The Use of Exosomes as Biomarkers for Evaluating and Monitoring Critically Ill Polytrauma Patients with Sepsis

Regarding genetic biomarkers for early assessment and monitoring the clinical course in polytrauma patients with sepsis, in recent years a remarkable evolution has been highlighted. One of the main representatives is the exosome miRNAs. In this paper, we would like to present in more details the various methods of using exosome miRNAs as a biomarker for monitoring polytrauma patients with sepsis, as well as establishing a belated outcome by aggregating the entire clinical aspects. The use of exosome miRNAs for late evaluating and monitoring the clinical evolution of polytrauma patients can bring significant improvements in current clinical practice through the optimization and modulation of intensive care according to the needs of each patient individually.     

Lack of association between TLR4 Asp299Gly and Thr399Ile polymorphisms and sepsis susceptibility: a meta-analysis

Sepsis, a condition of systemic inappropriate inflammation response to the invasion of microorganisms, results in considerable morbidity and mortality in patients. Some, but not all, epidemiological studies have suggested that Toll-like receptor 4 (TLR4) polymorphisms, Asp299Gly and Thr399Ile, may influence the risk of at-risk patients for sepsis. Our work tried to further study the association of the two common polymorphisms with sepsis susceptibility by performing a meta-analysis of previous data. Electronic searches of MEDLINE, EMBASE and Web of Science databases were performed. Original observational studies dealing with the association between polymorphisms Asp299Gly and/or Thr399Ile and sepsis risk were selected. Pooled odds ratios (ORs) and 95% confidence intervals (CIs) were calculated with random-effects model or fixed-effects model based on the heterogeneity analysis. Seventeen studies including 2212 cases and 3880 controls were included with most subjects of Caucasian ethnicity. The odds ratio for the association of Asp299Gly polymorphism with sepsis risk was 1.22 (95% CI: 0.90-1.65, P=0.21), and the association of Thr399Ile polymorphism was 1.16 (95%CI: 0.70-1.91, P=0.57). Subgroup analysis and sensitivity analysis did not change the results. Our meta-analysis revealed that the two common TLR4 polymorphisms, Asp299Gly and Thr399Ile, have no strong association with the likelihood of sepsis in Caucasian populations. Further studies are needed to investigate the effect of genetic networks and their mutual interactions in TLR4 signaling pathway on sepsis susceptibility.     

A CARD9 Polymorphism Is Associated with Decreased Likelihood of Persistent Conjugated Hyperbilirubinemia in Intestinal Failure

Recently, genetic associations have been described in intestinal transplants. Namely, Crohn''s disease susceptibility gene NOD2 polymorphisms have been reported to be more prevalent in patients with graft failure following intestinal transplantation (IT). Therefore, we sought to determine if polymorphisms in the NOD2 signaling cascade, including NOD2, CARD9, RAC1 and ATG16L1 are associated with intestinal failure (IF) or its complications. We carried out a cross-sectional study of 59 children with IF and 500 healthy Caucasian controls. Using the Taqman platform we determined the prevalence of NOD2 as well as ATG16L1, RAC1 and CARD9 SNPs. NOD2 pathway polymorphisms were evaluated in relation to outcomes of episodes of sepsis, ICU admissions, hyperbilirubinemia and need for IT. We found that the minor allele of a CARD9 SNP was associated with protection from developing IF when compared to healthy controls and was also associated with decreased odds of sustained conjugated hyperbilirubinemia. Therefore, IF patients with CARD9 polymorphism are less likely to develop progressive liver disease and suggests that host innate immunity may play a role in IF associated liver disease.     

