-parasite interactions. Curr Opin Microbiol 1998;1:455-460.
11. Scharfstein J, Schechter M, Senna M, Peralta J, Mendonca-Previato L, Miles M. Trypanosoma cruzi: characterization and isolation of a 57/51,000 m.w. surface glycoprotein (GP57/51) expressed by epimastigotes and bloodstream trypomastigotes. J Immunol 1986;137:1336-1341.
12. Campetella O, Martinez J, Cazzulo J. A major cysteine proteinase is developmentally regulated in Trypanosoma cruzi. FEMS Microbiol Lett 1990;55:145-149.
13. Souto-Padron T, Campetella O, Cazzulo J, de Souza W. Cysteine proteinase in Trypanosoma cruzi: immunocytochemical localization and involvement in parasite-host cell interaction. J Cell Sci 1990;96:485-490.
14. dos Reis F, Judice W, Juliano M, Juliano L, Scharfstein J, Lima A. The substrate specificity of cruzipain 2, a cysteine protease isoform from Trypanosoma cruzi. FEMS Microbiol Lett 2006;259:215-220.
15. McKerrow J, Caffrey C, Kelly B, Loke P, Sajid M. Proteases in parasitic diseases. Annual Review Pathology Mechanisms Disease 2006;1:497- 536.
16. Bontempi E, Cazzulo J. Digestion of human immunoglobulin G by the major cysteine proteinase (cruzipain) from Trypanosoma cruzi. FEMS Microbiol Lett 1990;58:337-341.
17. Meirelles M, Juliano L, Carmona E, Silva S, Costa E, Murta A, et al. Inhibitors of the major cysteinyl proteinase (GP57/51) impair host cell invasion and arrest the intracellular development of Trypanosoma cruzi in vitro. Mol Biochem Parasitol 1992;52:175-184.
18. Mendonca-Previato L, Gorin P, Braga A, Scharfstein J, Previato J. Chemical structure and antigenic aspects of complexes obtained from epimastigotes of Trypanosoma cruzi. Biochemistry 1983;22:4980- 4987.
19. Aslund L, Henriksson J, Campetella O, Frasch A, Pettersson U, Cazzulo J. The C-terminal extension of the major cysteine proteinase (cruzipain) from Trypanosoma cruzi. Mol Biochem Parasitol 1991;45:345-347.
20. Parodi A, Labriola C, Cazzulo J. The presence of complex-type oligosaccharides at the C-terminal domain glycosylation site of some molecules of cruzipain. Mol Biochem Parasitol 1995;69:247-255.
21. Barboza M, Duschak VG, Fukuyama Y, Nonami H, Erra-Balsells R, Cazzulo JJ, et al. Structural analysis of the N-glycans of the major cysteine proteinase of Trypanosoma cruzi: Identification of sulfated high-mannose type oligosaccharides. FEBS J 2005;272:3803-3815.
22. Cazzulo J. [Cruzipain, major cysteine proteinase of Trypanosoma cruzi: sequence and genomic organization of the codifying genes]. Medicina (B Aires) 1999;59(Suppl 2):7-10.
23. Del Nery E, Juliano MA, Lima APCA, Scharfstein J, Juliano L. Kininogenase Activity by the Major Cysteinyl Proteinase (Cruzipain) from Trypanosoma cruzi. 272.41.25713. J Biol Chem 1997;272:25713-25718.
24. Scharfstein J, Schmitz V, Morandi V, Capella MM, Lima AP, Morrot A, et al. Host cell invasion by Trypanosoma cruzi is potentiated by activation of bradykinin B(2) receptors. J Exp Med 2000;192:1289-1300.
25. Scharfstein J, Abrahamson M, de Souza C, Barrall Silva A. Antigenicity of cystatin-binding proteins from parasitic protozoan. Detection by a proteinase inhibitor based capture immunoassay (PINC-ELISA). J Immunol Methods 1995;182:63-72.
26. Stoka V, Nycander M, Lenarcic B, Labriola C, Cazzulo J, Bjork I, et al. Inhibition of cruzipain, the major cysteine proteinase of the protozoan parasite, Trypanosoma cruzi, by proteinase inhibitors of the cystatin superfamily. FEBS Lett 1995;370:101-104.
27. Ramos A, Duschak V, Gerez de Burgos N, Barboza M, Remedi M, Vides M, et al. Trypanosoma cruzi: cruzipain and membrane-bound cysteine proteinase isoform(s) interacts with human alpha(2)-macroglobulin and pregnancy zone protein. Exp Parasitol 2002;100:121-130.
