Cancer immunotherapy with chemoattractant peptides

Seminars in Cancer Biology

Volumn 14, Issue 3, 2004, Pages 209-218

  Coscia, Marta ^a,c   Biragyn, Arya ^b  

^a UNIVERSITY OF TURIN   (Italy)

^b NATIONAL INSTITUTE ON AGING   (United States)

^c SAIC FREDERICK INC   (United States)

Author keywords

Cancer immunotherapy; Chemoattractant peptides; Chemokine chemokine receptor network

Indexed keywords

1,1' [1,4 PHENYLENEBIS(METHYLENE)]BIS(1,4,8,11 TETRAAZACYCLOTETRADECANE); ALPHA CHEMOKINE; ANTIVIRUS AGENT; BETA DEFENSIN 2; BETA DEFENSIN 3; BETA DEFENSIN 4; CANCER VACCINE; CHEMOATTRACTANT; CHEMOKINE; CHEMOKINE RECEPTOR; CHEMOKINE RECEPTOR CCR6; DEFENSIN; FIBROBLAST GROWTH FACTOR 1; FRAMYCETIN; HAPTEN; INTERLEUKIN 10; INTERLEUKIN 12; MACROPHAGE INFLAMMATORY PROTEIN 3BETA; METALLOPROTEINASE; MONOCLONAL ANTIBODY; MONOCYTE CHEMOTACTIC PROTEIN 3; POLYMER; PR 39; RECEPTOR BLOCKING AGENT; SINGLE CHAIN FRAGMENT VARIABLE ANTIBODY; THROMBOCYTE FACTOR 4; TUMOR VACCINE; UNINDEXED DRUG; VASCULOTROPIN; VASCULOTROPIN ANTIBODY;

ANGIOGENESIS; ANTIBODY PRODUCTION; ANTIGEN PRESENTING CELL; ANTINEOPLASTIC ACTIVITY; CANCER; CANCER CELL; CANCER IMMUNOTHERAPY; CANCER INHIBITION; CANCER THERAPY; CLINICAL TRIAL; HOMEOSTASIS; HUMAN; HUMORAL IMMUNITY; IMMUNE RESPONSE; IMMUNOSURVEILLANCE; LEUKOCYTE; METASTASIS; NEOVASCULARIZATION (PATHOLOGY); NONHUMAN; REVIEW; TREATMENT OUTCOME; TUMOR GROWTH;

EID: 2342514244     PISSN: 1044579X     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.semcancer.2003.10.008     Document Type: Article

References (91)