Protein C -1641A/-1654C haplotype is associated with organ dysfunction and the fatal outcome of severe sepsis in Chinese Han population

Activation of protein C plays an important role in modulating coagulation as well as inflammation during severe sepsis. The baseline of activated protein C level in patients with severe sepsis showed interindividual variability between survivors and nonsurvivors, and the decreased level of protein C correlated with organ dysfunction and poor outcome. However, there are limited data concerning the genetic predisposition of individuals carrying two functional polymorphisms -1641A>G and -1654C>T within protein C gene to sepsis. Here we investigated the impact of these two variations on the development of severe sepsis in 240 patients with severe sepsis and 323 healthy controls using direct sequencing. After Bonferroni correction for multiple comparisons, -1641A/-1654C haplotype was significantly associated with the fatal outcome of severe sepsis (P = 0.008, OR 1.739, 95% CI 1.165–2.595), which was confirmed by multiple logistic regression analysis (P = 0.024, OR 2.090, 95% CI 1.101–3.967). Compared to patients without carrying -1641A/-1654C haplotype, the -1641A/-1654C haplotype carriers showed higher SOFAmax scores (10.3 ± 5.2 vs. 9.0 ± 4.5; P = 0.014) and more hepatic dysfunction (P = 0.004, OR 2.270, 95% CI 1.312–3.930). These findings suggest that protein C haplotype -1641A/-1654C is associated with organ dysfunction and is an independent risk factor for the fatal outcome of severe sepsis in Chinese Han population.     

Toll样受体5基因多态性与中国人群脓毒症的相关性研究

目的探讨Toll样受体5（Toll-likereceptor5,TLR5）基因多态性位点与脓毒症发生风险及疾病严重程度的相关性。方法采用病例一对照研究设计,募集了255例脓毒症患者和260例对照个体。应用贝克曼公司的商用SNPstream分型技术和PCR—RFLP方法对TLR5基因的3个编码区多态性位点进行分型。采用Logistic回归分析,校正性别、年龄、吸烟和饮酒、慢性病状态、APACHEⅡ评分和脓毒症病因等混杂因素的影响,评价多态性位点与脓毒症的发生风险,以及脓毒症性休克、死亡和器官功能障碍等表型的遗传相关性。结果TLR5基因的3个多态性位点在病例和对照组中的基因型分布均呈哈．温平衡状态。这3个编码区的多态性位点与脓毒症的发生风险和疾病严重程度均无遗传学关联。结论TLR5基因的多态性位点可能在脓毒症的发生、发展和病程转归中不发挥重要作用。     

Interleukin-10 rs2227307 and CXCR2 rs1126579 polymorphisms modulate the predisposition to septic shock

Despite major improvements in its treatment and diagnosis, sepsis is still a leading cause of death and admittance to the intensive care unit (ICU). Failure to identify patients at high risk of developing septic shock contributes to an increase in the sepsis burden and rapid molecular tests are currently the most promising avenue to aid in patient risk determination and therapeutic anticipation. The primary goal of this study was to evaluate the genetic susceptibility that affects sepsis outcome in 72 sepsis patients admitted to the ICU. Seven polymorphisms were genotyped in key inflammatory response genes in sepsis, including tumour necrosis factor-α, interlelukin (IL)-1β, IL-10, IL-8, Toll-like receptor 4, CXCR1 and CXCR2. The primary finding showed that patients who were homozygous for the major A allele in IL-10 rs1800896 had almost five times higher chance to develop septic shock compared to heterozygotes. Similarly, selected clinical features and CXCR2 rs1126579 single nucleotide polymorphisms modulated septic shock susceptibility without affecting survival. These data support the hypothesis that molecular testing has clinical usefulness to improve sepsis prognostic models. Therefore, enrichment of the ICU portfolio by including these biomarkers will aid in the early identification of sepsis patients who may develop septic shock.     

