臺灣博碩士論文加值系統

Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are related life-threatening cutaneous adverse reactions most often caused by medication. Carbamazepine (CBZ), a commonly prescribed anticonvulsant, is the number one culprit drug associated with SJS in Taiwan, accounts for 25% of all drug-induced SJS.
Susceptibility to drug induced idiosyncratic reactions is thought to be genetically determined and immune-mediated. Previous studies suggest that the pathogenesis of the severe cutaneous adverse drug reactions involves MHC-restricted presentation of a drug or its metabolites for T-cell activation. However, the specific MHC molecules involved are not known until our study of carbamazepine-induced SJS/TEN in which we identified HLA-B*1502 as the MHC molecule. To further investigate the pathogenesis mechanism, we hypothesized that CBZ or its metabolites bind to endogenous proteins through processing and complexed with HLA-B*1502 or directly bind to the HLA-B*1502-bound peptides, which are then recognized by T-lymphocytes. The specific aims of my thesis are:
1. Establishment of the soluble HLA-B*1502-producing stable clones.
2. Identification of the peptides bind to the HLA-B*1502.
3. Identification of the drug-modified peptides involved in CBZ-SJS.
Using soluble HLA-B*1502 molecule as a bait, I identified over 100 peptides bind to the HLA-B*1502. An unusual and an unique feature of these peptides were that many of these endogenous bound peptides (up to 20.9 %) contained polyproline, ranged from 4 to 9 continuous proline residues, but typically 6, 7 or 8 proline residues in the peptides. There were a total of 38 different proline-rich peptides been identified. Among these peptides, the existence of continuous proline residues in the center was a consistent finding; amino acid residues at the N- or C- terminal of peptides were variable. Interestingly, in the presence of CBZ, these proline-rich peptides decreased dramatically. We hypothesized that CBZ may bind to the polyproline peptides covalently, which modify them to become unnatural peptides thus unable to be recognized as the proline-rich peptides under analysis of LC/MS/MS and SEQUEST program. Further functional studies will be needed to demonstrate that these CBZ-modified peptides are indeed involved in the induction of T cell activation in both drug and peptide-specific manners.
This is the first study in identification of the MHC-bound peptides associated with adverse drug reactions. The discovery opened a door to understanding the complete mechanism of this life-threatening condition.

Abstract 1
Introduction 3
Materials&Methods 15
Results 30
Discussion 45
Figures 54
Tables 74
References 91

1.Goldstein, D.B., Tate, S.K. & Sisodiya, S.M. Pharmacogenetics goes genomic. Nat Rev Genet 4, 937-47 (2003).
2.Phillips, K.A., Veenstra, D.L., Oren, E., Lee, J.K. & Sadee, W. Potential role of pharmacogenomics in reducing adverse drug reactions: a systematic review. Jama 286, 2270-9 (2001).
3.Lazarou, J., Pomeranz, B.H. & Corey, P.N. Incidence of adverse drug reactions in hospitalized patients: a meta-analysis of prospective studies. Jama 279, 1200-5 (1998).
4.Pirmohamed, M., Naisbitt, D.J., Gordon, F. & Park, B.K. The danger hypothesis--potential role in idiosyncratic drug reactions. Toxicology 181-182, 55-63 (2002).
5.Pirmohamed, M. & Park, B.K. Genetic susceptibility to adverse drug reactions. Trends Pharmacol Sci 22, 298-305 (2001).
6.Roujeau, J.C. Clinical heterogeneity of drug hypersensitivity. Toxicology 209, 123-9 (2005).
7.Ingelman-Sundberg, M., Oscarson, M. & McLellan, R.A. Polymorphic human cytochrome P450 enzymes: an opportunity for individualized drug treatment. Trends Pharmacol Sci 20, 342-9 (1999).
8.Yuan, H.Y. et al. A novel functional VKORC1 promoter polymorphism is associated with inter-individual and inter-ethnic differences in warfarin sensitivity. Hum Mol Genet (2005).
9.Roujeau, J.C. et al. HLA phenotypes and bullous cutaneous reactions to drugs. Tissue Antigens 28, 251-4 (1986).
10.Roujeau, J.C. et al. Genetic susceptibility to toxic epidermal necrolysis. Arch Dermatol 123, 1171-3 (1987).
11.Chan, S.H. & Tan, T. HLA and allopurinol drug eruption. Dermatologica 179, 32-3 (1989).
12.Hung, S.I. et al. HLA-B*5801 allele as a genetic marker for severe cutaneous adverse reactions caused by allopurinol. Proc Natl Acad Sci U S A 102, 4134-9 (2005).
13.Martin, A.M. et al. Predisposition to abacavir hypersensitivity conferred by HLA-B*5701 and a haplotypic Hsp70-Hom variant. Proc Natl Acad Sci U S A 101, 4180-5 (2004).
14.Chung, W.H. et al. Medical genetics: a marker for Stevens-Johnson syndrome. Nature 428, 486 (2004).
15.Roujeau, J.C. & Stern, R.S. Severe adverse cutaneous reactions to drugs. N Engl J Med 331, 1272-85 (1994).
