摘要
【目的】
脓毒症预后的早期预测有助于采取更为恰当的治疗措施以改善脓毒症患者的预后,但目前仍缺乏有效的预测手段。本文应用代谢组学方法构建一个能早期、快速、有效预测脓毒症预后的判别模型,筛选与脓毒症预后相关的生物标志物,以便早期采取积极措施以改善脓毒症预后。
【方法】
75只SD大鼠随机分为盲肠结扎穿孔组(CLP,n=50)和假手术组(n=25),术后12 h在乙醚麻醉下采用断尾法采集外周血1.5ml,室温下静置120min后,3000 g离心20 min,收集上清液,-80℃冻存。继续观察CLP大鼠生存状况6日,然后根据其预后不同,将其分为生存组(存活期＞6日,n=23)、死亡组(存活期24 h～6日,n=22)和废弃标本(存活期＜24 h,n=5)。将三组大鼠血清标本经不同的预处理后,分别行高效液相色谱/质谱仪(HPLC/MS)和核磁共振波谱仪(NMR)分析,获得HPLC/MS谱和~1H NMR谱。对HPLC/MS谱和~1H NMR谱进行数据处理和多元统计分析后,分别获得与各组区分的标志物,然后采用径向基函数神经网络算法(RBFNN)分别构建脓毒症的预后判别模型,并对其预测效能进行评估。
【结果】
基于HPLC/MS谱图信息,主成分分析法(PCA)可完全区分三组大鼠的生理特征。代谢物鉴定发现,与假手术组相比,CLP组,尤其是死亡组大鼠,其血清中多种游离脂肪酸如棕榈油酸、棕榈酸、亚油酸、软脂酸和硬脂酸的相对浓度显著降低(P＜0.05或0.01)。这表明,脓毒症后外周血中用于氧化供能的多种游离脂肪酸被大量消耗,以弥补机体能量生成的不足,其降低程度与预后相关;与上述5个游离脂肪酸不同,CLP组大鼠尤其是生存组大鼠血清中多个多不饱和脂肪酸,如亚麻酸、二十二碳六烯酸和二十二碳五烯醇相对浓度显著升高(P＜0.05或0.01),这表明脓毒症后,外周血中具有抗炎作用的多不饱和脂肪大量释放,以抑制过度激活的炎症反应,其外周血浓度越高,预后越好。采用RBFNN构建的脓毒症预后判别模型准确预测的敏感性为(96.1±3.6)%,特异性为(91.0±4.3)%。
利用~1H NMR谱图信息,正交信号校正偏最小二乘-判别分析法(OPLS-DA)也可较好区分三组大鼠生理特征。代谢物鉴定发现,与假手术组相比,CLP组大鼠外周血羟丁酸、乳酸、丙氨酸、醋酸和乙酰乙酸水平显著增加,而甲酸水平显著下降(P＜0.05),尤其是死亡组大鼠,其变化程度更为显著,这表明脓毒症后糖、蛋白质、脂肪与核酸的代谢明显异常,且其程度与脓毒症预后相关。采用RBFNN构建的脓毒症预后判别模型准确预测的敏感性为(91.0±5.9)%(n=10),特异性为(87.9±1.1)%(n=10)。
【结论】
脓毒症后,糖、蛋白质、脂肪和核酸的代谢明显异常,应用基于HPLC/MS和NMR的代谢组学方法可全面了解上述应激代谢的变化规律,采用代谢信息构建的模型可早期、快速、有效地预测脓毒症大鼠的预后,有望用于临床。HPLC/MS与NMR侧重点不同,前者侧重于大分子物质,如脂肪酸代谢,而后者侧重于小分子物质,如糖和氨基酸代谢,综合两者信息有利于全面理解脓毒症后物质代谢的变化规律,进而判断脓毒症的严重程度。
Objective
Early prognostic evaluation of sepsis is an attractive strategy to decrease the mortality of septic patients,but presently there are no satisfactory approaches.This study is to establish an early,rapid and efficient metabonomic approach for prognostic evaluation of sepsis,and then to explore some potential biomarkers related with the outcome.
Methods
Seventy-five rats were randomly allocated to sham-operated group(n=25) and CLP group(n=50).According to the difference in survival duration during the following six days,CLP rats were divided into surviving(n=23,survival duration exceeding 6 d) and nonsurviving group(n=22,survival duration between 24 h and 6 d),excluding five undesirable ones(dead during the first day).At 12 h after surgery, approximately 1.5 ml blood was drawn using tail shearing method in these groups, then left to clot for 2 hours at room tempreture.The serum was separated by centrifugation at 3000 g for 20 min,and the aliquot was stored at -80℃until metabonomic analysis.After special pretreatments,serum samples were analyzed to acquire metabolic profiles using HPLC/MS and NMR spectroscopy respectively. Principle component analysis(PCA) and orthogonal single collection partial least squares discriminant analysis(OPLS-DA) were employed to visualize the changes in the metabolic profiles of sera from surviving,nonsurviving and sham-operated rats, then to discover potential biomarkers related with the otcome of septic rats.Radial basis function neural network(RBFNN) was employed to build predictive model for prognostic evaluation of sepsis
Results
Based on HPLC/MS data,PCA allows a clear discrimination of the pathologic characteristics among rats from three groups.The results from metabolin identifiction that the levels of palmitoleic acid,palmitic acid,linoleic acid,oleic acid and stearic acid are lower in septic rats,especially in nonsurvivors.It suggests that these important free fatty acids mainly used for energy metabolism are consumed greatly to raise energy supply for organism and their levers may be related with the prognosis of septic rats.Moreover,the levers of another three identified metabolin,including acid and docosapentaenoic acid are higher in septic rats,especially in survivors.It suggests that the levers of these polyunsaturated fatty acids(PUFA) with anti-inflammatory effect increase greatly against the excessive inflammatory reaction and might be related with the prognosis of septic rats.A RBFNN model for outcome predication was built upon the metabolic profile data from rat sera with the sensitivity of(96.1±3.6)%(n=10) and specificity of(91.0±4.3)%(n=10).