28. Araujo-Jorge TC, Lage MJ, Rivera MT, Carlier Y, Van Leuven F. Trypanosoma cruzi: enhanced alpha-macroglobulin levels correlate with the resistance of BALB/cj mice to acute infection. Parasitol Res 1992;78:215-221.
29. Coutinho C, Cavalcanti G, van Leuven F, Araujo-Jorge T. Alpha-2-macroglobulin binds to the surface of Trypanosoma cruzi. Parasitol Res 1997;83:144-150.
30. Irvine J, Coombs G, North M. Cystatin-like cysteine proteinase inhibitors of parasitic protozoa. FEMS Microbiol Lett 1992;75:67-72.
31. Monteiro ACS, Abrahamson M, Lima APCA, Vannier-Santos MA, Scharfstein J. Identification, characterization and localization of chagasin, a tight-binding cysteine protease inhibitor in Trypanosoma cruzi. J Cell Sci 2001;114:3933-3942.
32. Sanderson S, Westrop G, Scharfstein J, Mottram J, Coombs G. Functional conservation of a natural cysteine peptidase inhibitor in protozoan and bacterial pathogens. FEBS Lett 2003;542:12-16.
33. Engel JC, Doyle PS, Hsieh I, McKerrow JH. Cysteine Protease Inhibitors Cure an Experimental Trypanosoma cruzi Infection. J Exp Med 1998;188:725-734.
34. Santos CC, Sant'Anna C, Terres A, Cunha-e-Silva NL, Scharfstein J, de A. Lima APC. Chagasin, the endogenous cysteine-protease inhibitor of Trypanosoma cruzi, modulates parasite differentiation and invasion of mammalian cells. J Cell Sci 2005;118:901-915.
35. Barr SC, Warner KL, Kornreic BG, Piscitelli J, Wolfe A, Benet L, et al. A Cysteine Protease Inhibitor Protects Dogs from Cardiac Damage during Infection by Trypanosoma cruzi. Antimicrob Agents Chemother 2005;49:5160- 5161.
36. Du X, Guo C, Hansell E, Doyle P, Caffrey C, Holler T, et al. Synthesis and structure-activity relationship study of potent trypanocidal thio semicarbazone inhibitors of the trypanosomal cysteine protease cruzain. J Med Chem 2002;45:2695-2707.
37. Yong V, Schmitz V, Vannier-Santos M, de Lima A, Lalmanach G, Juliano L, et al. Altered expression of cruzipain and a cathepsin B-like target in a Trypanosoma cruzi cell line displaying resistance to synthetic inhibitors of cysteine-proteinases. Mol Biochem Parasitol 2000;109:47-59.
38. Ferreira D, Cortez M, Atayde VD, Yoshida N. Actin Cytoskeleton-Dependent and -Independent Host Cell Invasion by Trypanosoma cruzi Is Mediated by Distinct Parasite Surface Molecules. Infect Immun 2006;74:5522-5528.
39. Woolsey AM, Sunwoo L, Petersen CA, Brachmann SM, Cantley LC, Burleigh BA. Novel PI 3-kinase-dependent mechanisms of trypanosome invasion and vacuole maturation. J Cell Sci 2003;116:3611-3622.
40. Todorov AG, Einicker-Lamas M, de Castro SL, Oliveira MM, Guilherme A. Activation of Host Cell Phosphatidylinositol 3-Kinases by Trypanosoma cruzi Infection. J Biol Chem 2000;275:32182-32186.
41. Aparicio IM, Scharfstein J, Lima APCA. A New Cruzipain-Mediated Pathway of Human Cell Invasion by Trypanosoma cruzi Requires Trypomastigote Membranes. Infect Immun 2004;72:5892-5902.
42. Harth G, Andrews N, Mills AA, Engel JC, Smith R, McKerrow JH. Peptide-fluoromethyl ketones arrest intracellular replication and intercellular transmission of Trypanosoma cruzi. Mol Biochem Parasitol 1993;58:17-24.
43. Todorov AG, Andrade D, Pesquero JB, Araujo Rde C, Bader M, Stewart J, et al. Trypanosoma cruzi induces edematogenic responses in mice and invades cardiomyocytes and endothelial cells in vitro by activating distinct kinin receptor (B1/B2) subtypes. Faseb J 2003;17:73-75.
44. Aliberti J, Viola JPB, Vieira-de-Abreu A, Bozza PT, Sher A, Scharfstein J. Cutting Edge: Bradykinin Induces IL-12 Production by Dendritic Cells: A Danger Signal That Drives Th1 Polarization. J Immunol 2003;170:5349-5353.
