Mechanisms of cell death in acute liver failure

Frontiers in Physiology

Volumn 3 APR, Issue , 2012, Pages

  Bantel, Heike ^a   Schulze Osthoff, Klaus ^b  

^a HANNOVER MEDICAL SCHOOL   (Germany)

^b UNIVERSITY OF TÜBINGEN   (Germany)

Author keywords

Acute liver failure; Apoptosis; ATP; Caspases; Death receptors; Necrosis; Oxidative stress; Tumor necrosis factor

Indexed keywords

EID: 84866396691     PISSN: None     EISSN: 1664042X     Source Type: Journal    
DOI: 10.3389/fphys.2012.00079     Document Type: Review

References (89)

1. Badmann,A.,Keough,A.,Kaufmann,T., Bouillet,P.,Brunner,T.,and Corazza, N. (2011). Role of TRAIL and proapoptotic Bcl2 homolog Bim in acetaminophen-induced liver damage. Cell Death Dis. 2, e171.
2. Bantel, H., Lügering, A., Heidemann, J., Volkmann, X., Poremba, C., Strassburg, C. P., Manns, M. P., and Schulze-Osthoff, K. (2004). Detection of apoptotic caspase activation in sera from patients with chronic HCV infection is associated with fibrotic liver injury. Hepatology 40, 1078-1087.
3. Bantel, H., Lügering, A., Poremba, C., Lügering, N., Held, J., Domschke, W., and Schulze-Osthoff, K. (2001a). Caspase activation correlates with the degree of inflammatory liver injury in chronic hepatitis C virus infection. Hepatology 34, 758-767.
4. Bantel, H., Ruck, P., Gregor, M., and Schulze-Osthoff, K. (2001b). Detection of elevated caspase activation and early apoptosis in liver diseases. Eur. J. Cell Biol. 80, 230-239.
5. Bantel, H., Ruck, P., and Schulze Osthoff, K. (2000). In situ monitoring of caspase activation in hepatobiliary diseases. Cell Death Differ. 7, 504-505.
6. Bantel, H., and Schulze-Osthoff, K. (2003). Apoptosis in hepatitis C virus infection. Cell Death Differ. 10, 48-58.
7. Bantel, H., and Schulze-Osthoff, K. (2012). Can cell death biomarkers predict the outcome of acute liver failure? Liver Int. 32, 346.
8. Bechmann, L. P., Jochum, C., Kocabayoglu, P., Sowa, J. P., Kassalik, M., Gieseler, R. K., Saner, F., Paul, A., Trautwein, C., Gerken, G., and Canbay, A. (2010). Cytokeratin 18-based modification of the MELD score improves prediction of spontaneous survival after acute liver injury. J. Hepatol. 53, 639-647.
9. Bechmann, L. P., Marquitan, G., Jochum, C., Saner, F., Gerken, G., and Canbay, A. (2008). Apoptosis versus necrosis rate as a predictor in acute liver failure following acetaminophen intoxication compared with acute-on-chronic liver failure. Liver Int. 28, 713-716.
10. Bell, C. W., Jiang, W., Reich, C. F. III, and Pisetsky, D. S. (2006). The extracellular release of HMGB1 during apoptotic cell death. Am. J. Physiol. Cell Physiol. 291, 1318-1325.
11. Ben Moshe, T., Barash, H., Kang, T. B., Kim, J. C., Kovalenko, A., Gross, E., Schuchmann, M., Abramovitch, R., Galun, E., and Wallach, D. (2007). Role of caspase-8 in hepatocyte response to infection and injury in mice. Hepatology 45, 1014-1024.
12. Bennett, B. L., Sasaki, D. T., Murray, B. W., O'Leary, E. C., Sakata, S. T., Xu, W., Leisten, J. C., Motiwala, A., Pierce, S., Satoh, Y., Bhagwat, S. S., Manning, A. M., and Anderson, D. W. (2001). SP600125, an anthrapyrazolone inhibitor of Jun N-terminal kinase. Proc. Natl. Acad. Sci. U.S.A. 98, 13681-11386.
13. Blazka, M. E., Elwell, M. R., Holladay, S. D., Wilson, R. E., and Luster, M. I. (1996). Histopathology of acetaminophen-induced liver changes: role interleukin 1 alpha and tumor necrosis factor alpha. Toxicol. Pathol. 24, 181-189.