Based on NMR spectra data,OPLS-DA also allows a clear discrimination of the pathologic characteristics among rats from three groups.Compared with sham-operated group,marked decrease in formate lever and increases in hydroxybutyrate,lactate,acetate,acetoacetate and alanine levers,were observed in septic rats,especially in nonsurvivors,which might due to the enhanced catabolisms of carbohydrate,protein,fatty acid and nucleic acid.Based on the integrity metabolic profile data from ~1H NMR data,another RBFNN model for outcome predication was constructed with the sensitivity of(91.1±5.6)%(n=10) and specificity of(87.9±2.1)%(n=10).
Conclusions
After sepsis,the metabolism of carbohydrate,protein,fat and nucleinic acid changes markedly.Both HPLC/MS-based and NMR-based metabonomic approachs combined with pattern recognition permit the overall monitoring for the stress metabolism,and allow accurate outcome prediction of septic rats in the early stage. The proposed approach has advantages of rapid,low-cost and efficiency,and is expected to be applied in clinical prognostic evaluation of septic patients.
The analytic range of HPLC/MS is different with NMR spectroscopy.The former lays particular emphasis on those metabolins with molecular mass＞100,such as free fatty acid.The latter emphasizes those metabolins with molecular mass＜100, such as carbohydrate and amino acid.Therefore,HPLC/MS coupled with NMR spectroscopy will allow overall metabolin information related with sepsis,resulting in more accurate evaluation on illness severity of sepsis.
引文
[1] Angus DC, Linde-Zwirble WT, Lidicker J, et al. Epidemiology of severe sepsis in the United States: Analysis of incidence, outcome, and associated costs of care. Crit Care Med, 2001, 29: 1303-10.
[2] Wang H, Liao H, Ochani M, et al. Cholinergic agonists inhibit HMGB1 release and improve survival in experimental sepsis. Nat Med, 2004,10(11): 1216-21.
[3] Fisher CJ, Agosti J M, Opal SM, et al. Treatment of septic shock with the tumor necrosis factor receptor: Fc fusion protein. N Engl J Med, 1996, 334:1697-1702.
[4] Abraham E. Cytokine modifiers: pipe dream or reality? Chest, 1998,113: 224S- 227S.
[5] Rivers E, Nguyen B, Havstad S, et al. Early goal-directed therapy in the treatment of severe sepsis and septic shock. N Engl J Med, 2001,345:1368-77.
[6] The Acute Respiratory Distress Syndrome Network. Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome. N Engl J Med, 2000,342:1301-08.
[7] van den Berghe G, Wouters P, Weekers F, et al. Intensive insulin therapy in critically ill patients. N Engl J Med 2001,345:1359-67.
[8] Annane D, Sebille V, Charpentier C, et al. Effect of treatment with low doses of hydrocortisone and fludrocortisone on mortality in patients with septic shock. JAMA, 2002, 288: 862-71.
[9] Bernard GR, Vincent JL, Laterre PF, et al. Efficacy and safety of recombinant human activated protein C for severe sepsis. N Engl J Med, 2001,344(10): 699-709.
[10] Holmes CL, Russell JA, Walley KR. Genetic polymorphisms in sepsis and septic shock: role in prognosis and potential for therapy. Chest 2003,124(3):1103-15.
[11] Chen QX, Lv C, Huang LX, et al. Genomic variations within DEFB1 are associated with the susceptibility to and the fatal outcome of severe sepsis in Chinese Han population. Genes Immun, 2007, 8(5): 439-43.
[12] Walley KR, Russell JA. Protein C -1641 AA is associated with decreased survival and more organ dysfunction in severe sepsis. Crit Care Med, 2007,35(1): 12-7.
[13]Arditi M, Manoque KR, Caplan M, Yoger R. Cerebrospinal fluid cachectin/tumour necrosis factor alpha and platelet-activating factor concentrations and severity of bacterial meningitis in children. J Infect Dis 1990,160:139-47.
[14]Mokart D, Merlin M, Sannini A, et al. Procalcitonin, interleukin 6 and systemic inflammatory response syndrome (SIRS): early markers of postoperative sepsis after major surgery. Br J Anaesth, 2005, 94: 767-773.
[15]Hamano K, Gohra H, Noda H, et al. Increased serum interleukin-8: correlation with poor prognosis in patients with postoperative multiple organ failure. World J Surg, 1998, 22: 1077-1081.
[16]Ashare A, Powers LS, Butler NS, et al. Anti-inflammatory response is associated with mortality and severity of infection in sepsis. Am J Physiol Lung Cell Mol Physiol, 2005, 288(4): L633-640.
[17] Shi C, Andersson R, Zhao X, et al. Potential role of reactive oxygen species in pancreatitis-associated multiple organ dysfunction. Pancreatology, 2005,5(4-5): 492-500.
[18]Povoa P, Coelho L, Almeida E, et al. C-reactive protein as a marker of infection in critically ill patients. Clin Microbiol Infect, 2005, 11(2):101-108.