Genomic insights into acute inflammatory lung injury

Acute lung injury (ALI) is a devastating syndrome (usually associated with sepsis) that represents a major healthcare burden in the United States. We have focused our studies on unraveling the genetic underpinnings of this syndrome utilizing a candidate gene approach to identify novel genes for ALI susceptibility. Two novel genes identified by this approach include pre-B cell colony-enhancing factor (PBEF) and the gene for myosin light chain kinase (MLCK). PBEF protein levels were elevated in human bronchoalveolar lavage and serum samples from patients with ALI, and DNA sequencing identified two single nucleotide polymorphisms in the PBEF promoter (T-1001G, C-1543T) that were overrepresented in patients with sepsis-induced ALI. More recently, we found MLCK single polymorphisms and haplotypes to be associated with human ALI with unique variants observed in African-Americans with ALI. Thus genomic and genetic approaches represent powerful strategies in the identification of novel candidate genes and potential targets for ALI therapies.     

基质金属蛋白酶基因多态性与主动脉夹层的研究进展*

摘要：主动脉夹层(Aortic dissection, AD)为最危险的主动脉疾病之一,病死率较高,且发病率呈逐年上升的趋势。越来越多的证据 表明遗传因素影响该疾病的发生及发展,基因多态性为该疾病的遗传易感因素之一。主动脉夹层患者可观察到主动脉中膜的退 化,当主动脉结构发生改变时,必然导致一系列的病理生理反应,进而影响其功能。细胞外基质（Extracellular matrix,ECM）是由弹 性纤维和胶原纤维组成的,可以保持主动脉管壁的稳定性。主动脉夹层的发生与ECM 的代谢平衡有关,降解ECM的酶为基质金 属蛋白酶（Matrix metalloproteinases,MMPs）,这种酶在主动脉的重塑过程中也发挥作用,与夹层的发生密切相关。单核苷酸多态 性(single nucleotide polymorphism,SNP)作为遗传学标记,可以预测该疾病的发生, 指导该疾病的临床研究方向,对于易感性较高 的患者可进行早期的预防及监测,在AD的预防及治疗方面发挥重大作用。本文对基质金属蛋白酶基因多态性与主动脉夹层之间 的关系做一综述。     

基质金属蛋白酶基因多态性与主动脉夹层的研究进展

主动脉夹层（Aorticdissection,AD）为最危险的主动脉疾病之一,病死率较高,且发病率呈逐年上升的趋势。越来越多的证据表明遗传因素影响该疾病的发生及发展,基因多态性为该疾病的遗传易感因素之一。主动脉夹层患者可观察到主动脉中膜的退化,当主动脉结构发生改变时,必然导致一系列的病理生理反应,进而影响其功能。细胞外基质（Extracellularmatrix,ECM）是由弹性纤维和胶原纤维组成的,可以保持主动脉管壁的稳定性。主动脉夹层的发生与ECM的代谢平衡有关,降解ECM的酶为基质金属蛋白酶（Matrixmetalloproteinases,MMPs）,这种酶在主动脉的重塑过程中也发挥作用,与夹层的发生密切相关。单核苷酸多态性（singlenucleotidepolymorphism,SNP）作为遗传学标记,可以预测该疾病的发生,指导该疾病的临床研究方向,对于易感性较高的患者可进行早期的预防及监测,在AD的预防及治疗方面发挥重大作用。本文对基质金属蛋白酶基因多态性与主动脉夹层之间的关系做一综述．     

Outcome prediction in sepsis combined use of genetic polymorphisms - A study in Japanese population

Genetic polymorphisms have recently been found to be related to clinical outcome in septic patients. The present study investigated to evaluate the influence of genetic polymorphisms in Japanese septic patients on clinical outcome and whether use of genetic polymorphisms as predictors would enable more accurate prediction of outcome. Effects of 16 genetic polymorphisms related to pro-inflammatory mediators and conventional demographic/clinical parameters (age, sex, past medical history, and APACHE II score) on ICU mortality as well as disease severity during ICU stay were examined in the septic patients (n=123) admitted to the ICU between October 2001 and November 2007 by multivariable logistic regression analysis. ICU mortality was significantly associated with TNF -308GA, IL1β -31CT/TT, and APACHE II score. Receiver-operating characteristics (ROC) analysis demonstrated that, compared with APACHE II score alone (ROC-AUC=0.68), use of APACHE II score and two genetic parameters (TNF -308 and IL1β -31) enabled more accurate prediction of ICU mortality (ROC-AUC=0.80). Significant association of two genetic polymorphisms, TNF -308 and IL1β -31, with ICU mortality was observed in septic patients. In addition, combined use of these genetic parameters with APACHE II score may enable more accurate prediction of outcome in septic patients.     