14. Blazka, M. E., Wilmer, J. L., Holladay, S. D., Wilson, R. E., and Luster, M. I. (1995). Role of proinflammatory cytokines in acetaminophen hepatotoxicity. Toxicol. Appl. Pharmacol. 133, 43-52.
15. Boess, F., Bopst, M., Althaus, R., Polsky, S., Cohen, S. D., Euster, H. P., and Boelsterli, U. A. (1998). Acetaminophen hepatotoxicity in tumor necrosis factor/lymphotoxin-alpha gene knockout mice. Hepatology 27, 1021-1029.
16. Borsello, T., Clarke, P. G., Hirt, L., Vercelli, A., Repici, M., Schorderet, D. F., Bogousslavsky, J., and Bonny, C. (2003). A peptide inhibitor of c-Jun N-terminal kinase protects against excitotoxicity and cerebral ischemia. Nat. Med. 9, 1180-1186.
17. Bourdi, M., Korrapati, M. C., Chakraborty, M., Yee, S. B., and Pohl, L. R. (2008). Protective role of c-Jun N-terminal kinase 2 in acetaminophen-induced liver injury. Biochem. Biophys. Res. Commun. 374, 6-10.
18. Canbay, A., Higuchi, H., Bronk, S. F., Taniai, M., Sebo, T. J., and Gores, G. J. (2003). Fas enhances fibrogenesis in the bile duct ligated mouse: a link between apoptosis and fibrosis. Gastroenterology 123, 1323-1330.
19. Chan, K., Truong, D., Shangari, N., and O'Brien, P. J. (2005). Drug-induced mitochondrial toxicity. Expert Opin. Drug Metab. Toxicol. 4, 655-669.
20. Chang, C. C., Wang, S. S., Huang, H. C., Chan, C. Y., Lee, F. Y., Lin, H. C., Nong, J. Y., Chuang, C. L., and Lee, S. D. (2011). Selective cyclooxygenase inhibition improves hepatic encephalopathy in fulminant hepatic failure of rat. Eur. J. Pharmacol. 666, 226-232.
21. Corcoran, G. B., Racz, W. J., Smith, C. V., and Mitchell, J. R. (1985). Effects of N-acetylcysteine on acetaminophen covalent binding and hepatic necrosis in mice. J. Pharmacol. Exp. Ther. 232, 864-872.
22. Craig, D. G., Lee, P., Pryde, E. A., Masterton, G. S., Hayes, P. C., and Simpson, K. J. (2011). Circulating apoptotic and necrotic cell death markers in patients with acute liver injury. Liver Int. 31, 1127-1136.
23. Dear, J. W., Simpson, K. J., Nicolai, M. P., Catterson, J. H., Street, J., Huizinga, T., Craig, D. G., Dhaliwal, K., Webb, S., Bateman, D. N., and Webb, D. J. (2011). Cyclophilin A is a damageassociated molecular pattern molecule that mediates acetaminopheninduced liver injury. J. Immunol. 187, 3347-3352.
24. Desbarats, J., and Newell, M. K. (2000). Fas engagement accelerates liver regeneration after partial hepatectomy. Nat. Med. 6, 920-923.
25. El-Hassan, H., Anwar, K., Macanas-Pirard, P., Crabtree, M., Chow, S. C., Johnson, V. L., Lee, P. C., Hinton, R. H., Price, S. C., and Kass, G. E. (2003). Involvement of mitochondria in acetaminopheninduced apoptosis and hepatic injury: roles of cytochrome c, bax, bid, and caspases. Toxicol. Appl. Pharmacol. 191, 118-129.
26. El-Mofty, S. K., Scrutton, M. C., Serroni, A., Nicolini, C., and Farber, J. L. (1975). Early, reversible plasma membrane injury in galactosamineinduced liver cell death. Am. J. Pathol. 79, 579-596.
27. Faouzi, S., Burckhardt, B. E., Hanson, J. C., Campe, C. B., Schrum, L. W., Rippe, R. A., and Maher, J. J. (2001). Anti-Fas induces hepatic chemokines and promotes inflammation by an NF-kappa Bindependent, caspase-3-dependent pathway. J. Biol. Chem. 276, 49077-49082.