[19]Castelli GP, Pognani C, Meisner M, et al. Procalcitonin and C-reactive protein during systemic inflammatory response syndrome, sepsis and organ dysfunction. Crit Care, 2004, 8: 234-242.
[20] Opal SM, Scannon PJ, Vincent JL, et al. Relationship between plasma levels of lipopolysaccharide (LPS) and LPS-binding protein in patients with severe sepsis and septic shock. J Infect Dis, 1999,180:1584-9.
[21]Ware LB, Eisner MD, Thompson BT, et al. Significance of von Willebrand factor in septic and nonseptic patients with acute lung injury. Am J Respir Crit Care Med, 2004, 170(7): 766-72.
[22]Osuchowski MF, Welch K, Siddiqui J, et al. Circulating cytokine/inhibitor profiles reshape the understanding of the SIRS/CARS continuum in sepsis and predict mortality. J Immunol, 2006,177(3): 1967-74.
[23]Dajak M, Ignjatovic S, Majkic-Singh N, et al. Prognostic value of phospholipase A2 group II, C-reactive protein and Simplified Acute Physiological Score II in intensive care patients. Clin Lab, 2006,52(7-8): 387-92.
[24]Sponholz C, Sakr Y, Reinhart K, Brunkhorst F. Diagnostic value and prognostic implications of serum procalcitonin after cardiac surgery: a systematic review of the literature. Crit Care, 2006,10: R145.
[25] Balc IC, Sungurtekin H, Gurses E, et al. Usefulness of procalcitonin for diagnosis of sepsis in the intensive care unit. Crit Care, 2003,7: 85-90.
[26]Preul MC, Caramanos Z, Collins DL, et al. Accurate, noninvasive diagnosis of human brain tumors by using proton magnetic resonance spectroscopy. Nat Med, 1996, 2(3): 323-5.
[27]Beckonert O, Monnerjahn J, Bonk U, Leibfritz D. Visualizing metabolic changes in breast-cancer tissue using 1H-NMR spectroscopy and self-organizing maps. NMR Biomed, 2003,16(1): 1-11.
[28]Makinen VP, Soininen P, Forsblom C, et al. Diagnosing diabetic nephropathy by 1H NMR metabonomics of serum. MAGMA, 2006,19(6): 281-96.
[29] Wu Y, Chai J, Li J. Changes in plasma free amino acid concentrations in rats during sepsis. Zhonghua Wai Ke Za Zhi, 2001,39:638-42.
[30] Nguyen HB, Rivers EP, Knoblich BP, et al. Early lactate clearance is associated with improved outcome in severe sepsis and septic shock. Crit Care Med, 2004, 32:1637-42.
[31] Revelly JP, Tappy L, Martinez A, et al. Lactate and glucose metabolism in severe sepsis and cardiogenic shock. Crit Care Med, 2005,33: 2235-40.
[32] Nicholson JK, Wilson ID. High resolution proton magnetic resonance spectroscopy of biological fluids. Prog Nucl Magn Reson Spectrosc, 1989,21(4): 449-501.
[33]Pham-Tuan H, Kaskavelis L, Daykin CA, Janssen HG. Method development in high-performance liquid chromatography for high-throughput profiling and metabonomic studies of biofluid samples. J Chromatogr B: Analyt Technol Biomed Life Sci, 2003, 789(2): 283-301.
[34]Shockcor JP, Holmes E, Metabonomic applications in toxicity screening and disease diagnosis. Curr Top Med Chem, 2002,2(1): 35-51.
[35] Nicholson JK, Lindon JC, Holmes E. Metabonomics: understanding the metabolic responses of living systems to pathophysiological stimuli via multivariate statistical analysis of biological NMR data. Xenobiotica, 1999,29(11): 1181-9.
[36]Lehtimaki KK, Valonen PK, Griffin JL, et al. Metabolite changes in BT4C rat gliomas undergoing ganciclovir-thymidine kinase gene therapy-induced programmed cell death as studied by ~1H NMR spectroscopy in vivo, ex vivo, and in vitro. J Biol Chem, 2003, 278(46): 45915-23.
[37]Sabatine MS, Liu E, Morrow DA, et al. Metabolomic identification of novel biomarkers of myocardial ischemia. Circulation, 2005,112(25): 3868-75.
[38] Jun Yang, Guowang Xua, Yufang Zheng, et al. Diagnosis of liver cancer using HPLC-based metabonomics avoiding false-positive result from hepatitis and hepatocirrhosis diseases. J Chromatogr B Analyt Technol Biomed Life Sci, 2004,813: 59-65.
[39]Coen M, O'Sullivan M, Bubb WA, et al. Proton nuclear magnetic resonance-based metabonomics for rapid diagnosis of meningitis and ventriculitis. Clin Infect Dis. 2005, 41(11): 1582-90.
[40]Wichterman KA, Baue AE, Chaudry IH. Sepsis and septic shock - a review of laboratory models and a proposal. J Surg Res, 1980,29(2): 189-201.
[41]Cobas JC, Sardina FJ. Nuclear magnetic resonance data processing. MestRe-C: A software package for desktop computers. Concepts Magn Res A, 2003,19A: 80-96.
[42]Dieterle F, Schlotterbeck G, Ross A, et al. Application of metabonomics in a compound ranking study in early drug development revealing drug-induced excretion of choline into urine. Chem Res Toxicol, 2006,19(9): 1175-81.
[43]Dieterle F, Ross A, Schlotterbeck G, Senn H. Metabolite projection analysis for fast identification of metabolites in metabonomics: application in an amiodarone study. Anal Chem, 2006,78(11): 3551-61.