45. Scharfstein J. Parasite cysteine proteinase interactions with alpha2-macroglobulin or kininogens: differential pathways modulating inflammation and innate immunity in infection by pathogenic trypanosomatids. Immunobiology 2006;211:117-125.
46. Peluffo G, Piacenza L, Irigoin F, Alvarez M, Radi R. L-arginine metabolism during interaction of Trypanosoma cruzi with host cells. Trends Parasitol 2004;20:363-369.
47. Mantovani A, Sica A, Sozzani S, Allavena P, Vecchi A, Locati M. The chemokine system in diverse forms of macrophage activation and polarization. Trends Immunol 2004;25:677-686.
48. Giordanengo L, Guinazu N, Stempin C, Fretes R, Cerban F, Gea S. Cruzipain, a major Trypanosoma cruzi antigen, conditions the host immune response in favor of parasite. Eur J Immunol 2002;32:1003-1011.
49. Guinazu N, Pellegrini A, Giordanengo L, Aoki MP, Rivarola H, Cano R, et al. Immune response to a major Trypanosoma cruzi antigen, cruzipain, is differentially modulated in C57BL/6 and BALB/c mice. Microbes Infect 2004;6:1250-1258.
50. Planelles L, Thomas M, Maranon C, Morell M, Lopez M. Differential CD86 and CD40 co-stimulatory molecules and cytokine expression pattern induced by Trypanosoma cruzi in APCs from resistant or susceptible mice. Clin Exp Immunol 2003;131:41-47.
51. Stempin C, Giordanengo L, Gea S, Cerban F. Alternative activation and increase of Trypanosoma cruzi survival in murine macrophages stimulated by cruzipain, a parasite antigen. J Leukoc Biol 2002;72:727-734.
52. Stempin C, Tanos T, Coso O, Cerban F. Arginase induction promotes Trypanosoma cruzi intracellular replication in Cruzipain-treated J774 cells through the activation of multiple signaling pathways. Eur J Immunol 2004;34:200-209.
53. Ropert C, Gazzinelli R. Regulatory role of Toll-like receptor 2 during infection with Trypanosoma cruzi. J Endotoxin Res 2004;10:425-430.
54. Campos MAS, Almeida IC, Takeuchi O, Akira S, Valente EP, Procopio DO, et al. Activation of Toll-Like Receptor-2 by Glycosylphosphaudylinositol Anchors from a Protozoan Parasite. J Immunol 2001;167:416-423.
55. Campos MA, Closel M, Valente EP, Cardoso JE, Akira S, Alvarez-Leite JI, et al. Impaired Production of Proinflammatory Cytokines and Host Resistance to Acute Infection with Trypanosoma cruzi in Mice Lacking Functional Myeloid Differentiation Factor 88. J Immunol 2004;172:1711-1718.
56. Burleigh BA, Woolsey AM. Cell signalling and Trypanosoma cruzi invasion. Cell Microbiol 2002;4:701-711.
57. Heussler VT, Kuenzi P, Rottenberg S. Inhibition of apoptosis by intracellular protozoan parasites. Int J Parasitol 2001;31:1166- 1176.
58. Chuenkova MV, Pereira MA. A trypanosomal protein synergizes with the cytokines ciliary neurotrophic factor and leukemia inhibitory factor to prevent apoptosis of neuronal cells. Mol Biol Cell 2000;11:1487-1498.
59. Chuenkova MV, Pereira MA. The T. cruzi trans-sialidase induces PC12 cell differentiation via MAPK/ERK pathway. Neuroreport 2001;12:3715-3718.
60. Chuenkova MV, Furnari FB, Cavenee WK, Pereira MA. Trypanosoma cruzi trans-sialidase: a potent and specific survival factor for human Schwann cells by means of phosphatidylinositol 3-kinase/Akt signaling. Proc Natl Acad Sci U S A 2001;98:9936-9941.
61. Aoki MP, Guinazu NL, Pellegrini AV, Gotoh T, Masih DT, Gea S. Cruzipain, a major Trypanosoma cruzi antigen, promotes arginase-2 expression and survival of neonatal mouse cardiomyocytes. Am J Physiol Cell Physiol 2004;286:C206-212.
62. Fichera LE, Albareda MC, Laucella SA, Postan M. Intracellular growth of Trypanosoma cruzi in cardiac myocytes is inhibited by cytokine-induced nitric oxide release. Infect Immun 2004;72:359-363.