16.Roujeau, J.C. The spectrum of Stevens-Johnson syndrome and toxic epidermal necrolysis: a clinical classification. J Invest Dermatol 102, 28S-30S (1994).
17.Paul, C. et al. Apoptosis as a mechanism of keratinocyte death in toxic epidermal necrolysis. Br J Dermatol 134, 710-4 (1996).
18.Rzany, B. et al. Epidemiology of erythema exsudativum multiforme majus, Stevens-Johnson syndrome, and toxic epidermal necrolysis in Germany (1990-1992): structure and results of a population-based registry. J Clin Epidemiol 49, 769-73 (1996).
19.Roujeau, J.C. et al. Medication use and the risk of Stevens-Johnson syndrome or toxic epidermal necrolysis. N Engl J Med 333, 1600-7 (1995).
20.Albani, F., Riva, R. & Baruzzi, A. Carbamazepine clinical pharmacology: a review. Pharmacopsychiatry 28, 235-44 (1995).
21.Soderpalm, B. Anticonvulsants: aspects of their mechanisms of action. Eur J Pain 6 Suppl A, 3-9 (2002).
22.Pearce, R.E., Vakkalagadda, G.R. & Leeder, J.S. Pathways of carbamazepine bioactivation in vitro I. Characterization of human cytochromes P450 responsible for the formation of 2- and 3-hydroxylated metabolites. Drug Metab Dispos 30, 1170-9 (2002).
23.Lillibridge, J.H. et al. Protein-reactive metabolites of carbamazepine in mouse liver microsomes. Drug Metab Dispos 24, 509-14 (1996).
24.Pirmohamed, M., Kitteringham, N.R., Guenthner, T.M., Breckenridge, A.M. & Park, B.K. An investigation of the formation of cytotoxic, protein-reactive and stable metabolites from carbamazepine in vitro. Biochem Pharmacol 43, 1675-82 (1992).
25.Park, B.K., Pirmohamed, M. & Kitteringham, N.R. Role of drug disposition in drug hypersensitivity: a chemical, molecular, and clinical perspective. Chem Res Toxicol 11, 969-88 (1998).
26.Pichler, W., Yawalkar, N., Schmid, S. & Helbling, A. Pathogenesis of drug-induced exanthems. Allergy 57, 884-93 (2002).
27.Kehren, J. et al. Cytotoxicity is mandatory for CD8(+) T cell-mediated contact hypersensitivity. J Exp Med 189, 779-86 (1999).
28.Yawalkar, N. et al. Infiltration of cytotoxic T cells in drug-induced cutaneous eruptions. Clin Exp Allergy 30, 847-55 (2000).
29.Pichler, W.J. Predictive drug allergy testing: an alternative viewpoint. Toxicology 158, 31-41 (2001).
30.Leyva, L. et al. Anticonvulsant-induced toxic epidermal necrolysis: monitoring the immunologic response. J Allergy Clin Immunol 105, 157-65 (2000).
31.Naisbitt, D.J. et al. Hypersensitivity reactions to carbamazepine: characterization of the specificity, phenotype, and cytokine profile of drug-specific T cell clones. Mol Pharmacol 63, 732-41 (2003).
32.Naisbitt, D.J. et al. Characterization of drug-specific T cells in lamotrigine hypersensitivity. J Allergy Clin Immunol 111, 1393-403 (2003).
33.Zanni, M.P. et al. Characterization of lidocaine-specific T cells. J Immunol 158, 1139-48 (1997).
34.Mauri-Hellweg, D. et al. Activation of drug-specific CD4+ and CD8+ T cells in individuals allergic to sulfonamides, phenytoin, and carbamazepine. J Immunol 155, 462-72 (1995).
35.Pichler, W.J. & Yawalkar, N. Allergic reactions to drugs: involvement of T cells. Thorax 55 Suppl 2, S61-5 (2000).
36.Nassif, A. et al. Drug specific cytotoxic T-cells in the skin lesions of a patient with toxic epidermal necrolysis. J Invest Dermatol 118, 728-33 (2002).
37.Schnyder, B., Mauri-Hellweg, D., Zanni, M., Bettens, F. & Pichler, W.J. Direct, MHC-dependent presentation of the drug sulfamethoxazole to human alphabeta T cell clones. J Clin Invest 100, 136-41 (1997).
38.Zanni, M.P. et al. HLA-restricted, processing- and metabolism-independent pathway of drug recognition by human alpha beta T lymphocytes. J Clin Invest 102, 1591-8 (1998).
39.Burkhart, C. et al. Influence of reduced glutathione on the proliferative response of sulfamethoxazole-specific and sulfamethoxazole-metabolite-specific human CD4+ T-cells. Br J Pharmacol 132, 623-30 (2001).
40.Shastri, N., Schwab, S. & Serwold, T. Producing nature''s gene-chips: the generation of peptides for display by MHC class I molecules. Annu Rev Immunol 20, 463-93 (2002).
41.Yewdell, J.W., Reits, E. & Neefjes, J. Making sense of mass destruction: quantitating MHC class I antigen presentation. Nat Rev Immunol 3, 952-61 (2003).