[44] Yap IKS, Clayton TA, Tang H, et al. An integrated metabonomic approach to describe temporal metabolic disregulation induced in the rat by the model hepatotoxin allyl formate. J proteome Res, 2006,5(10): 2675-84.
[45]Trygg J, Wold S. Orthogonal projections to latent structures (O-PLS). J Chemometr, 2002,16: 119-28.
[46]Hotelling H. The Generalization of Student's Ratio The Annals of Mathematical Statistics, 1931, 2:360-78.
[47] Feng J, Li X, Pei F, et al. ~1H NMR analysis for metabolites in serum and urine from rats administrated chronically with La(NO3)3. Anal Biochem, 2002,301:1-7.
[48] Wu H, Zhang X, Li X, et al. Comparison of metabolic profiles from serum from hepatotoxin-treated rats by nuclear-magnetic-resonance-spectroscopy-based metabonomic analysis. Anal Biochem, 2005,340: 99-105.
[49]Liao P, Wei L, Zhang X, et al. Metabolic profiling of serum from gadolinium chloride-treated rats by 1H NMR spectroscopy. Anal Biochem 2007,364:112-121.
[50]Brereton RG Chemometrics: data analysis for the laboratory and chemical plant. London: John Wiley & Sons, 2003.
[51]Sarimveis H, Doganis P, Alexandridis A. A classification technique based on radial basis function neural networks. Advances in Engineering Software, 2006,37(4): 218-21.
[52] Jones CM, Athanasiou T. Summary receiver operating characteristic curve analysis techniques in the evaluation of diagnostic tests. Ann Thorac Surg, 2005,79:16-20.
[53] Martin G, Mannino D, Eaton S, Moss M. The epidemiology of sepsis in the United States from 1979 through 2000. N Engl J Med, 2003, 348(16): 1546-54.
[54]Adrie C, Alberti C, Chaix-Couturier C, et al. Epidemiology and economic evaluation of severe sepsis in France: Age, severity, infection site, and place of acquisition (community, hospital, or intensive care unit) as determinants of workload and cost. J Crit Care, 2005, 20(1): 46-58.
[55]Moerer O, Schmid A, Hofmann M, et al. Direct costs of severe sepsis in three German intensive care units based on retrospective electronic patient record analysis of resource use. Intensive Care Med, 2002,28(10): 1440-6.
[56] Warren H. Strategies for the treatment of sepsis. N Engl J Med, 1997,336(13): 952-3.
[57]Parrillo JE, Parker MM, Natanson C, et al. Septic shock in humans. Advances in the understanding of pathogenesis, cardiovascular dysfunction, and therapy. Ann Intern Med, 1990,113(3): 227-42.
[58]Pittet D, Rangel-Frausto S, Li N, T et al. Systemic inflammatory response syndrome, sepsis, severe sepsis and septic shock: incidence, morbidities and outcomes in surgical ICU patients. Intensive Care Med, 1995,21(4): 302-9.
[59]Kollef M, Sherman G, Ward S, Fraser V. Inadequate antimicrobial treatment of infections: a risk factor for hospital mortality among critically ill patients. Chest 1999,115(2): 462-74.
[60] Kim YJ, Chung HY. Antioxidative and anti-inflammatory actions of docosahexaenoic acid and eicosapentaenoic acid in renal epithelial cells and macrophages. J Med Food, 2007,10(2): 225-31.
[61]Kazda A. Sepsis-problems of meeting energy needs. Cas Lek Cesk 1986,125:1086-9.
[62]Joynt GM, Lipman J, Gomersall CD, et al. Gastric intramucosal pH and blood lactate in severe sepsis. Anaesthesia, 1997,52:726-32.
[63]Kazda A. Meeting the energy requirements in sepsis. Czech Med, 1988,11:1-9.
[64]Kirschenbaum LA, Astiz ME, Rackow EC. Interpretation of blood lactate concentrations in patients with sepsis. Lancet 1998,352: 921-2
[65]Brealey D, Karyampudi S, Jacques TS, et al. Mitochondrial dysfunction in a long-term rodent model of sepsis and organ failure. Am J Physiol Regul Integr Comp Physiol, 2004, 286: R491-7.
[66] Friedman G, Berlot G, Kahn RJ, Vincent JL. Combined measurebments of blood lactate concentrations and gastric intramucosal pH in patients with severe sepsis. Crit Care Med, 1995,23:1184-93.
[67] Gore DC, Jahoor F, Hibbert J, DeMaria EJ. Except for alanine, muscle protein catabolism is not influenced by alterations in glucose metabolism during sepsis. Arch Surg, 1995, 130: 1171-6.
[68]Samra JS, Summers LK, Frayn KN. Sepsis and fat metabolism. Br J Surg, 1996, 83(9): 1186-96.
[69] Martinez A, Chiolero R, Bollman M, et al. Assessment of adipose tissue metabolism by means of subcutaneous microdialysis in patients with sepsis or circulatory failure. Clin Physiol Funct Imaging, 2003,23(5):286-92.
[70]Memon RA, Feingold KR, Moser AH, et al. Regulation of fatty acid transport protein and fatty acid translocase mRNA levels by endotoxin and cytokines. Am J Physiol Endocrinol Metab, 1998, 274(2): E210-7.
[71]Brealey D, Brand M, Hargreaves I, et al. Association between mitochondrial dysfunction and severity and outcome of septic shock. Lancet, 2002,360(9328): 219-23.
[72]Calder PC. n-3 Fatty acids, inflammation, and immunity: Relevance to postsurgical and critically ill patients. Lipids 2004,39(12): 1147-61.