Diagnostic Accuracy of NT-ProBNP for Heart Failure with Sepsis in Patients Younger than 18 Years

This clinical study investigated plasma NT-proBNP levels as a potential predictor of heart failure in pediatric patients with sepsis. Plasma NT-ProBNP levels of 211 pediatric patients with sepsis and 126 healthy children were measured. Patients were stratified as with heart failure (HF) or without heart failure (non-HF). Patients were graded as having sepsis, severe sepsis, or septic shock. The optimal cut-off values of plasma NT-ProBNP for heart failure were determined by analyzing the receiver operating characteristic (ROC). In the HF, non-HF and control groups, the median plasma NT-proBNP levels were 3640, 656, and 226 ng/L, respectively. For all patients with sepsis, the optimal diagnostic cut-off value was 1268 ng/L for differentiating heart failure. In the severe sepsis patients and septic shock patients, the optimal diagnostic cut-off values were 1368 ng/L and 1525 ng/L, respectively. This report is the first one to reveal that NT-proBNP may predict heart failure in children with sepsis. It provides an important clinical reference for the diagnosis of heart failure in pediatric patients with sepsis, and enables monitoring septic children for cardiac involvement.     

Oxidation-reduction states of NADH in vivo: from animals to clinical use

Mitochondrial dysfunction is part of many pathological states in patients, such as sepsis or stroke. Presently, the monitoring of mitochondrial function in patients is extremely rare, even though NADH redox state is routinely measured in experimental animals. In this article, we describe the scientific backgrounds and practical use of mitochondrial NADH fluorescence measurement that was applied to patients in the past few years. In addition to NADH, we optically measured the microcirculatory blood flow and volume, as well as HbO(2) oxygenation, from the same tissue area. The four detected parameters provide real time data on tissue viability, which is critical for patients monitoring.     

Effect of CYP17 and PSA gene polymorphisms on prostate cancer risk and circulating PSA levels in the Slovak population

Cytochrome P-450c17α (CYP17) and prostate-specific antigen (PSA) genes, which are involved in the androgen metabolism cascade, have been studied as possible candidates for genetic influences on prostate cancer development. Contradictory results prompted us to evaluate the frequencies of polymorphisms in the CYP17 and PSA genes as well as the association between these genetic variants and serum PSA levels in prostate cancer patients and men routinely screened for prostate cancer with PSA in the Slovak male population. The CYP17 and PSA polymorphisms were determined by the PCR-RFLP analysis in 197 Caucasian prostate cancer patients and 256 Caucasian controls. We did not find any association between the CYP17 and PSA genotypes and prostate cancer risk overall, or by grade. Also the total serum PSA levels in the cases with the AG or AA genotype were not significantly higher than in the men with the GG genotype (P > 0.05). Our study did not provide support for the hypothesized relationship between CYP17 and PSA gene polymorphisms and prostate cancer in the Slovak male population.     

Plasminogen Activator Inhibitor-2 Polymorphism Associates with Recurrent Coronary Event Risk in Patients with High HDL and C-Reactive Protein Levels