28. Ferrari, D., Stepzynska,A., Los, M.,Wesselborg, S., and Schulze-Osthoff, K. (1998). Differential regulation and ATP requirement for caspase-8 and-3 activation during CD95-and anti-cancer drug-induced apoptosis. J. Exp. Med. 188, 979-984.
29. Fischer, U., Jänicke, R. U., and Schulze Osthoff, K. (1998). Many cuts to ruin: a comprehensive update of caspase substrates. Cell Death Differ. 10, 76-100.
30. Fischer, U., and Schulze-Osthoff, K. (2005). New approaches and therapeutics targeting apoptosis in disease. Pharmacol. Rev. 57, 187-215.
31. Galanos, C., Freudenberg, M. A., and Reutter, W. (1979). Galactosamineinduced sensitization to the lethal effects of endotoxin. Proc. Natl. Acad. Sci. U.S.A. 76, 5939-5943.
32. Gujral, J. S., Knight, T. R., Farhood, A., Bajt, M. L., and Jaeschke, H. (2002). Mode of cell death after acetaminophen overdose in mice: apoptosis or oncotic necrosis? Toxicol. Sci. 67, 322-328.
33. Gunawan, B. K., Liu, Z. X., Han, D., Hanawa, N., Gaarde, W. A., and Kaplowitz, N. (2006). C-Jun N-terminal kinase plays a major role in murine acetaminophen hepatotoxicity. Gastroenterology 131, 165-178.
34. Henderson, N. C., Pollock, K. J., Frew, J., Mackinnon, A. C., Flavell, R. A., Davis, R. J., Sethi, T., and Simpson, K. J. (2007). Critical role of c-jun (NH2) terminal kinase in paracetamol-induced acute liver failure. Gut 56, 982-990.
35. Hikita, H., Takehara, T., Kodama, T., Shimizu, S., Shigekawa, M., Hosui, A., Miyagi, T., Tatsumi, T., Ishida, H., Li, W., Kanto, T., Hiramatsu, N., Shimizu, S., Tsujimoto, Y., and Shimizu, N. (2011). Delayed-onset caspase-dependent massive hepatocyte apoptosis upon Fas activation in Bak/Bax-deficient mice. Hepatology 54, 240-251.
36. Hinson, J. A., Roberts, D. W., and James, L. P. (2010). Mechanisms of acetaminophen-induced liver necrosis. Handb. Exp. Pharmacol. 196, 369-405.
37. Hiramatsu, N., Hayashi, N., Katayama, K., Mochizuki, K., Kawanishi, Y., Kasahara, A., Fusamoto, H., and Kamada, T. (1994). Immunohistochemical detection of Fas antigen in liver tissue of patients with chronic hepatitis C. Hepatology 19, 1354-1359.
38. Hu, B., and Colletti, L. M. (2010). CXC receptor-2 knockout genotype increases X-linked inhibitor of apoptosis protein and protects mice from acetaminophen hepatotoxicity. Hepatology 52, 691-702.
39. Jaeschke, H., Cover, C., and Bajt, M. L. (2006). Role of caspases in acetaminophen-induced liver injury. Life Sci. 78, 1670-1676.
40. Jaeschke, H., Williams, C. D., Ramachandran, A., and Bajt, M. L. (2012). Acetaminophen hepatotoxicity and repair: the role of sterile inflammation and innate immunity. Liver Int. 32, 8-20.
41. Joka, D., Wahl, K., Moeller, S., Schlue, J., Vaske, B., Bahr, M. J., Manns, M. P., Schulze-Osthoff, K., and Bantel, H. (2012). Prospective biopsy-controlled evaluation of cell death biomarkers for prediction of liver fibrosis and nonalcoholic steatohepatitis. Hepatology 55, 455-464.
42. Keppler, D., Lesch, R., Reutter, W., and Decker, K. (1968). Experimental hepatitis induced by Dgalactosamine. Exp. Mol. Pathol. 9, 279-290.
43. Kerr, J. F. R.,Wyllie,A. H., and Currie,A. R. (1972). Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 26, 239-257.
44. Knight, T. R., and Jaeschke, H. (2002). Acetaminophen-induced inhibition of Fas receptor-mediated liver cell apoptosis: mitochondrial dysfunction versus glutathione depletion. Toxicol. Appl. Pharmacol. 181, 133-141.