1. Bacon K., Baggiolini M., Broxmeyer H., Horuk R., Lindley I., Mantovani A., et al. Chemokine/chemokine receptor nomenclature. J. Interferon Cytokine Res. 22(10):2002;1067-1068
2. Moser B., Loetscher P. Lymphocyte traffic control by chemokines. Nat. Immunol. 2(2):2001;123-128
3. Homey B., Muller A., Zlotnik A. Chemokines: agents for the immunotherapy of cancer? Nat. Rev. Immunol. 2(3):2002;175-184
4. Rosenberg S.A. A new era for cancer immunotherapy based on the genes that encode cancer antigens. Immunity. 10(3):1999;281-287
5. Melief C.J., Kast W.M. T-cell immunotherapy of tumors by adoptive transfer of cytotoxic T lymphocytes and by vaccination with minimal essential epitopes. Immunol. Rev. 145:1995;167-177
6. Wang R.F. The role of MHC class II-restricted tumor antigens and CD4+ T cells in antitumor immunity. Trends Immunol. 22(5):2001;269-276
7. Sallusto F., Palermo B., Lenig D., Miettinen M., Matikainen S., Julkunen I., et al. Distinct patterns and kinetics of chemokine production regulate dendritic cell function. Eur. J. Immunol. 29(5):1999;1617-1625
8. Dieu M.C., Vanbervliet B., Vicari A., Bridon J.M., Oldham E., Ait-Yahia S., et al. Selective recruitment of immature and mature dendritic cells by distinct chemokines expressed in different anatomic sites. J. Exp. Med. 188(2):1998;373-386
9. Campbell M.J., Esserman L., Byars N.E., Allison A.C., Levy R. Idiotype vaccination against murine B cell lymphoma. Humoral and cellular requirements for the full expression of antitumor immunity. J. Immunol. 145(3):1990;1029-1036
10. Biragyn A., Tani K., Grimm M.C., Weeks S.D., Kwak L.W. Genetic fusion of chemokines to a self tumor antigen induces protective, T-cell dependent antitumor immunity. Nat. Biotechnol. 17:1999;253-258
11. Coussens L.M., Werb Z. Inflammation and cancer. Nature. 420(6917):2002;860-867
12. Zilberberg L, Shinkaruk S, Lequin O, Rousseau B, Hagedorn M, Costa F, et al. Structure and inhibitory effects on angiogenesis and tumor development of a new vascular endothelial growth inhibitor. J Biol Chem 2003.
13. Glade-Bender J., Kandel J.J., Yamashiro D.J. VEGF blocking therapy in the treatment of cancer. Expert Opin. Biol. Ther. 3(2):2003;263-276
14. Bernardini G., Spinetti G., Ribatti D., Camarda G., Morbidelli L., Ziche M., et al. I-309 binds to and activates endothelial cell functions and acts as an angiogenic molecule in vivo. Blood. 96(13):2000;4039-4045
15. Salcedo R., Young H.A., Ponce M.L., Ward J.M., Kleinman H.K., Murphy W.J., et al. Eotaxin (CCL11) induces in vivo angiogenic responses by human CCR3+ endothelial cells. J. Immunol. 166(12):2001;7571-7578
16. Volin M.V., Woods J.M., Amin M.A., Connors M.A., Harlow L.A., Koch A.E. Fractalkine: a novel angiogenic chemokine in rheumatoid arthritis. Am. J. Pathol. 159(4):2001;1521-1530
17. Wierda W.G., Johnson M.M., Do K.A., Manshouri T., Dey A., O'Brien S., et al. Plasma interleukin 8 level predicts for survival in chronic lymphocytic leukaemia. Br. J. Haematol. 120(3):2003;452-456
18. White E.S., Flaherty K.R., Carskadon S., Brant A., Iannettoni M.D., Yee J., et al. Macrophage migration inhibitory factor and CXC chemokine expression in non-small cell lung cancer: role in angiogenesis and prognosis. Clin. Cancer Res. 9(2):2003;853-860
19. Muller A., Homey B., Soto H., Ge N., Catron D., Buchanan M.E., et al. Involvement of chemokine receptors in breast cancer metastasis. Nature. 410(6824):2001;50-56
20. Belperio J.A., Keane M.P., Arenberg D.A., Addison C.L., Ehlert J.E., Burdick M.D., et al. CXC chemokines in angiogenesis. J. Leukoc. Biol. 68(1):2000;1-8
21. Arenberg D.A., Kunkel S.L., Polverini P.J., Glass M., Burdick M.D., Strieter R.M. Inhibition of interleukin-8 reduces tumorigenesis of human non-small cell lung cancer in SCID mice. J. Clin. Invest. 97(12):1996;2792-2802