42.Hickman, H.D. et al. Toward a definition of self: proteomic evaluation of the class I peptide repertoire. J Immunol 172, 2944-52 (2004).
43.Hickman, H.D. et al. C-terminal epitope tagging facilitates comparative ligand mapping from MHC class I positive cells. Hum Immunol 61, 1339-46 (2000).
44.Prilliman, K.R. et al. HLA-B15 peptide ligands are preferentially anchored at their C termini. J Immunol 162, 7277-84 (1999).
45.Hiraki, D.D. et al. Bioengineered soluble HLA-B7. Genesis, characterization, and occurrence of dimerization. Hum Immunol 40, 235-46 (1994).
46.Prilliman, K. et al. Large-scale production of class I bound peptides: assigning a signature to HLA-B*1501. Immunogenetics 45, 379-85 (1997).
47.Barnea, E. et al. Analysis of endogenous peptides bound by soluble MHC class I molecules: a novel approach for identifying tumor-specific antigens. Eur J Immunol 32, 213-22 (2002).
48.Margulies, D.H., Ramsey, A.L., Boyd, L.F. & McCluskey, J. Genetic engineering of an H-2Dd/Q10b chimeric histocompatibility antigen: purification of soluble protein from transformant cell supernatants. Proc Natl Acad Sci U S A 83, 5252-6 (1986).
49.Dal Porto, J. et al. A soluble divalent class I major histocompatibility complex molecule inhibits alloreactive T cells at nanomolar concentrations. Proc Natl Acad Sci U S A 90, 6671-5 (1993).
50.Prilliman, K.R. et al. Complexity among constituents of the HLA-B*1501 peptide motif. Immunogenetics 48, 89-97 (1998).
51.Mage, M.G. et al. A recombinant, soluble, single-chain class I major histocompatibility complex molecule with biological activity. Proc Natl Acad Sci U S A 89, 10658-62 (1992).
52.Zemmour, J., Little, A.M., Schendel, D.J. & Parham, P. The HLA-A,B "negative" mutant cell line C1R expresses a novel HLA-B35 allele, which also has a point mutation in the translation initiation codon. J Immunol 148, 1941-8 (1992).
53.Brodsky, F.M. & Parham, P. Monomorphic anti-HLA-A,B,C monoclonal antibodies detecting molecular subunits and combinatorial determinants. J Immunol 128, 129-35 (1982).
54.Syka, J.E., Coon, J.J., Schroeder, M.J., Shabanowitz, J. & Hunt, D.F. Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry. Proc Natl Acad Sci U S A 101, 9528-33 (2004).
55.Van Hove, J.L., Yang, H.W., Wu, J.Y., Brady, R.O. & Chen, Y.T. High-level production of recombinant human lysosomal acid alpha-glucosidase in Chinese hamster ovary cells which targets to heart muscle and corrects glycogen accumulation in fibroblasts from patients with Pompe disease. Proc Natl Acad Sci U S A 93, 65-70 (1996).
56.Hsu, J.L., Huang, S.Y., Chow, N.H. & Chen, S.H. Stable-isotope dimethyl labeling for quantitative proteomics. Anal Chem 75, 6843-52 (2003).
57.Prilliman, K.R. et al. Alpha-2 domain polymorphism and HLA class I peptide loading. Tissue Antigens 54, 450-60 (1999).
58.Hausch, F., Shan, L., Santiago, N.A., Gray, G.M. & Khosla, C. Intestinal digestive resistance of immunodominant gliadin peptides. Am J Physiol Gastrointest Liver Physiol 283, G996-G1003 (2002).
59.Lemmel, C. et al. Differential quantitative analysis of MHC ligands by mass spectrometry using stable isotope labeling. Nat Biotechnol 22, 450-4 (2004).
60.Kay, B.K., Williamson, M.P. & Sudol, M. The importance of being proline: the interaction of proline-rich motifs in signaling proteins with their cognate domains. Faseb J 14, 231-41 (2000).
61.van der Burg, S.H. et al. An HLA class I peptide-binding assay based on competition for binding to class I molecules on intact human B cells. Identification of conserved HIV-1 polymerase peptides binding to HLA-A*0301. Hum Immunol 44, 189-98 (1995).
62.Fruci, D. et al. HLA class I binding of synthetic nonamer peptides carrying major anchor residue motifs of HLA-B27 (B*2705)-binding peptides. Immunogenetics 38, 41-6 (1993).
63.Gnjatic, S., Bressac-de Paillerets, B., Guillet, J.G. & Choppin, J. Mapping and ranking of potential cytotoxic T epitopes in the p53 protein: effect of mutations and polymorphism on peptide binding to purified and refolded HLA molecules. Eur J Immunol 25, 1638-42 (1995).
64.Silver, M.L., Parker, K.C. & Wiley, D.C. Reconstitution by MHC-restricted peptides of HLA-A2 heavy chain with beta 2-microglobulin, in vitro. Nature 350, 619-22 (1991).
65.Buchli, R. et al. Real-time measurement of in vitro peptide binding to soluble HLA-A*0201 by fluorescence polarization. Biochemistry 43, 14852-63 (2004).