臺灣博碩士論文加值系統

中文摘要-1

慢性肝病病人的matrix metalloproteinase-2活動性與cystatin C血清濃度的相關性
Relationship between matrix metalloproteinase-2 activity and cystatin C levels in patients with hepatic disease

目的：先前吾等研究已顯示cystatin c的濃度與肝病嚴重度有直接相關性。本研究則在探討matrix metalloproteinase-2（先前已被証明與肝纖維化有關）與cystatin在肝病嚴重度的關係。

設計與方法：連續收集154位肝病病人的血清，測量其cystatin C，matrix metalloproteinase-2（MMP-2）及metalloproteinase-9的濃度，並測量其他肝病參數。與40位正常對照組比較。

結果：病人的MMP-2及cystatin C濃度顯著比對照組要高，而MMP-9則較低。線性回歸分析顯示cystatin C與MMP-2有直接關係（Y=83.39+270.56X，R=0.38, P＜0.001）。MMP-2及肝病嚴重度亦有同樣關係。

結論：本研究首次展示cystatin C及MMP-2的關係。表示在肝病病人中兩者有某種相互關係。

中文摘要-2

慢性C型肝炎細胞激素（cytokines）的血清濃度及基因多樣性（polymorphism）的衝激（impact）

造成C型肝炎病毒（HCV）清除或者持續的因素尚無法完全清楚界定，其中宿主的免疫反應的重要性特別受到注意。
尤其幾個細胞激素（cytokines）的血清濃度及基因多樣性（polymorphism）在C型肝炎病毒感染的結果值得多加研究。

利用酵素連結免疫吸附分析法（Enzyme-linked immunosorbent assay, ELISA）來測量72個C型肝炎病人及180個健康對照者的腫瘤壞死因子-α（tumor necrosis factor alpha, TNF-α）介白素-4（interleukin-4, IL-4）及介白素-10 (IL-10)的濃度。同時更進一步，利用聚合酶鏈反應（polymerase chain reaction, PCR）限制酶斷片長度多樣性（restriction fragment length polymorphism, RFLP）來分析這些細胞激素的基因的單一核苷酸多樣性（single nucleotide polymorphism, SNP）及C型肝炎病毒感染的相關性。

這些分析的細胞激素的SNPs包括TNF-αG238A, TNF-αG308A, IL-4C589T, IL-10A1082G, IL-10T819C及IL-10A592C。

C型肝炎病人的TNF-α,IL-4及IL-10的血清濃度比對照組的值要高（P＜0.001）。再者，TNF-α G308A基因型（genotype）及對偶質（allele）在二組之間有意義的差別（P＜0.05），但其他的細胞激素就沒有差別。這些資料顯示在健康人及慢性C型肝炎病人之間，TNF-αG308A的多樣性的分佈可能是不一樣的。

Abstract-1

Objectives: A direct relationship between cystatin C levels and the severity of hepatic disease has been revealed in our previous study. This study was aimed to consider whether a correlation exists between matrix metalloproteinases (MMPs), which have been proven to be involved in liver cirrhosis, and cystatin C to reflect the severity of hepatic disease.
Design and Methods: A total of 154 consecutive patients with various liver diseases were recruited to determine their serum levels of cystatin C, MMP-2 and-9, together with other hepatic parameters. These were compared with 40 normal controls.
Results: Average levels of MMP-2 and cystatin C were significantly higher in patients while MMP-9 was significantly lower, as compared to controls. A linear regression analysis has revealed a direct relationship between cystatin C and MMP-2 (Y = 83.39 + 270.56X, R = 0.38, P < 0.001), as well as between MMP-2 and the severity of liver diseases.
Conclusion: This is the first study to demonstrate a correlation between cystatin C and MMP-2, suggesting that there may be certain interactions between cystatin C and MMP-2 in patients with hepatic diseases.
○C 2005 The Canadian Society of Clinical Chemists. All rights reserved.