22. Arenberg D.A., Keane M.P., DiGiovine B., Kunkel S.L., Morris S.B., Xue Y.Y., et al. Epithelial-neutrophil activating peptide (ENA-78) is an important angiogenic factor in non-small cell lung cancer. J. Clin. Invest. 102(3):1998;465-472
23. Luan J., Shattuck-Brandt R., Haghnegahdar H., Owen J.D., Strieter R., Burdick M., et al. Mechanism and biological significance of constitutive expression of MGSA/GRO chemokines in malignant melanoma tumor progression. J. Leukoc. Biol. 62(5):1997;588-597
24. Choi S.J., Cruz J.C., Craig F., Chung H., Devlin R.D., Roodman G.D., et al. Macrophage inflammatory protein 1-alpha is a potential osteoclast stimulatory factor in multiple myeloma. Blood. 96(2):2000;671-675
25. Abe M., Hiura K., Wilde J., Moriyama K., Hashimoto T., Ozaki S., et al. Role for macrophage inflammatory protein (MIP)-1alpha and MIP-1beta in the development of osteolytic lesions in multiple myeloma. Blood. 100(6):2002;2195-2202
26. Choi S.J., Oba Y., Gazitt Y., Alsina M., Cruz J., Anderson J., et al. Antisense inhibition of macrophage inflammatory protein 1-alpha blocks bone destruction in a model of myeloma bone disease. J. Clin. Invest. 108(12):2001;1833-1841
27. Nanki T., Hayashida K., El Gabalawy H.S., Suson S., Shi K., Girschick H.J., et al. Stromal cell-derived factor-1-CXC chemokine receptor 4 interactions play a central role in CD4+ T cell accumulation in rheumatoid arthritis synovium. J. Immunol. 165(11):2000;6590-6598
28. Corcione A., Ottonello L., Tortolina G., Facchetti P., Airoldi I., Guglielmino R., et al. Stromal cell-derived factor-1 as a chemoattractant for follicular center lymphoma B cells. J. Natl. Cancer Inst. 92(8):2000;628-635
29. Burger J.A., Burger M., Kipps T.J. Chronic lymphocytic leukemia B cells express functional CXCR4 chemokine receptors that mediate spontaneous migration beneath bone marrow stromal cells. Blood. 94(11):1999;3658-3667
30. Feng Y., Broder C.C., Kennedy P.E., Berger E.A. HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor. Science. 272(5263):1996;872-877
31. Hatse S., Princen K., Bridger G., De Clercq E., Schols D. Chemokine receptor inhibition by AMD3100 is strictly confined to CXCR4. FEBS Lett. 527(1-3):2002;255-262
32. Matthys P., Hatse S., Vermeire K., Wuyts A., Bridger G., Henson G.W., et al. AMD3100, a potent and specific antagonist of the stromal cell-derived factor-1 chemokine receptor CXCR4, inhibits autoimmune joint inflammation in IFN-gamma receptor-deficient mice. J. Immunol. 167(8):2001;4686-4692
33. Catani M.V., Corasaniti M.T., Ranalli M., Amantea D., Litovchick A., Lapidot A., et al. The Tat antagonist neomycin B hexa-arginine conjugate inhibits gp-120-induced death of human neuroblastoma cells. J. Neurochem. 84(6):2003;1237-1245
34. Bertolini F., Dell'Agnola C., Mancuso P., Rabascio C., Burlini A., Monestiroli S., et al. CXCR4 neutralization, a novel therapeutic approach for non-Hodgkin's lymphoma. Cancer Res. 62(11):2002;3106-3112
35. Addison C.L., Arenberg D.A., Morris S.B., Xue Y.Y., Burdick M.D., Mulligan M.S., et al. The CXC chemokine, monokine induced by interferon-gamma, inhibits non-small cell lung carcinoma tumor growth and metastasis. Hum. Gene Ther. 11(2):2000;247-261
36. Salcedo R., Resau J.H., Halverson D., Hudson E.A., Dambach M., Powell D., et al. Differential expression and responsiveness of chemokine receptors (CXCR1-3) by human microvascular endothelial cells and umbilical vein endothelial cells. FASEB J. 14(13):2000;2055-2064
37. Li A., Dubey S., Varney M.L., Dave B.J., Singh R.K. IL-8 directly enhanced endothelial cell survival, proliferation, and matrix metalloproteinases production and regulated angiogenesis. J. Immunol. 170(6):2003;3369-3376