45. Kon, K., Kim, J. S., Jaeschke, H., and Lemasters, J. J. (2004). Mitochondrial permeability transition in acetaminophen-induced necrosis and apoptosis of cultured mouse hepatocytes. Hepatology 40, 1170-1179.
46. Larson, A. M., Polson, J., Fontana, R. J., Davern, T. J., Lalani, E., Hynan, L. S., Reisch, J. S., Schiødt, F. V., Ostapowicz, G., Shakil, A. O., and Lee, W. M. (2005). Acute Liver Failure Study Group. Acetaminopheninduced acute liver failure: results of a United States multicenter, prospective study. Hepatology 42, 1364-1372.
47. Lawson, J. A., Fisher, M. A., Simmons, C. A., Farhood, A., and Jaeschke, H. (1999). Inhibition of Fas receptor (CD95)-induced hepatic caspase activation and apoptosis by acetaminophen in mice. Toxicol. Appl. Pharmacol. 156, 179-186.
48. Lee, J. Y., Chae, D. W., Kim, S. M., Nam, E. S., Jang, M. K., Lee, J. H., Kim, H. Y., and Yoo, J. Y. (2004). Expression of FasL and perforin/granzyme B mRNA in chronic hepatitis B virus infection. J. Viral Hepat. 11, 130-135.
49. Leers, M. P. G., Kölgen, W., Björklund, V., Bergmann, T., Gordon, T., Persson, B., Björklund, P., Ramaekers, F. C., Björklund, B., Nap, M., Jörnvall, H., and Schutte, B. (1999). Immunohistochemical detection and mapping of cytokeratin 18 neo-epitope exposed during early apoptosis. J. Pathol. 187, 567-572.
50. Leist, M., Gantner, F., Bohlinger, I., Germann, P. G., Tiegs, G., and Wendel, A. (1994). Murine hepatocyte apoptosis induced in vitro and in vivo by TNF-alpha requires transcriptional arrest. J. Immunol. 153, 1778-1788.
51. Leist, M., Gantner, F., Bohlinger, I., Tiegs, G., Germann, P. G., and Wendel, A. (1995). Tumor necrosis factor-induced hepatocyte apoptosis precedes liver failure in experimental murine shock models. Am. J. Pathol. 146, 1220-1234.
52. Libert, C., Brouckaert, P., and Fiers, W. (1994). Protection by alpha 1-acid glycoprotein against tumor necrosis factor-induced lethality. J. Exp. Med. 180, 1571-1575.
53. Liong, E. C., Xiao, J., Lau, T. Y., Nanji, A. A., and Tipoe, G. L. (2012). Cyclooxygenase inhibitors protect d-galactosamine/lipopolysaccharide induced acute hepatic injury in experimental mice model. Food Chem. Toxicol. 50, 861-866.
54. Liu, Z. X., Govindarajan, S., and Kaplowitz, N. (2004). Innate immune system plays a critical role in determining the progression and severity of acetaminophen hepatotoxicity. Gastroenterology 127, 1760-1774.
55. Los, M., Mozoluk, M., Ferrari, D., Stepczynska, A., Stroh, C., Renz, A., Herceg, Z., Wang, Z. Q., and Schulze-Osthoff, K. (2002). Activation and caspase-mediated inhibition of PARP: a molecular switch between necrosis and apoptosis in death receptor signaling. Mol. Biol. Cell 13, 978-988.
56. Los, M., Wesselborg, S., and Schulze Osthoff, K. (1999). The role of caspases in development, immunity and apoptotic signal transduction: lessons from knock-out mice. Immunity 10, 629-639.
57. Mita, E., Hayashi, N., Iio, S., Takehara, T., Hijioka, T., Kasahara, A., Fusamoto, H., and Kamada, T. (1994). Role of Fas ligand in apoptosis induced by hepatitis C virus infection. Biochem. Biophys. Res. Commun. 204, 468-474.
58. Nakae, H., Narita, K., and Endo, S. (2001). Soluble Fas and soluble Fas ligand levels in patients with acute hepatic failure. J. Crit. Care 16, 59-63.