The objective of this work was to investigate whether fibrinolysis plays a role in establishing recurrent coronary event risk in a previously identified group of postinfarction patients. This group of patients was defined as having concurrently high levels of high-density lipoprotein cholesterol (HDL-C) and C-reactive protein (CRP) and was previously demonstrated to be at high-risk for recurrent coronary events. Potential risk associations of a genetic polymorphism of plasminogen activator inhibitor-2 (PAI-2) were probed as well as potential modulatory effects on such risk of a polymorphism of low-density lipoprotein receptor related protein (LRP-1), a scavenger receptor known to be involved in fibrinolysis in the context of cellular internalization of plasminogen activator/plansminogen activator inhibitor complexes. To this end, Cox multivariable modeling was performed as a function of genetic polymorphisms of PAI-2 (SERPINB, rs6095) and LRP-1 (LRP1, rs1800156) as well as a set of clinical parameters, blood biomarkers, and genetic polymorphisms previously demonstrated to be significantly and independently associated with risk in the study population including cholesteryl ester transfer protein (CETP, rs708272), p22phox (CYBA, rs4673), and thrombospondin-4 (THBS4, rs1866389). Risk association was demonstrated for the reference allele of the PAI-2 polymorphism (hazard ratio 0.41 per allele, 95% CI 0.20-0.84, p=0.014) along with continued significant risk associations for the p22phox and thrombospondin-4 polymorphisms. Additionally, further analysis revealed interaction of the LRP-1 and PAI-2 polymorphisms in generating differential risk that was illustrated using Kaplan-Meier survival analysis. We conclude from the study that fibrinolysis likely plays a role in establishing recurrent coronary risk in postinfarction patients with concurrently high levels of HDL-C and CRP as manifested by differential effects on risk by polymorphisms of several genes linked to key actions involved in the fibrinolytic process.     

Advances in endophenotyping schizophrenia

The search for the genetic architecture of schizophrenia has employed multiple, often converging strategies. One such strategy entails the use of tracing the heritability and neurobiology of endophenotypes. Endophenotypes are quantifiable traits not visible to the eye, which are thought to reflect an intermediate place on the path from genes to disorder. Endophenotype abnormalities in domains such as neurophysiology or neurocognition occur in schizophrenia patients as well as their clinically “unaffected” relatives, and reflect polymorphisms in the DNA of schizophrenia spectrum subjects which create vulnerability to developing schizophrenia. By identifying the single nucleotide polymorphisms (SNPs) associated with endophenotypes in schizophrenia, psychiatric neuroscientists can select new strong inference based molecular targets for the treatment of schizophrenia.     

Allelic and haplotype frequencies of the p53 polymorphisms in brain tumor patients

The polymorphisms of the tumor suppressor gene p53 in exon 4 (p53 BstUI) and in intron 6 (p53 MspI) have been suggested to be associated with the genetically determined susceptibility in diverse types of human cancer. In our hospital-based case-control study, we examined the allele and genotype incidence of these polymorphisms as well as their haplotype combinations in 60 brain tumor patients (27 males and 33 females) and 183 controls without malignancies. The genotype characteristics were determined by the PCR-based RFLP method using DNA extracted from peripheral blood. In this study we show that the p53 BstUI and the p53 MspI polymorphisms are not associated with increased risk of brain tumors. Thus, we conclude that the p53 BstUI and the p53 MspI polymorphic sites within the tumor suppressor gene p53 do not represent genetic determinants of susceptibility to brain tumors.     

Protein C promoter polymorphisms associate with sepsis in children with systemic meningococcemia

Meningococcal disease may present as sepsis, meningitis or a combination of both. Protein C (PC) is an important regulator of thrombin activity. Two polymorphisms in the promoter region of PC (C-1654T, A-1641G) have been shown to affect PC levels. In patients with meningococcal sepsis, low PC levels have been correlated with increased severity and poor outcome. We established a multicenter case-control study to determine whether PC promoter polymorphisms are associated with occurrence and outcome of meningococcal disease and sepsis. 288 previously healthy children with meningococcal infection from 97 pediatric hospitals in Germany, Switzerland, Italy, and Austria and 309 healthy controls were included in the study. A strong age-dependant effect was found. Patients younger than 1 year carried significantly more often the CG-CG genotype than healthy controls (28.6% vs. 17.8%, P = 0.04). Carriers of the CG allele showed a 3.43-fold increased odds ratio (OR) to develop sepsis (95% CI: 1.05-11.20; 85.7% vs. 63.6%, P = 0.036). The TA-TA genotype conferred a protective role for the development of sepsis (P = 0.017) with a Haldane OR of 0.09 (95% CI: 0.01-0.94). Systolic blood pressure values were significantly decreased in patients carrying the CG-CG genotype (70 vs. 86 mmHg, P = 0.005), and the need for adrenergic support significantly higher (70% vs. 26%, P = 0.018), resulting in an OR of 6.61 (95% CI: 1.28-34.14). These findings show that in young children PC promoter genotype is associated with susceptibility for meningococcal disease, the development of meningococcal sepsis, lower blood pressure, and need for adrenergic support.     