38. Bello L., Lucini V., Carrabba G., Giussani C., Machluf M., Pluderi M., et al. Simultaneous inhibition of glioma angiogenesis, cell proliferation, and invasion by a naturally occurring fragment of human metalloproteinase-2. Cancer Res. 61(24):2001;8730-8736
39. Maione T.E., Gray G.S., Petro J., Hunt A.J., Donner A.L., Bauer S.I., et al. Inhibition of angiogenesis by recombinant human platelet factor-4 and related peptides. Science. 247(4938):1990;77-79
40. Perollet C., Han Z.C., Savona C., Caen J.P., Bikfalvi A. Platelet factor 4 modulates fibroblast growth factor 2 (FGF-2) activity and inhibits FGF-2 dimerization. Blood. 91(9):1998;3289-3299
41. Jouan V., Canron X., Alemany M., Caen J.P., Quentin G., Plouet J., et al. Inhibition of in vitro angiogenesis by platelet factor-4-derived peptides and mechanism of action. Blood. 94(3):1999;984-993
42. Gupta S.K., Singh J.P. Inhibition of endothelial cell proliferation by platelet factor-4 involves a unique action on S phase progression. J. Cell Biol. 127(4):1994;1121-1127
43. Tanaka T., Manome Y., Wen P., Kufe D.W., Fine H.A. Viral vector-mediated transduction of a modified platelet factor 4 cDNA inhibits angiogenesis and tumor growth. Nat. Med. 3(4):1997;437-442
44. Bello L., Giussani C., Carrabba G., Pluderi M., Lucini V., Pannacci M., et al. Suppression of malignant glioma recurrence in a newly developed animal model by endogenous inhibitors. Clin. Cancer Res. 8(11):2002;3539-3548
45. Dorsey R., Kundu N., Yang Q., Tannenbaum C.S., Sun H., Hamilton T.A., et al. Immunotherapy with interleukin-10 depends on the CXC chemokines inducible protein-10 and monokine induced by IFN-gamma. Cancer Res. 62(9):2002;2606-2610
46. Yao L., Pike S.E., Setsuda J., Parekh J., Gupta G., Raffeld M., et al. Effective targeting of tumor vasculature by the angiogenesis inhibitors vasostatin and interleukin-12. Blood. 96(5):2000;1900-1905
47. Arenberg D.A., Kunkel S.L., Polverini P.J., Morris S.B., Burdick M.D., Glass M.C., et al. Interferon-gamma-inducible protein 10 (IP-10) is an angiostatic factor that inhibits human non-small cell lung cancer (NSCLC) tumorigenesis and spontaneous metastases. J. Exp. Med. 184(3):1996;981-992
48. Braun S.E., Chen K., Foster R.G., Kim C.H., Hromas R., Kaplan M.H., et al. The CC chemokine CK beta-11/MIP-3 beta/ELC/Exodus 3 mediates tumor rejection of murine breast cancer cells through NK cells. J. Immunol. 164(8):2000;4025-4031
49. Luster A.D., Leder P. IP-10, a -C-X-C-chemokine, elicits a potent thymus-dependent antitumor response in vivo. J. Exp. Med. 178(3):1993;1057-1065
50. Vicari A.P., Ait-Yahia S., Chemin K., Mueller A., Zlotnik A., Caux C. Antitumor effects of the mouse chemokine 6Ckine/SLC through angiostatic and immunological mechanisms. J. Immunol. 165(4):2000;1992-2000
51. Cravens P.D., Lipsky P.E. Dendritic cells, chemokine receptors and autoimmune inflammatory diseases. Immunol. Cell Biol. 80(5):2002;497-505
52. McColl S.R. Chemokines and dendritic cells: a crucial alliance. Immunol. Cell Biol. 80(5):2002;489-496
53. Dieu-Nosjean M.C., Massacrier C., Homey B., Vanbervliet B., Pin J.J., Vicari A., et al. Macrophage inflammatory protein 3alpha is expressed at inflamed epithelial surfaces and is the most potent chemokine known in attracting Langerhans cell precursors. J. Exp. Med. 192(5):2000;705-718
54. Kumamoto T., Huang E.K., Paek H.J., Morita A., Matsue H., Valentini R.F., et al. Induction of tumor-specific protective immunity by in situ Langerhans cell vaccine. Nat. Biotechnol. 20(1):2002;64-69
55. Xia D.J., Zhang W.P., Zheng S., Wang J., Pan J.P., Wang Q., et al. Lymphotactin cotransfection enhances the therapeutic efficacy of dendritic cells genetically modified with melanoma antigen gp100. Gene Ther. 9(9):2002;592-601