[73] Ren J, Chung SH. Anti-inflammatory effect of alpha-linolenic acid and its mode of action through the inhibition of nitric oxide production and inducible nitric oxide synthase gene expression via NF-kappaB and mitogen-activated protein kinase pathways. J Agric Food Chem, 2007,55(13): 5073-80.
[74]Solakivi T,Jaakkola O,Kalela A,et al.Lipoprotein docosapentaenoic acid is associated with serum matrix metalloproteinase-9 concentration Lipids Health Dis,2005,4(1):8.
[75]Kim YJ,Chung HY.Antioxidative and anti-inflammatory actions of docosahexaenoic acid and eicosapentaenoic acid in renal epithelial cells and macrophages.J Med Food,2007,10(2):225-31.
[76]Bosscha K,Reijnders K,Hulstaert PF,et al.Prognostic scoring systems to predict outcome in peritonitis and intra-abdominal sepsis.Br J Surg,1997,84:1532-34.
[77]Arabi Y,Al Shirawi N,Memish Z,et al.Assessment of six mortality prediction models in patients admitted with severe sepsis and septic shock to the intensive care unit:a prospective cohort study.Crit Care,2003,7:R116-22.
[78]Taylor SL,Morgan DL,Denson KD,et al.A comparison of the Ranson,Glasgow,and APACHE Ⅱ scoring systems to a multiple organ system score in predicting patient outcome in pancreatitis.Am J Surg 2005,189:219-22.
[79]Dahaba AA,Hagara B,Fall A,et al.Procalcitonin for early prediction of survival outcome in postoperative critically ill patients with severe sepsis.Br J Anaesth,2006,97:503-8.
[80]Davis BH,Olsen SH,Ahmad E,Bigelow NC.Neutrophil CD64 is an improved indicator of infection or sepsis in emergency department patients.Arch Pathol Lab Med,2006,130:654-61.
[1] Garcia JG, Moreno Vinasco L: Genomic insights into acute inflammatory lung injury. Am J Physiol Lung Cell Mol Physiol 2006, 291:L1113-L1117
[2] Arcaroli J, Fessler MB, Abraham E: Genetic polymorphisms and sepsis. Shock 2005, 24: 300-312
[3] Mira JP, Cariou A, Grall F, et al: Association of TNF2, a TNF-alpha promoter polymorphism, with septic shock susceptibility and mortality: a multicenter study. JAMA 1999,282:561-568
[4] Arbour NC, Lorenz E, Schutte BC, et al: TLR4 mutations are associated with endotoxin hyporesponsiveness in humans. Nat Genet 2000,25:187-191
[5] Lorenz E, Mira JP, Frees KL, et al: Relevance of mutations in the TLR4 receptor in patients with Gram-negative septic shock. Arch Intern Med 2002,162:1028-1032
[6] Arnalich F, Garcia-Palomero E, Lopez J, et al: Predictive value of nuclear factor kappaB activity and plasma cytokine levels in patients with sepsis. Infect Immun 2000, 68: 1942-1945
[7] Yang KY, Arcaroli JJ, Abraham E: Early alterations in neutrophil activation are associated with outcome in acute lung injury. Am J Respir Crit Care Med 2003,167:1567-1574
[8] Arcaroli J, Silva E, Maloney JP, et al: Variant IRAK-1 haplotype is associated with increased nuclear factor-kappaB activation and worse outcomes in sepsis. Am J Respir Crit Care Med 2006,173:1335-1341
[9] Vincent JL, Abraham E: The last 100 years of sepsis. Am J Respir Crit Care Med 2006, 173:256-263
[10] Wang H, Yang H, Tracey KJ: Extracellular role of HMGB1 in inflammation and sepsis. J Intern Med 2004,255:320-331
[11] Yang H, Wang H, Czura CJ, et al: The cytokine activity of HMGB1. J Leukoc Biol 2005, 78:1-8
[12] Sadikot RT, Christman JW, Blackwell TS: Molecular targets for modulating lung inflammation and injury. Curr Drug Targets 2004,5:581-588
[13] Park JS, Gamboni-Robertson F, He Q, et al: High mobility group box 1 protein interacts with multiple Toll-like receptors. Am J Physiol Cell Physiol 2006,290:C917-C924
[14] Yang H, Ochani M, Li J, et al: Reversing established sepsis with antagonists of endogenous high-mobility group box 1. Proc Nat Acad Sci U S A 2004,101:296-301
[15] Cobb JP: Nitric oxide synthase inhibition as therapy for sepsis: A decade of promise. Surg Infect (Larchmt) 2001, 2:93-100
[16] Lopez A, Lorente JA, Steingrub J, et al: Multiple-center, randomized, placebocontrolled, double-blind study of the nitric oxide synthase inhibitor 546C88: Effect on survival in patients with septic shock. Crit Care Med 2004,32:21-30
[17] Farley KS, Wang LF, Razavi HM, et al: Effects of macrophage inducible nitric oxide synthase in murine septic lung injury. Am J Physiol Lung Cell Mol Physiol 2006, 290:L1164-L1172
[18] Fink MP: Reactive oxygen species as mediators of organ dysfunction caused by sepsis, acute respiratory distress syndrome, or hemorrhagic shock: Potential benefits of resuscitation with Ringer's ethyl pyruvate solution. Curr Opin Clin Nutr Metab Care 2002,5:167-174
[19] Angstwurm MW, Engelmann L, Ximmermann T, et al: Selenium in Intensive Care (SIC): Results of a prospective randomized, placebo-controlled, multiple-center study in patients with severe systemic inflammatory response syndrome, sepsis, and septic shock. Crit Care Med 2007,35:118-126