Keywords: MMP-2; Cystatin C; Hepatic disease

Abstract-2

The factors leading to clearance or persistent infection of hepatitis C virus (HCV) were not well defined, and the importance of the host immune response has been highlighted. Therefore, the impact of the serum concentration and genetic polymorphism of several cytokines on the outcome of HCV infection warrant additional study.
Enzyme-linked immunosorbent assay was employed to measure serum tumor necrosis factor alpha (TNF), interleukin (IL)-4, and IL-10 concentrations on 72 hepatitis C patients and 180 controls. Furthermore, the association between single nucleotide polymorphism (SNP) of genes of these cytokines and hepatitis C infection was evaluated by polymerase chain reaction- restriction fragment length polymorphism and statistical analysis.
These analyzed SNPs of cytokines included TNF-αG238A, TNF-α G308A, IL-4 C589T, IL-10 A1082G, IL-10 T819C, and IL-10 A592C. Serum levels of TNF-μ α, IL-4, and IL-10 were significantly higher in hepatitis C patients than that of controls (P < 0.001). Furthermore, the distribution of TNF-αG308A genotypes and alleles, but not others, was statistically different between patients and controls (P < 0.05). These data suggested the distribution of polymorphism at TNF-αG308A may be different between normal subjects and patients with chronic infection of hepatitis C. (Translational Research 2007;150:116-121)

Abbreviations: ALT = alanine aminotransferase; AST = aspartate aminotransferase; ELISA =enzyme-linked immunosorbent assay; HBV = hepatitis B virus; HCV = hepatitis C virus; HIV =human immunodeficiency virus; IFN = interferon; IL =interleukin; NK =natural killer; OR =odds ratio; PCR = polymerase chain reaction; RFLP =restriction fragment length polymorphism;RT =reverse transcriptase; TNF-α= tumor necrosis factor alpha.

Contents

Introduction in Chinese ---------------------------------------------------------------------- 4
Introduction in English----------------------------------------------------------------------- 5
Abstract in Chinese （cystatin C and MMP-2）--------------------------------------- 6
（serum levels and gene polymorphism of cytokines）------ 7

Abstract in English （cystatin C and MMP-2）----------------------------------------8
（serum levels and gene polymorphism of cytokines）------9
Chapter 1 Introduction part Ⅰ（matrix metalloproteinase and cystatin C）---------11
Introduction part Ⅱ（TNF-α,IL-4,IL-10）-----------------------------------13
Chapter 2 Materials and Methods-----------------------------------------------------------15
2. Ⅰ-1 Materials ----------------------------------------------------------------------------- 15
2. Ⅰ-2 Quantitative determination of serum MMP-2 and MMP-9 --------------------17
2. Ⅰ-3 Determination of serum cystatin C ----------------------------------------------- 17
2. Ⅰ-4 Statistical analysis--------------------------------------------------------------------18
2. Ⅱ-1 General design and study subjects ------------------------------------------------ 19
2. Ⅱ-2 Preparation of RNA and RT-PCR -------------------------------------------------19
2. Ⅱ-3 Measurements of INF-α, IL-4, IL-10 by ELISA---------------------------------20
2. Ⅱ-4 PCR-RILP for genetic polymorphism within promoter regions of TNF-α, IL-4 and IL-10------------------------------------------------------------------------------20
2. Ⅱ-5 Statistical analysis---------------------------------------------------------------------20

Chapter 3 Results
3.Ⅰ Results Ⅰ（cystatin and MMP-2 , MMP-9）-------------------------------------21
3.Ⅰ-1 Basic characteristics of study Subjects----------------------------------------------21
3.Ⅰ-2 Significantly higher cystatin C levels in hepatic disease -------------------------21
3 Ⅰ-3 Quantitative determinations of MMP-2 and MMP-9 and their correlations to cystatin C values in hepatic diseases ----------------------------------------------24
3 Ⅰ-4 Relationship between the severity of liver disease and MMP-2 -----------------27
3 Ⅱ-1 Characteristics of study subjects ----------------------------------------------------29
3 Ⅱ-2 ELISA analysis for serum levels of TNF-α, IL-4 and IL-10 -------------------- 29
3 Ⅱ-3 Genotype and allele distribution of promoter of TNF-α , IL-4 and
IL-10 gene -----------------------------------------------------------------------------------31

Chapter 4 Discussion Ⅰ-----------------------------------------------------------------------36
Discussion Ⅱ-----------------------------------------------------------------------40
Tables Ⅰ
Table 1 Clinical parameters of controls and patients with various hepatic diseases---16
Table 2 Cystatin C concentrations , MMP-2 and MMP-9 of patients with hepatic diseases compared with normal controls------------------------------------------23