56. Bruna-Romero O., Schmieg J., Del Val M., Buschle M., Tsuji M. The dendritic cell-specific chemokine, dendritic cell-derived CC chemokine 1, enhances protective cell-mediated immunity to murine malaria. J. Immunol. 170(6):2003;3195-3203
57. Kershaw M.H., Wang G., Westwood J.A., Pachynski R.K., Tiffany H.L., Marincola F.M., et al. Redirecting migration of T cells to chemokine secreted from tumors by genetic modification with CXCR2. Hum. Gene Ther. 13(16):2002;1971-1980
58. Ludewig B., Ochsenbein A.F., Odermatt B., Paulin D., Hengartner H., Zinkernagel R.M. Immunotherapy with dendritic cells directed against tumor antigens shared with normal host cells results in severe autoimmune disease. J. Exp. Med. 191(5):2000;795-804
59. Cui Y., Kelleher E., Straley E., Fuchs E., Gorski K., Levitsky H., et al. Immunotherapy of established tumors using bone marrow transplantation with antigen gene-modified hematopoietic stem cells. Nat. Med. 9(7):2003;952-958
60. Wright S.D., Ramos R.A., Tobias P.S., Ulevitch R.J., Mathison J.C. CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science. 249(4975):1990;1431-1433
61. Sweet M.J., Hume D.A. Endotoxin signal transduction in macrophages. J. Leukoc. Biol. 60(1):1996;8-26
62. Tsutsumi-Ishii Y., Nagaoka I. NF-kappa B-mediated transcriptional regulation of human beta-defensin-2 gene following lipopolysaccharide stimulation. J. Leukoc. Biol. 71(1):2002;154-162
63. Engering A.J., Cella M., Fluitsma D., Brockhaus M., Hoefsmit E.C., Lanzavecchia A., et al. The mannose receptor functions as a high capacity and broad specificity antigen receptor in human dendritic cells. Eur. J. Immunol. 27(9):1997;2417-2425
64. Guyre P.M., Graziano R.F., Goldstein J., Wallace P.K., Morganelli P.M., Wardwell K., et al. Increased potency of Fc-receptor-targeted antigens. Cancer Immunol. Immunother. 45(3/4):1997;146-148
65. Hawiger D., Inaba K., Dorsett Y., Guo M., Mahnke K., Rivera M., et al. Dendritic cells induce peripheral T cell unresponsiveness under steady state conditions in vivo. J. Exp. Med. 194(6):2001;769-779
66. Sallusto F., Schaerli P., Loetscher P., Schaniel C., Lenig D., Mackay C.R., et al. Rapid and coordinated switch in chemokine receptor expression during dendritic cell maturation. Eur. J. Immunol. 28(9):1998;2760-2769
67. Kraft K., Olbrich H., Majoul I., Mack M., Proudfoot A., Oppermann M. Characterization of sequence determinants within the carboxyl-terminal domain of chemokine receptor CCR5 that regulate signaling and receptor internalization. J. Biol. Chem. 276(37):2001;34408-34418
68. Le Y., Wetzel M.A., Shen W., Gong W., Rogers T.J., Henderson E.E., et al. Desensitization of chemokine receptor CCR5 in dendritic cells at the early stage of differentiation by activation of formyl peptide receptors. Clin. Immunol. 99(3):2001;365-372
69. Biragyn A., Kwak L.W. B-cell malignancies as a model for cancer vaccines: from prototype protein to next generation genetic chemokine fusions. Immunol. Rev. 170:1999;115-126
70. Biragyn A., Surenhu M., Yang D., Ruffini P.A., Haines B.A., Klyushnenkova E., et al. Mediators of innate immunity that target immature, but not mature, dendritic cells induce antitumor immunity when genetically fused with nonimmunogenic tumor antigens. J. Immunol. 167(11):2001;6644-6653
71. Imai T., Nagira M., Takagi S., Kakizaki M., Nishimura M., Wang J., et al. Selective recruitment of CCR4-bearing Th2 cells toward antigen-presenting cells by the CC chemokines thymus and activation-regulated chemokine and macrophage-derived chemokine. Int. Immunol. 11(1):1999;81-88
72. Karpus W.J., Lukacs N.W., Kennedy K.J., Smith W.S., Hurst S.D., Barrett T.A. Differential CC chemokine-induced enhancement of T helper cell cytokine production. J. Immunol. 158(9):1997;4129-4136
73. Gu L., Tseng S., Horner R.M., Tam C., Loda M., Rollins B.J. Control of TH2 polarization by the chemokine monocyte chemoattractant protein-1. Nature. 404(6776):2000;407-411