59. Ni, H. M., Bockus, A., Boggess, N., Jaeschke, H., and Ding, W. X. (2012). Activation of autophagy protects against acetaminopheninduced hepatotoxicity. Hepatology 55, 222-232.
60. Ogasawara, J., Watanabe-Fukunaga, R., Adachi, M., Matsuzawa, A., Kasugai, T, Kitamura,Y.,Itoh,N.,Suda,T.,and Nagata, S. (1993). Lethal effect of the anti-Fas antibody in mice. Nature 364, 806-809.
61. Pianko, S., Patella, S., Ostapowicz, G., Desmond, P., and Sievert, W. (2001). Fas-mediated hepatocyte apoptosis is increased by hepatitis C virus infection and alcohol consumption, and may be associated with hepatic fibrosis: mechanisms of liver cell injury in chronic hepatitis C virus infection. J. Viral Hepat. 8, 406-413.
62. Ray, S. D., Mumaw, V. R., Raje, R. R., and Fariss, M. W. (1996). Protection of acetaminophen-induced hepatocellular apoptosis and necrosis by cholesteryl hemisuccinate pretreatment. J. Pharmacol. Exp. Ther. 279, 1470-1483.
63. Rivero, M., Crespo, J., Fabrega, E., Casafont,F.,Mayorga,M.,Gomez-Fleitas, M., and Pons-Romero, F. (2002). Apoptosis mediated by the Fas system in the fulminant hepatitis by hepatitis B virus. J. Viral Hepat. 9, 107-113.
64. Roth, G. A., Lubsczyk, B. A., Pilz, J., Faybik, P., Hetz, H., and Krenn, C. G. (2009). Nucleosome serum levels in acute hepatic failure and MARS treatment. Transplant. Proc. 41, 4207-4210.
65. Russell, S. W., Rosenau, W., and Lee, J. C. (1972). Cytolysis induced by human lymphotoxin. Am. J. Pathol. 69, 103-118.
66. Rutherford, A. E., Hynan, L. S., Borges, C. B., Forcione, D. G., Blackard, J. T., Lin, W., Gorman, A. R., Obaid, S. S., Reuben, A., Harrison, E., Reddy, K. R., Le, W. M., Chung, R. T., and for the ALF Study Group. (2007). Serum apoptosis markers in acute liver failure: a pilot study. Clin. Gastroenterol. Hepatol. 5, 1477-1483.
67. Ryo, K., Kamogawa, Y., Ikeda, I., Yamauchi, K.,Yonehara, S., Nagat, S., and Hayashi, N. (2000). Significance of Fas antigen-mediated apoptosis in human fulminant hepatic failure. Am. J. Gastroenterol. 95, 2047-2055.
68. Saito, C., Zwingmann, C., and Jaeschke, H. (2010). Novel mechanisms of protection against acetaminophen hepatotoxicity in mice by glutathione and N-acetylcysteine. Hepatology 51, 246-254.
69. Schulze-Osthoff, K., and Bantel, H. (2011). Necrosis versus apoptosis in acetaminophen-induced hepatotoxicity. Hepatology 53, 1070.
70. Schulze-Osthoff, K., Ferrari, D., Los, M., Wesselborg, S., and Peter, M. E. (1998). Apoptosis signaling by death receptors. Eur. J. Biochem. 254, 439-459.
71. Schulze-Osthoff, K., Krammer, P. H., and Dröge, W. (1994). Divergent signaling via APO-1/Fas and the TNF receptor,two homologous molecules involved in physiological cell death. EMBO J. 13, 4587-4596.
72. Schwerk, C., and Schulze-Osthoff, K. (2005). Regulation of apoptosis by alternative pre-mRNA splicing. Mol. Cell 19, 1-13.
73. Seidel, N., Volkmann, X., Länger, F., Flemming, P., Manns, M. P., Schulze-Osthoff, K., and Bantel, H. (2005). The extent of liver steatosis in chronic hepatitis C virus infection is mirrored by caspase activity in serum. Hepatology 42, 113-120.
74. Sharma,A. D.,Narain,N.,Händel,E. M., Iken, M., Singhal, N., Cathomen, T., Manns, M. P., Schöler, H. R., Ott, M., and Cantz, T. (2011). MicroRNA221 regulates FAS-induced fulminant liver failure. Hepatology 53, 1651-1661.