[20] Salvemini D, Cuzzocrea S: Therapeutic potential of superoxide dismutase mimetics as therapeutic agents in critical care medicine. Crit Care Med 2003,31(1 Suppl):S29-S38
[21] Asehnoune K, Strassheim D, Mitra S, et al: Involvement of reactive oxygen species in tolllike receptor 4-dependent activation of NFkappaB. J Immunol 2004,172:2522-2529
[22] Meng X, Harken AH: The interaction between Hsp70 and TNF-alpha expression: A novel mechanism for protection of the myocardium against post-injury depression. Shock 2002, 17:345-353
[23] Hotchkiss RS, Karl IE: The pathophysiology and treatment of sepsis. N Engl J Med 2003, 348:138-150
[24] Brown KA, Brain SD, Pearson JD, et al: Neutrophils in development of multiple organ failure in sepsis. Lancet 2006,368:157-169
[25] Abraham E: Alterations in cell signaling in sepsis. Clin Infect Dis 2005, 41(Suppl 7): S459-S464
[26] Nussler NC, Stange B, Nussler AK, et al: Upregulation of intraepithelial lymphocyte function in the small intestinal mucosa in sepsis. Shock 2001,16:454-458
[27] Chinnaiyan AM, Huber-Lang M, Kumar Sinha C, et al: Molecular signatures of sepsis: Multiorgan gene expression profiles of systemic inflammation. Am J Pathol 2001, 159:1199-1209
[28] Cobb JP, Laramie JM, Stormo GD, et al: Sepsis gene expression profiling: Murine splenic compared with hepatic responses determined by using complementary DNA microarrays. Crit Care Med 2002, 30:2711-2721
[29] Adib-Conquy M, Adrie C, Moine P, et al: NF-kappaB expression in mononuclear cells of patients with sepsis resembles that observed in lipopolysaccharide tolerance. Am J Respir Crit Care Med 2000,162:1877-1883
[30] Adib-Conquy M, Asehnoune K, Moine P, et al: Long-term-impaired expression of nuclear factor-kappa B and I kappa B alpha in peripheral blood mononuclear cells of trauma patients. J Leukoc Biol 2001,70: 30-38
[31] Moore FA, Moore EE, Poggetti R, et al: Gut bacterial translocation via the portal vein: A clinical perspective with major torso trauma. J Trauma 1991,31:629-636
[32] Murphy DB, Cregg N, Tremblay L, et al: Adverse ventilatory strategy causes pulmonary-to -systemic translocation of endotoxin. Am J Respir Crit Care Med 2000,162:27-33
[33] Huston JM, Ochani M, Rosas-Ballina M, et al: Splenectomy inactivates the cholinergic antiinflammatory pathway during lethal endotoxemia and polymicrobial sepsis. J Exp Med 2006, 203:1623-1628
[34] Aird WC: The role of the endothelium in severe sepsis and multiple organ dysfunction syndrome. Blood 2003,101:3765-3777
[35] Boos CJ, Goon PK, Lip GY: The endothelium, inflammation, and coagulation in sepsis. Clin Pharmacol Ther 2006, 79:20-22
[36] Martin TR, Nakamura M, Matute-Bello G: The role of apoptosis in acute lung injury. Crit Care Med 2003,31(4 Suppl):S184-S188
[37] Cavaillon JM, Adib-Conquy M: Monocytes/Garcia JG, Moreno Vinasco L: Genomic insights into acute inflammatory lung injury. Am J Physiol Lung Cell Mol Physiol 2006, 291:L1113-L1117
[38] Koh IH, liberatore AM, Menchaca-Diaz JL, et al: Bacterial translocation, microcirculation injury and sepsis. Endocr Metab Immune Disord Drug Targets 2006,6:143-150
[39] Cavaillon JM, Annane D: Compartmentalization of the inflammatory response in sepsis and SIRS. J Endotoxin Res 2006,12:151-170
[40] Reddy RC, Chen GH, Newstead MW, et al: Alveolar macrophage deactivation in murine septic peritonitis: Role of interleukin 10. Infect Immun 2001, 69:1394-1401
[41] Cavaillon JM, Adrie C, Fitting C, et al: Reprogramming of circulatory cells in sepsis and SIRS. J Endotoxin Res 2005,11: 311-320
[42] Burnham EL, Taylor WR, Quyyumi AA, et al: Increased circulating endothelial progenitor cells are associated with survival in acute lung injury. Am J Respir Crit Care Med 2005, 172:854-860
[43] Doerschuk CM: Circulating endothelial progenitor cells in pulmonary inflammation. Thorax 2005, 60:362-364
[44] Zeraans RL, Matthay MA: Bench-to-bedside review: The role of the alveolar epithelium in the resolution of pulmonary edema in acute lung injury. Crit Care 2004, 8:469-477
[45] Matthay MA, Robriquet L, Fang X: Alveolar epithelium: Role in lung fluid balance and acute lung injury. Proc Am Thorac Soc 2005, 2:206-213
[46] Levi M, Keller TT, van Gorp E, et al: Infection and inflammation and the coagulation system. Cardiovasc Res 2003,60:26-39
[47] Sakr Y, Dubois MJ, De Backer D, et al: Persistent microcirculatory alterations are associated with organ failure and death in patients with septic shock. Crit Care Med 2004, 32:1825-1831
[48] Singer M, De Santis V, Vitale D, et al: Multiorgan failure is an adaptive, endocrinemediated, metabolic response to overwhelming systemic inflammation. Lancet 2004, 364:545-548