Tables Ⅱ
Table 3 Sequences of primers and restriction enzymes used in cytokine gene polymorphism and expected sizes of resultant PCR-RFLP products ------- 32
Table 4 Genotype frequencies of TNF-α, IL-4 and IL-10 polymorphic site in controls and HCV patients--------------------------------------------------------------------34
Table 5 Allele frequencies of IL-4, IL-10 and TNF-α polymorphic site in controls and HCV patients-------------------------------------------------------------------------35

Figures Ⅰ
Figure 1 Cystatin C concentrations in normal controls and patients with hepatic diseases of various severity--------------------------------------------------------25
Figure 2 Shows representative serum MMPs zymographs of the subjects ------------26
Figure 3 Linear regression analysis of the relationship between cystatin concentrations and MMP-2 activity ------------------------------------------------------------ 27
Figure 4 MMP-2 activity in normal controls and patients with various severity of liver disease-----------------------------------------------------------------------------28
Figures Ⅱ
Figure 5 Serum levels of TNF-α, IL-4 and IL-10 in hepatitis c patiens and controls determined by ELISA kits------------------------------------------------------30
Figure 6 PCR-RFLP analysis of genetic polymorphism for various cytokines ----- 33

References ------------------------------------------------------------------------------------ 43

Published papers-------------------------------------------------------------------------------51