75. Simpson, K. J., Lukas, N. W., McGregor, A. H., Harrison, D. J., Strieter, R. M., and Kunkel, S. L. (2000). Inhibition of tumor necrosis factor alpha does not prevent experimental paracetamol-induced hepatic necrosis. J. Pathol. 190, 489-494.
76. Song, E., Lee, S. K., Wang, J., Ince, N., Ouyang, N., Min, J., Chen, J., Shankar, P., and Lieberman, J. (2003). RNA interference targeting Fas protects mice from fulminant hepatitis. Nat. Med. 3, 347-351.
77. Strand, S., Hofmann, W. J., Grambihler, A., Hug, H., Volkmann, M., Otto, G., Wesch, H., Mariani, S. M., Hack, V., Krammer, P. H., and Galle, P. R. (1998). Hepatic failure and liver cell damage in acute Wilson's disease involve CD95 (APO-1/Fas) mediated apoptosis. Nat. Med. 4, 588-593.
78. Streetz, K., Leifeld, L., Grundmann, D., Ramakers, J., Eckert, K., Spengler, U., Brenner, D., Manns, M., and Trautwein, C. (2000). Tumor necrosis factor alpha in the pathogenesis of human and murine fulminant hepatic failure. Gastroenterology 119, 446-460.
79. Tagami, A., Ohnishi, H., and Huges, R. D. (2003). Increased serum soluble Fas in patients with acute liver failure due to paracetamol overdose. Hepatogastroenterology 5, 1477-1483.
80. Tinel, M., Berson,A.,Vadrot, M., Descatoire, V., Grodet, A., Feldmann, G., Thénot, J. P., and Pessayre, D. (2004). Subliminal Fas stimulation increases the hepatotoxicity of acetaminophen and bromobenzene in mice. Hepatology 39, 655-666.
81. Tokushige, K., Yamaguchi, N., Ikeda, I., Hashimoto, E., Yamauchi, K., and Hayashi, N. (2000). Significance of soluble TNF receptor I in acute type fulminant heaptitis. Am.J.Gastroenterol. 95, 2040-2046.
82. Vandenabeele, P., Galluzzi, L., Berghe, T. V., and Kroemer, G. (2010). Molecular mechanisms of necroptosis: an ordered cellular explosion. Nat. Rev. Mol. Cell Biol. 11, 700-714.
83. Volkmann, X., Ansteatt, M., Hadem, J., Stiefel, P., Bahr, M. J., Lehner, F., Manns, M. P., Schulze-Osthoff, K., and Bantel, H. (2008). Caspase activation is associated with spontaneous recovery from acute liver failure. Hepatology 47, 1624-1633.
84. Volkmann, X., Cornberg, M., Wedemeyer, H., Lehner, F., Manns, M. P., Schulze-Osthoff, K., and Bantel, H. (2006). Caspase activation is required for antiviral treatment response in chronic hepatitis C virus infection. Hepatology 43, 1311-1316.
85. Volkmann, X., Fischer, U., Bahr, M. J., Ott, M., Lehner, F., Macfarlane, M., Cohen,G. M.,Manns,M. P.,Schulze Osthoff, K., and Bantel, H. (2007). Increased hepatotoxicity of tumor necrosis factor-related apoptosisinducing ligand in diseased human liver. Hepatology 46, 1498-1508.
86. Wullaert, A., van Loo, G., Heynick, K., and Beyaert, R. (2007). Hepatic tumor necrosis factor signaling and nclear factor-kappaB: effects on liver homeostasis and beyond. Endocr. Rev. 28, 365-386.
87. Yoon, J. H., and Gores, G. J. (2002). Death receptor-mediated apoptosis and the liver. J. Hepatol. 37, 400-410.
88. Zender, L., Hutker, S., Liedtke, C., Tillmann, H. L., Zender, S., Mundt, B., Waltemathe, M., Gosling, T., Flemming, P., Malek, N. P., Trautwein, C., Manns, M. P., Kuhnel, F., and Kubicka, S. (2003). Caspase [8] small interfering RNA prevents acute liver failure in mice. Proc. Natl. Acad. Sci. U.S.A. 105, 9053-9058.
89. Zhang, H., Cook, J., Nickel, J., Yu, R., Stecker, K., Myers, K., and Dean, N. M. (2000). Reduction of liver Fas expression by antisense oligonucleotide protects mice from fulminant hepatitis. Nat. Biotechnol. 18, 862-867.