74. Boyer J.D., Kim J., Ugen K., Cohen A.D., Ahn L., Schumann K., et al. HIV-1 DNA vaccines and chemokines. Vaccine. 17(Suppl. 2):1999;S53-S64
75. Biragyn A., Belyakov I.M., Chow Y.H., Dimitrov D.S., Berzofsky J.A., Kwak L.W. DNA vaccines encoding HIV-1 gp120 fusions with proinflammatory chemoattractants induce systemic and mucosal immune responses. Blood. 100:2002;1153-1159
76. Kikuchi T., Crystal R.G. Antigen-pulsed dendritic cells expressing macrophage-derived chemokine elicit Th2 responses and promote specific humoral immunity. J. Clin. Invest. 108(6):2001;917-927
77. Lalani A.S., Barrett J.W., McFadden G. Modulating chemokines: more lessons from viruses. Immunol. Today. 21(2):2000;100-106
78. Murphy P.M. Viral exploitation and subversion of the immune system through chemokine mimicry. Nat. Immunol. 2(2):2001;116-122
79. Boshoff C., Endo Y., Collins P.D., Takeuchi Y., Reeves J.D., Schweickart V.L., et al. Angiogenic and HIV-inhibitory functions of KSHV-encoded chemokines. Science. 278(5336):1997;290-294
80. Endres M.J., Garlisi C.G., Xiao H., Shan L., Hedrick J.A. The Kaposi's sarcoma-related herpesvirus (KSHV)-encoded chemokine vMIP-I is a specific agonist for the CC chemokine receptor (CCR)8. J. Exp. Med. 189:1999;1993-1998
81. Benelli R., Barbero A., Buffa A., Aluigi M.G., Masiello L., Morbidelli L., et al. Distinct chemotactic and angiogenic activities of peptides derived from Kaposi's sarcoma virus encoded chemokines. Int. J. Oncol. 17(1):2000;75-81
82. Damon I., Murphy P.M., Moss B. Broad spectrum chemokine antagonistic activity of a human poxvirus chemokine homolog. Proc. Natl. Acad. Sci. U.S.A. 95:1998;6403-6407
83. Yamashita T., Saito K. Purification, primary structure, and biological activity of guinea pig neutrophil cationic peptides. Infect. Immun. 57:1989;2405-2409
84. Lillard J.W.J., Boyaka P.N., Chertov O., Oppenheim J.J., McGhee J.R. Mechanisms for induction of acquired host immunity by neutrophil peptide defensins. Proc. Natl. Acad. Sci. U.S.A. 96:1999;651-656
85. Territo M.C., Ganz T., Selsted M.E., Lehrer R. Monocyte-chemotactic activity of defensins from human neutrophils. J. Clin. Invest. 84(6):1989;2017-2020
86. Niyonsaba F., Iwabuchi K., Matsuda H., Ogawa H., Nagaoka I. Epithelial cell-derived human beta-defensin-2 acts as a chemotaxin for mast cells through a pertussis toxin-sensitive and phospholipase C-dependent pathway. Int. Immunol. 14:2002;421-426
87. Garcia J.R., Jaumann F., Schulz S., Krause A., Rodriguez-Jimenez J., Forssmann U., Adermann K., Kluver E., Vogelmeier C., Becker D., Hedrich R., Forssmann W.G., Bals R. Identification of a novel, multifunctional beta-defensin (human beta-defensin 3) with specific antimicrobial activity. Its interaction with plasma membranes of Xenopus oocytes and the induction of macrophage chemoattraction. Cell Tissue Res. 306(2):2001;257-264
88. Garcia J.R., Krause A., Schulz S., Rodriguez-Jimenez F.J., Kluver E., Adermann K., et al. Human beta-defensin 4: a novel inducible peptide with a specific salt-sensitive spectrum of antimicrobial activity. FASEB J. 15(10):2001;1819-1821
89. Yang D., Chertov O., Bykovskaia S.N., Chen Q., Buffo M.J., Shogan J., et al. Beta-defensins: linking innate and adaptive immunity through dendritic and T cell CCR6. Science. 286(5439):1999;525-528
90. Biragyn A., Ruffini P.A., Leifer C.A., Klyushnenkova E., Shakhov A., Chertov O., et al. Toll-like receptor 4-dependent activation of dendritic cells by beta-defensin 2. Science. 298:2002;1025-1029
91. Bendandi M., Gocke C.D., Kobrin C.B., Benko F.A., Sternas L.A., Pennington R., et al. Complete molecular remissions induced by patient-specific vaccination plus granulocyte-monocyte colony-stimulating factor against lymphoma. Nat. Med. 5:1999;1171-1177