[49] Hotchkiss RS, Swanson PE, Freeman BD, et al: Apoptotic cell death in patients with sepsis, shock, and multiple organ dysfunction. Crit Care Med 1999, 27:1230-1251
[50] Landry DW, Oliver JA: The pathogenesis of vasodilatory shock. N Engl J Med 2001, 345: 588-595
[51] Barrett LK, Singer M, Clapp LH: Vasopressin: Mechanisms of action on the vasculature in health and in septic shock. Crit Care Med 2007,35:33-40
[52] Court O, Kumar A, Parrillo JE, et al: Clinical review: Myocardial depression in sepsis and septic shock. Crit Care 2002,6:500-508
[53] Rivers E, Bryant MPH, Havstad S, et al: Early goal directed therapy in the treatment of severe sepsis and septic shock: The Early Goal-Directed Therapy Collaborative Group. N Engl J Med 2001,345:1368-1377
[54] Dahn MS, Mitchell RA, Lange MP, et al: Hepatic metabolic response to injury and sepsis. Surgery 1995,117:520-530
[55] Tugtekin IF, Radermacher P, Theisen M, et al: Increased ileal-mucosal-arterial PCO2 gap is associated with impaired villus microcirculation in endotoxic pigs. Intensive Care Med 2001, 27:757-766
[56] Cunnion RE, Schaer GL, Parker MM, et al: The coronary circulation in human septic shock. Circulation 1986, 73:637-644
[57] Langenberg C, Bellomo R, May C, et al: Renal blood flow in sepsis. Crit Care 2005, 9:R363-R374
[58] Patel BM, Chittock DR, Russell JA, et al: Beneficial effects of short-term vasopressin infusion during severe septic shock. Anesthesiology 2002,96:576-582
[59] Spronk PE, Zandstra DF, Ince C: Bench-tobedside review: Sepsis is a disease of the microcirculation. Crit Care 2004, 8:462-468
[60] VanderMeer TJ, Wang H, Fink MP: Endotoxemia causes Deal mucosal acidosis in the absence of mucosal hypoxia in a normodynamic porcine model of septic shock. Crit Care Med 1995, 23:1217-1226
[61] Rosser DM, Stidwill RP, Jacobson D, et al: Oxygen tension in the bladder epithelium rises in both high and low cardiac output endotoxemic sepsis. J Appl Physiol 1995, 79:1878-1882
[62] Van den Berghe G, Wouters P, Weekers F, et al: Intensive insulin therapy in critically ill patients. N Engl J Med 2001,345:1359-1367
[63] Van den Berghe G: How does blood glucose control with insulin save lives in intensive care? J Clin Invest 2004,114:1187-1195
[64] Vanhorebeek I, De Vos R, Mesotten D, et al: Protection of hepatocyte mitochondrial ultrastructure and function by strict blood glucose control with insulin in critically ill patients. Lancet 2005,365:53-59
[65] Arnold J, Campbell IT, Samuels TA, et al: Increased whole body protein breakdown predominates over increased whole body protein synthesis in multiple organ failure. Clin Sci (Lond) 1993,84:655-661
[66] Chiolero R, Revelly JP, Tappy L: Energy metabolism in sepsis and injury. Nutrition 1997, 13(9 Suppl):45S-51S
[67] Trager K, DeBacker D, Radermacher P: Metabolic alterations in sepsis and vasoactive drug-related metabolic effects. Curr Opin Crit Care 2003,9:271-278
[68] McClave SA, Snider HL: Dissecting the energy needs of the body. Curr Opin Clin Nutr Metab Care 2001, 4:143-147
[69] Wischmeyer PE: The glutamine story: Where are we now? Curr Opin Crit Care 2006, 12:142-148
[70] Bellingan GJ: The pathogenesis of ALI/ARDS. Thorax 2002,57:540-546
[71] Moloney ED, Griffiths MJ: Protective ventilation of patients with acute respiratory distress syndrome. Br J Anaesth 2004, 92: 261-267
[72] Bernard GR: Acute respiratory distress syndrome: A historical perspective. Am J Respir Crit Care Med 2005,172:798-806
[73] Bachofen M, Weibel ER: Structural alterations of lung parenchyma in the adult respiratory distress syndrome. Clin Chest Med 1982,3:35-56
[74] Martin TR, Hagimoto N, Nakamura M, et al: Apoptosis and epithelial injury in the lungs. Proc Am Thorac Soc 2005,2:214-220
[75] Vlahakis NE, Hubmayr RD: Cellular stress failure in ventilator-injured lungs. Am J Respir Crit Care Med 2005,171:1328-1342
[76] Rieux-Laucat F, Fischer A, Deist FL: Celldeath signaling and human disease. Curr Opin Immunol 2003,15:325-331
[77] Riedl SJ, Shi Y: Molecular mechanisms of caspase regulation during apoptosis. Nat Rev Mol Cell Biol 2004,5:897-907
[78] Matute-Bello G, Liles WC, Steinberg KP, et al: Soluble Fas ligand induces epithelial cell apoptosis in humans with acute lung injury (ARDS). J Immunol 1999,163:2217-2225
[79] Bardales RH, Xie SS, Schaefer RF, et al: Apoptosis is a major pathway responsible for the resolution of type II pneumocytes in acute lung injury. Am J Pathol 1996,149: 845-852
[80] Haddad JJ: Science review: Redox and oxygen-sensitive transcription factors in the regulation of oxidant-mediated lung injury. Role for hypoxia-inducible factor-lalpha. Crit Care 2003,7:47-54