[1] Murawaki J, Yamamoto H, Kawasaki H, Shima H. Serum tissue inhibitor of metalloproteinase in patients with chronic liver disease and with hepatocellular carcinoma. Clin Chim Acta 1993;218:47–58.
[2] Muzzillo DA, Imoto M, Fukuda Y, et al. Clinical evaluation of serum tissue inhibitor of metalloproteinase-1 levels in patients with liver diseases. J Gastroenterol Hepatol 1993;8:437– 41.
[3] Filler G, Bokenkamp A, Hofmann W, Le Bricon T, Martinez-Bru C, Grubb A. Cystatin C as a marker of GFR-history, indications, and future research. Clin Biochem 2005;38:1–8.
[4] Shimode K, Fujihara S, Nakamura M, Kobayashi S, Tsunematsu T. Diagnosis of cerebral amyloid angiopathy by enzyme-linked immunosorbent assay of cystatin C in cerebrospinal fluid. Stroke 1991;22:860–6.
[5] Bollengier F, Cystatin C. Alias post-[gamma]-globulin: a marker for multiple sclerosis? J Clin Chem Clin Biochem 1987;25: 589– 93.
[6] Keppler D, Waridel P, Abrahamson M, Bachmann D, Berdoz J, Sordat B. Latency of cathepsin B secreted by human colon carcinoma cells is not linked to secretion of cystatin C and is relieved by neutrophil elastase. Biochim Biophys Acta 1994;1226:117– 25.
[7] Corticchiato O, Cajot JF, Abrahamson M, Chan SJ, Keppler D, Sordat B. Cystatin C and cathepsin B in human colon carcinoma: expression by cell lines and matrix degradation. Int J Cancer 1992;52:645–52.
[8] Kos J, Stabuc B, Schweiger A, et al. Cathepsins B, H and L and their inhibitors stefin A and cystatin C in sera of melanoma patients. Clin Cancer Res 1997;3:1815–22.
[9] Takeuchi M, Fukuda Y, Nakano I, Katano Y, Hayakawa T. Elevation of serum cystatin C concentration in patients with chronic liver disease. Eur J Gastroenterol Hepatol 2001;13: 951– 5.
[10] Chu SC, Wang CP, Chang YH, et al. Elevated cystatin C serum concentrations in patients with hepatic diseases of various severities. Clin Chim Acta 2004;341:133– 8.
[11] Sherwood P, Lyburn I, Brown S, Ryder S. How are abnormal results for liver function tests dealt with in primary care? Audit of yield and impact. BMJ 2001;32:276– 8.
[12] Matrisian LM. Metalloproteinases and their inhibitors in matrix remodeling. Trends Genet 1990;6:121– 5.
[13] Stetler-Stevenson WG, Aznavoorian S, Liotta LA. Tumor cell interactions with the extracellular matrix during invasion and metastasis. Annu Rev Cell Biol 1993;9:541 – 73.
[14] Walsh KM, Timms P, Campbell S, MacSween RN, Morris AJ. Plasma activities of matrix metalloproteinase-2 (MMP-2) and tissue inhibitors of metalloproteinases-1 and-2 (TIMP-1 and TIMP-2) as noninvasive markers of liver disease in chronic hepatitis C: comparison using ROC analysis. Dig Dis Sci 1999;44:624– 30.
[15] Murawaki Y, Yamada S, Ikuta Y, Kawasaki H. Clinical usefulness of serum matrix metalloproteinase-2 activity in patients with chronic viral liver disease. J Hepatol 1999;26:1521–9.
[16] Hayasaka A, Suzuki N, Fujimoto N, et al. Elevated plasma activities of matrix metalloproteinase-9 (92-kd type IV collagenase/ gelatinase in hepatocellular carcinoma. Hepatology 1996;24: 1058– 62.
[17] Arii S, Mise M, Harada T, et al. Overexpression of matrix metalloproteinase 9 gene in hepatocellular carcinoma with invasive potential. Hepatology 1996;24:316–22.
[18] Yata Y, Takahara T, Furui K, Zhang LP, Jin B, Watanabe A. Spatial distribution of tissue inhibitor of metalloproteinase- 1 mRNA in chronic liver diseases. J Hepatol 1999;30: 425– 32.
[19] Iredale JP. Tissue inhibitors of metalloproteinases in liver fibrosis. Int J Biochem Cell Biol 1997;29:43 – 54.
[20] Kuo WH, Chou FP, Lu SC, Chu SC, Hsieh YS. Significant differences in serum activities of matrix metalloproteinase-2 and-9 between HCVand HBV-infected patients and carriers. Clin Chim Acta 2000;294: 157– 68.
[21] Kasahara A, Hayashi N, Mochizuki K, et al. Circulating matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase- 1 as serum markers of fibrosis in patients with chronic hepatitis C. Relationship to interferon response. J Hepatol 1997;26:574–83.
[22] Lichtinghagen R, Huegel O, Seifert T, et al. Matrix metalloproteinases and their inhibitors in acute viral hepatitis. Clin Chem 2000;46: 183– 92.
[23] Murawaki Y, Ikuta Y, Okamoto K, Mimura K, Koda M, Kawasaki H. Plasma matrix metalloproteinase-9 (gelatinase B) in patients with hepatocellular carcinoma. Res Commun Mol Pathol Pharmacol 2000;108:351– 7.
[24] Koulentaki M, Valatas V, Xidakis K, et al. Matrix metalloproteinases and their inhibitors in acute viral hepatitis. J Viral Hepat 2002;9:189–93.
[25] Chung TW, Kim JR, Suh JI, et al. Correlation between plasma levels of matrix metalloproteinase (MMP)-9/MMP-2 ratio and alpha-fetoproteins in chronic hepatitis carrying hepatitis B virus. Gastroenterol Hepatol 2004;19:565– 71.
[26] Leroy V, Monier F, Bottari S, et al. Circulating matrix metalloproteinases1, 2, 9 and their inhibitors TIMP-1 and TIMP-2 as serum markers of liver fibrosis in patients with chronic hepatitis C: comparison with PIIINP and hyaluronic acid. Am J Gastroenterol 2004;99:271–9.
[27] Ebata M, Fukuda Y, Nakano I, Katano Y, Fujimoto N, Hayakawa T. Serum activities of tissue inhibitor of metalloproteinases-2 and of precursor form of matrix metalloproteinase-2 in patients with liver disease. Liver 1997;17:293– 9.
[28] Murawaki Y, Yamada S, Koda M, Hirayama C. Collagenase and collagenolytic cathepsin in normal and fibrotic rat liver. J Biochem 1990;108:241– 4.
[29] Leto G, Tumminello FM, Pizzolanti G, Montalto G, Soresi T.-Y. Chen et al. / Clinical Biochemistry 38 (2005) 632–638 637 M, Gebbia N. Lysosomal cathepsins B and L and stefin A blood levels in patients with hepatocellular carcinoma and/or liver cirrhosis: potential clinical implication. Oncology 1997;54: 79– 83.
[30] Asanuma K, Shirato I, Ishidoh K, Kominami E, Tomino Y. Selective modulation of the secretion of proteinases and their inhibitors by growth factors in cultured differentiated podocytes. Kidney Int 2002;62:822– 31.
[31] Ray S, Lukyanov P, Ochieng J. Members of the cystatin superfamily interact with MMP-9 and protect it from autolytic degradation without affecting its gelatinolytic activities. Biochim Biophys Acta 2003; 1652:91–102.