63. Machado FS, Martins GA, Aliberti JC, Mestriner FL, Cunha FQ, Silva JS. Trypanosoma cruzi-infected cardiomyocytes produce chemokines and cytokines that trigger potent nitric oxide-dependent trypanocidal activity. Circulation 2000;102:3003-3008.
64. Aoki MP, Cano RC, Pellegrini AV, Tanos T, Guinazu NL, Coso OA, et al. Different signaling pathways are involved in cardiomyocyte survival induced by a Trypanosoma cruzi glycoprotein. Microbes Infect 2006.
65. Hashimoto M, Nakajima-Shimada J, Aoki T. Trypanosoma cruzi posttranscriptionally up-regulates and exploits cellular FLIP for inhibition of death-inducing signal. Mol Biol Cell 2005;16:3521-3528.
66. Petersen CA, Krumholz KA, Carmen J, Sinai AP, Burleigh BA. Trypanosoma cruzi infection and nuclear factor kappa B activation prevent apoptosis in cardiac cells. Infect Immun 2006;74:1580-1587.
67. Moore-Lai D, Rowland E. Microarray data demonstrate that Trypanosoma cruzi downregulates the expression of apoptotic genes in BALB/c fibroblasts. J Parasitol 2004;90:893-895.
68. Dosreis GA, Freire-de-Lima CG, Nunes MP, Lopes MF. The importance of aberrant T-cell responses in Chagas disease. Trends Parasitol 2005;21:237-243.
69. de Souza EM, Araujo-Jorge TC, Bailly C, Lansiaux A, Batista MM, Oliveira GM, et al. Host and parasite apoptosis following Trypanosoma cruzi infection in in vitro and in vivo models. Cell Tissue Res 2003;314:223-235.
70. Laderach D, Cerban F, Motran C, Vottero de Cima E, Gea S. Trypanosoma cruzi: the major cysteinyl proteinase (cruzipain) is a relevant immunogen of parasite acidic antigens (FIII). Int J Parasitol 1996;26:1249- 1254.
71. Sparwasser T, Hultner L, Koch ES, Luz A, Lipford GB, Wagner H. Immunostimulatory CpG-oligodeoxynucleotides cause extramedullary murine hemopoiesis. J Immunol 1999;162:2368-2374.
72. Hoft DF, LynchL RG, Kirchhoff V. Kinetic analysis of antigen-specific immune responses in resistant and susceptible mice during infection with Trypanosoma cruzi. J Immunol 1993;151:7038-7047.
73. Piacenza L, Peluffo G, Radi R. L-arginine-dependent suppression of apoptosis in Trypanosoma cruzi: contribution of the nitric oxide and polyamine pathways. Proc Natl Acad Sci U S A 2001;98:7301-7306.
74. Frank FM, Petray PB, Cazorla SI, Munoz MC, Corral RS, Malchiodi EL. Use of a purified Trypanosoma cruzi antigen and CpG oligodeoxynucleotides for immunoprotection against a lethal challenge with trypomastigotes. Vaccine 2003;22:77-86.
75. Schnapp AR, Eickhoff CS, Scharfstein J, Hoft DF. Induction of B-and T-cell responses to cruzipain in the murine model of Trypanosoma cruzi infection. Microbes Infect 2002;4:805-813.
76. Schnapp AR, Eickhoff CS, Sizemore D, Curtiss R, Hoft DF. Cruzipain induces both mucosal and systemic protection against Trypanosoma cruzi in mice. Infect Immun 2002;70:5065-5074.
77. Ruiz AM, Esteva M, Cabeza Meckert P, Laguens RP, Segura EL. Protective immunity and pathology induced by inoculation of mice with different subcellular fractions of Trypanosoma cruzi. Acta Trop 1985;42:299-309.
78. Lopez Bergami P, Cabeza Meckert P, Kaplan D, Levitus G, Elias F, Quintana F, et al. Immunization with recombinant Trypanosoma cruzi ribosomal P2beta protein induces changes in the electrocardiogram of immunized mice. FEMS Immunol Med Microbiol 1997;18:75-85.
79. Motran CC, Cerban FM, Rivarola HW, Vottero de Cima E. Characterization of autoantibodies generated in mice by immunization with the C-terminal region of Trypanosoma cruzi ribosomal P1 and P2 proteins. Clin Immunol 1999;91:17-24.