[81] Quinlan GJ, Evans TW, Gutteridge JM: Iron and the redox status of the lungs. Free Radic Biol Med 2002,33:1306-1313
[82] Maxwell PH, Ratcliffe PJ: Oxygen sensors and angiogenesis. Semin Cell Dev Biol 2002, 13:29-37
[83] Dennery PA, Visner G, Weng YH, et al: Resistance to hyperoxia with heme oxygenase-1 disruption: Role of iron. Free Radic Biol Med 2003,34:124-133
[84] Anning PB, Chen Y, Lamb NJ: Iron overload upregulates haem oxygenase 1 in the lung more rapidly than in other tissues. FEBS Lett 1999,447:111-114
[85] Sharshar T, Annane D, de la Grandmaison GL, et al: The neuropathology of septic shock. Brain Pathol 2004,14:21-33
[86] Sharshar T, Hopkinson NS, Orlikowski D, et al: Science review: The brain in sepsis. Culprit and victim. Crit Care 2005,9:37-44
[87] Carrasco GA, van de Kar LD: Neuroendocrine pharmacology of stress. Eur J Pharmacol 2003, 463:235-272
[88] Sharshar T, Gray F, Lorin de la Grandmaison G, et al: Apoptosis of neurons in cardiovascular autonomic centres triggered by inducible nitric oxide synthase after death from septic shock. Lancet 2003,362:1799-1805
[89] Messaris E, Memos N, Chatzigianni E, et al: Time-dependent mitochondrial-mediated programmed neuronal cell death prolongs survival in sepsis. Crit Care Med 2004, 32: 1764-1770
[90] Fink MP, Delude RL: Epithelial barrier dysfunction: A unifying theme to explain the pathogenesis of multiple organ dysfunction at the cellular level. Crit Care Clin 2005,21:177-196
[91] Magnotti LJ, Upperman JS, Xu DZ, et al: Gut-derived mesenteric lymph but not portal blood increases endothelial cell permeability and promotes lung injury after hemorrhagic shock. Ann Surg 1998,228:518-527
[92] Liu S, Stolz DB, Sappington PL, et al: HMGB1 is secreted by immunostimulated enterocytes and contributes to cytomixinduced hyperpermeability of Caco-2 monolayers. Am J Physiol Cell Physiol 2006, 290: C990-C999
[93] Sugi K, Musch MW, Field M, et al: Inhibition of Na~(?) ,K~(?) -ATPase by interferon gamma down-regulates intestinal epithelial transport and barrier function. Gastroenterology 2001, 120:1393-1403
[94] Pastor CM, Billiar TR, Losser MR, et al: Liver injury during sepsis. J Crit Care 1995, 10:183-197
[95] Carbonell LF, Nadal JA, Llanos MC, et al: Depletion of liver glutathione potentiates the oxidative stress and decreases nitric oxide synthesis in a rat endotoxin shock model. Crit Care Med 2000, 28:2002-2006
[96] Moreno R, Vincent JL, Matos R, et al: The use of maximum SOFA score to quantify organ dysfunction/failure in intensive care: Results of a prospective, multicentre study. Intensive Care Med 1999,25:686-696
[97] Wan L, Bellomo R, Di Giantomasso D, et al: The pathogenesis of septic acute renal failure. Curr Opin Crit Care 2003, 9:496-502
[98] Di Giantomasso D, Morimatsu H, May CN, et al: Intrarenal blood flow distribution in hyperdynamic septic shock: Effect of norepinephrine. Crit Care Med 2003,31: 2509-2513
[99] Cohen RI, Hassell AM, Marzouk K, et al: Renal effects of nitric oxide in endotoxemia. Am J Respir Crit Care Med 2001,164:1890-1895
[100] Carraway MS, Welty-Wolf KE, Miller DL, et al: Blockade of tissue factor: Treatment for organ injury in established sepsis. Am J Respir Crit Care Med 2003,167:1200-1209
[101] Rabuel C, Mebazaa A: Septic shock: A heart story since the 1960s. Intensive Care Med 2006, 32:799-807
[102] Parker MM, Shelhamer JH, Bacharach SL, et al: Profound but reversible myocardial depression in patients with septic shock. Ann Intern Med 1984,100:483-490
[103] Mehta NJ, Khan IA, Gupta V, et al: Cardiac troponin I predicts myocardial dysfunction and adverse outcome in septic shock. Int J Cardiol 2004, 95:13-17
[104] Parrillo JE, Burch C, Shelhamer JH, et al: A circulating myocardial depressant substance in humans with septic shock: Septic shock patients with a reduced ejection fraction have a circulating factor that depresses in vitro myocardial cell performance. J Clin Invest 1985, 76:1539-1553
[105]Zhu X,Bernecker OY,Manohar NS,et al:Increased leakage of sarcoplasmic reticulum Ca2」 contributes to abnormal myocyte Ca2」 handling and shortening in sepsis.Crit Care Med 2005,33:598-604
[106]Barth E,Radermacher P,Thiemermann C,et al:Role of inducible nitric oxide synthase in the reduced responsiveness of the myocardium to catecholamines in a hyperdynamic,murine model of septic shock.Crit Care Med 2006,34:307-313
[107]Lancel S,Joulin O,Favory R,et al:Ventricular myocyte caspases are directly responsible for endotoxin-induced cardiac dysfunction.Circulation 2005,111:2596-2604
[108]Singer M,Glynne P:Treating critical illness:The importance of first doing no harm.PLoS Med 2005,2:e167