[32] Van der Poel CL, Cuypers HT, Reesink HW, Weiner AJ, Quan S, Di Nello R, et al. Confirmation of hepatitis C virus infection by second generation four-antigen recombinant immunoblot assay and polymerase chain reaction. Lancet 1991;337:317–9.
[33] Seeff LB. Natural history of hepatitis C. Hepatology 1997;26: 21S–8S.
[34] Boyer N, Marcellin P. Pathogenesis, diagnosis and management of hepatitis C. J Hepatol 2000;32:98 –112.
[35] Barrett S, Goh J, Coughlan B, Ryan E, Stewart S, Cockram A, et al. The natural course of hepatitis C virus infection after 22 years in a unique homogenous cohort: spontaneous viral clearance and chronic HCV infection. Gut 2001;49:423–30.
[36] Zucker SD. Whomsoever ignores the natural history of the hepatitis C virus is doomed to treat it. Gastroenterology 2002;122: 578–9.
[37] Hohler T, Kruger A, Gerken G, Schneider PM, Meyer zum Buschenfelde KH, Rittner C. Tumor necrosis factor alpha promoter polymorphism at position-238 is associated with chronic active hepatitis C. J Med Virol 1998;54:173–7.
[38] Barrett S, Ryan E, Crowe J. Association of the HLA-DRB1*01 allele with spontaneous viral clearance in an Irish cohort infected with hepatitis C virus via contaminated anti-D immunoglobulin. J Hepatol 1999;30:979–83.
[39] Powell EE, Edwards-Smith CJ, Hay JL, Clouston AD, Crawford DH, Shorthouse C, et al. Host genetic factors influence disease progression in chronic hepatitis C. Hepatology 2000;31:828 –33.
[40] Tsutsumi T, Suzuki T, Shimoike T, Suzuki R, Moriya K, Shintani Y, et al. Interaction of hepatitis C virus core protein with retinoid X receptor alpha modulates its transcriptional activity. Hepatology 2002;35:937– 46.
[41] Quiroga JA, Martin J, Pardo M, Carreno V. Serum levels of soluble immune factors and pathogenesis of chronic hepatitis C, and their relation to therapeutic response to interferon-alpha. Dig Dis Sci 1994;39:2485–96.
[42] Cacciarelli TV, Martinez OM, Gish RG, Villanueva JC, Krams SM. Immunoregulatory cytokines in chronic hepatitis C virus infection: pre- and posttreatment with interferon alpha. Hepatology 1996;24:6 –9.
[43] Mosmann TR, Coffman RL. Heterogeneity of cytokine secretion patterns and functions of helper T cells. Adv Immunol 1989;46: 111–47.
[44] Ferrari C, Valli A, Galati L, Penna A, Scaccaglia P, Giuberti T, et al. T-cell response to structural and nonstructural hepatitis C virus antigens in persistent and self-limited hepatitis C virus infections. Hepatology 1994;19:286 –95.
[45] Lasarte JJ, Garcia-Granero M, Lopez A, Casares N, Garcia N, Civeira MP, et al. Cellular immunity to hepatitis C virus core protein and the response to interferon in patients with chronic hepatitis C. Hepatology 1998;28:815–22.
[46] Sher A, Gazzinelli RT, Oswald IP, Clerici M, Kullberg M, Pearce EJ, et al. Role of T-cell derived cytokines in the downregulation of immune responses in parasitic and retroviral infection. Immunol Rev 1992;127:183–204.
[47] Turner DM, Williams DM, Sankaran D, Lazarus M, Sinnott PJ, Hutchinson IV. An investigation of polymorphism in the interleukin- 10 gene promoter. Eur J Immunogenet 1997;24:1– 8.
[48] Wilson AG, Symons JA, McDowell TL, McDevit HO, Duff GW. Effects of a polymorphism in the human tumor necrosis factor-a Translational Research Proc Natl Acad Sci U S A 1997;94:3195–9.
[49] Fishman D, Faulds G, Jeffery R, Mohamed-Ali V, Yudkin JS, Humphries S, et al. The effect of novel polymorphisms in the interleukin-6 (IL-6) gene on IL-6 transcription and plasma IL-6 levels, and an association with systemic-onset juvenile chronic arthritis. J Clin Invest 1998;102:1369 –76.