Clinical relevance of TLR2, TLR4, CD14 and FcgammaRIIA gene polymorphisms in Streptococcus pneumoniae infection

Streptococcus pneumoniae is the most common cause of community-acquired pneumonia and a major cause of morbidity and mortality throughout the world. It has been a major research priority to identify gene polymorphisms responsible for/associated with susceptibility and severity of S. pneumoniae infection to gain a better understanding of host genetic variants and their influence and clinical relevance to pneumococcal infections. In the present study, polymorphisms in several candidate genes, including TLR2-Arg/Gln753, TLR4-Asp/Gly299, TLR4-Thr/Ile399, CD14-159C/T and FcgammaRIIA-R/H131, were examined in 85 children with pneumococcal sepsis as an invasive pneumococcal disease and 409 healthy blood donors as controls. The prevalence of the TLR4-299/399 polymorphisms was significantly lower in the patient population than in controls (4 vs 11%; P<0.05; odds ratio (OR) 0.3; 95% confidence interval (CI) 0.1-1), while the prevalence of the CD14-159CC and FcgammaRIIA-R/R131 genotypes was significantly higher (35 vs 25%; P<0.05; OR 1.7; 95% CI 1-2.8 and 39 vs 21%; P<0.001; OR 2.5; 95% CI 1.4-4, respectively). Further, only 35% of patients carried either low-risk genotypes or protective genotypes in contrast to 61% of controls (P<0.0001; OR 2.8; 95% CI 1.7-4.6). We conclude that genetic variability in the TLR4, CD14 and FcgammaRIIA genes is associated with an increased risk of developing invasive disease in patients who are infected with S. pneumoniae.     

Direct amplification of length polymorphisms (DALP), or how to get and characterize new genetic markers in many species.

Direct amplification of length polymorphisms (DALP) uses an arbitrarily primed PCR (AP-PCR) to produce genomic fingerprints and to enable sequencing of DNA polymorphisms in virtually any species. Oligonucleotide pairs were designed to each produce a specific multi-banded pattern and all the fragments thus generated can be directly sequenced with the same two universal M13 sequencing primers. This strategy combines the advantages of a high resolution fingerprint technique and the possibility of characterizing the polymorphisms. The use of family members as templates in the multi-locus detection step allows a direct test of allele transmission, as well as early mapping of the markers or selection of loci associated with some traits or diseases. We used this method to detect micro-deletions/insertions and microsatellite DNA loci useful in population genetics studies, but it could be applied in many other fields of biology, such as genome mapping for definition of polymorphic sequence tagged sites, directly localized on a genetic map.     

Gene Polymorphisms and Pharmacogenetics in Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a systemic, chronic and inflammatory disease of unknown etiology with genetic predisposition. The advent of new biological agents, as well as the more traditional disease-modifying antirheumatic drugs, has resulted in highly efficient therapies for reducing the symptoms and signs of RA; however, not all patients show the same level of response in disease progression to these therapies. These variations suggest that RA patients may have different genetic regulatory mechanisms. The extensive polymorphisms revealed in non-coding gene-regulatory regions in the immune system, as well as genetic variations in drug-metabolizing enzymes, suggest that this type of variation is of functional and evolutionary importance and may provide clues for developing new therapeutic strategies. Pharmacogenetics is a rapidly advancing area of research that holds the promise that therapies will soon be tailored to an individual patient’s genetic profile.