80. Giordanengo L, Fretes R, Diaz H, Cano R, Bacile A, Vottero-Cima E, et al. Cruzipain induces autoimmune response against skeletal muscle and tissue damage in mice. Muscle Nerve 2000;23:1407-1413.
81. Mirkin GA, Jones M, Sanz OP, Rey R, Sica RE, Gonzalez Cappa SM. Experimental Chagas' disease: electrophysiology and cell composition of the neuromyopathic inflammatory lesions in mice infected with a myotropic and a pantropic strain of Trypanosoma cruzi. Clin Immunol Immunopathol 1994;73:69-79.
82. Giordanengo L, Maldonado C, Rivarola H, Iosa D, Girones N, Fresno M, et al. Induction of antibodies reactive to cardiac myosin and development of heart alterations in cruzipain-immunized mice and their offspring. Eur J Immunol 2000;30:3181-3189.
83. Sterin-Borda L, Giordanengo L, Joensen L, Gea S. Cruzipain induces autoantibodies against cardiac muscarinic acetylcholine receptors. Functional and pathological implications. Eur J Immunol 2003;33:2459-2468.
84. Sterin-Borda L, Joensen L, Bayo-Hanza C, Esteva M, Borda E. Therapeutic use of muscarinic acetylcholine receptor peptide to prevent mice chagasic cardiac dysfunction. J Mol Cell Cardiol 2002;34:1645-1654.
85. Leon JS, Godsel LM, Wang K, Engman DM. Cardiac Myosin Autoimmunity in Acute Chagas' Heart Disease. Infect Immun 2001;69:5643-5649.
86. Martinez J, Campetella O, Frasch AC, Cazzulo JJ. The major cysteine proteinase (cruzipain) from Trypanosoma cruzi is antigenic in human infections. Infect Immun 1991;59:4275-4277.
87. Scharfstein J, Luquetti A, Murta AC, Senna M, Rezende JM, Rassi A, et al. Chagas' disease: serodiagnosis with purified Gp25 antigen. Am J Trop Med Hyg 1985;34:1153-1160.
88. Gazzinelli RT, Leme VM, Cancado JR, Gazzinelli G, Scharfstein J. Identification and partial characterization of Trypanosoma cruzi antigens recognized by T cells and immune sera from patients with Chagas' disease. Infect Immun 1990;58:1437-1444.
89. Martinez J, Campetella O, Frasch AC, Cazzulo JJ. The reactivity of sera from chagasic patients against different fragments of cruzipain, the major cysteine proteinase from Trypanosoma cruzi, suggests the presence of defined antigenic and catalytic domains. Immunol Lett 1993;35:191-196.
90. Duschak VG, Riarte A, Segura EL, Laucella SA. Humoral immune response to cruzipain and cardiac dysfunction in chronic Chagas disease. Immunol Lett 2001;78:135-142.
91. Laucella SA, de Titto EH, Segura EL. Epitopes common to Trypanosoma cruzi and mammalian tissues are recognized by sera from Chagas' disease patients: prognosis value in Chagas disease. Acta Trop 1996;62:151-162.
92. Gea S, Gruppi A, Basso B, Menso E, Vottero-Cima E. Antibodies to T. cruzi cytosol acidic antigens (FTV) in Chagas' disease recognize parasite cell surface and human heart epitopes. J Clin Lab Immunol 1990;31:183- 187.
93. Morrot A, Strickland DK, Higuchi Mde L, Reis M, Pedrosa R, Scharfstein J. Human T cell responses against the major cysteine proteinase (cruzipain) of Trypanosoma cruzi: role of the multifunctional alpha 2-macroglobulin receptor in antigen presentation by monocytes. Int Immunol 1997;9:825-834.
94. Arnholdt A, Piuvezam M, Russo D, Lima A, Pedrosa R, Reed S, et al. Analysis and partial epitope mapping of human T cell responses to Trypanosoma cruzi cysteinyl proteinase. J Immunol 1993;151:3171-3179.
95. Fonseca S, Moins-Teisserenc H, Clave E, Ianni B, Nunes V, Mady C, et al. Identification of multiple HLA-A*0201-restricted cruzipain and FL-160 CD8+ epitopes recognized by T cells from chronically Trypanosoma cruzi-infected patients. Microbes Infect 2005;7:688-697.
96. Martinez J, Henriksson J, Ridaker M, Pettersson U, Cazzulo J. Polymorphisms of the genes encoding cruzipain, the major cysteine proteinase of Trypanosoma cruzi, in the region encoding the C-terminal domain. FEMS Microbiol Lett 1998;159:35-39.