[50] Pravica V, Asderakis A, Perrey C, Hajeer A, Sinnott PJ, Hutchinson IV. In vitro production of IFN-gamma correlates with CA repeat polymorphism in the human IFN-gamma gene. Eur J Immunogenet 1999;26:1–3.
[51] Mulcahy B, Waldron-Lynch F, McDermott MF. Genetic variability in the tumor necrosis factor-lymphotoxin region influences susceptibility to rheumatoid arthritis. Am J Hum Genet 1996;59:676–83.
[52] Turner D, Grant SC, Yonan N, Sheldon S, Dyer PA, Sinnott PJ, et al. Cytokine gene polymorphisms and heart transplant rejection. Transplantation 1997;64:776 –9.
[53] Awad MR, El Gamel A, Hasleton P, Turner DM, Sinnott PJ, Hutchinson IV. Genotype variation in the transforming growth factor-1 gene: association with transforming growth factor-1 production, fibrotic lung disease, and graft fibrosis after lung transplantation. Transplantation 1998;66:1014 –20.
[54] Awad M, Pravica V, Perrey C. CA repeat allele polymorphism in the first intron of the human interferon-gamma gene is associated with lung allograft fibrosis. Hum Immunol 1999;60:43–346.
[55] Hohler T, Kruger A, Gerken G. A tumor necrosis factor-alpha (TNF-alpha) promoter polymorphism is associated with chronic hepatitis B infection. Clin Exp Immunol 1998;111:579–82.
[56] Vickers MA, Green FR, Terry C, Mayosi BM, Julier C, Lathrop M, et al. Genotype at a promoter polymorphism of the interleukin- 6 gene is associated with baseline levels of plasma C-reactive protein. Cardiovasc Res 2002;53:1029 –34.
[57] Yee LJ, Tang J, Herrera J, Kaslow RA, Van Leeuwen DJ. Tumor necrosis factor gene polymorphisms in patients with cirrhosis from chronic hepatitis C virus infection. Genes Immun 2000;1: 386–90.
[58] Yee LJ, Tang J, Gibson AW, Kimberly R, Van Leeuwen DJ, Kaslow RA. Interleukin 10 polymorphisms as predictors of sustained response in antiviral therapy for chronic hepatitis C infection. Hepatology 2001;33:708 –12.
[59] Tambur AR, Ortegel JW, Ben-Ari Z, Shabtai E, Klein T, Michowiz R, et al. Role of cytokine gene polymorphism in hepatitis C recurrence and allograft rejection among liver transplant recipients. Transplantation 2001;71:1475– 80.
[60] Rosen HR, McHutchison JG, Conrad AJ, Lentz JJ, Marousek G, Rose SL, et al. Tumor necrosis factor genetic polymorphisms and response to antiviral therapy in patients with chronic hepatitis C. Am J Gastroenterol 2002;3:714 –20.
[61] Vidigal PG, Germer JJ, Zein NN. Polymorphisms in the interleukin- 10, tumor necrosis factor-alpha, and transforming growth factorbeta1 genes in chronic hepatitis C patients treated with interferon and ribavirin. J Hepatol 2002;36:271–7.
[62] Dai CY, Chuang WL, Chang WY, Chen SC, Lee LP, Hsieh MY, et al. Tumor necrosis factor- alpha promoter polymorphism at position -308 predicts response to combination therapy in hepatitis C virus infection. J Infect Dis 2006;193:98 –101.
[63] Kusumoto K, Uto H, Hayashi K, Takahama Y, Nakao H, Suruki R, et al. Interleukin-10 or tumor necrosis factor-alpha polymorphisms and the natural course of hepatitis C virus infection in a hyperendemic area of Japan. Cytokine. 2006;34:24 –31.
[64] Park YS, Lee KO, Oh MJ, Chai YG. Distribution of genotypes in the 5= untranslated region of hepatitis C virus in Korea. J Med Microbiol 1998;47:643–7.
[65] Zhang Y, Li J, Zhan Y, Wu L, Yu X, Zhang W, et al. Analysis of serum cytokines in patients with severe acute respiratory syndrome. Infect Immun 2004;72:4410 –5.