Sepsis Diagnostics in the Era of "Omics" Technologies.

Sepsis is a multifactorial clinical syndrome with an extremely dynamic clinical course and with high diverse clinical phenotype. Early diagnosis is crucial for the final clinical outcome. Previous studies have not identified a biomarker for the diagnosis of sepsis which would have sufficient sensitivity and specificity. Identification of the infectious agents or the use of molecular biology, next gene sequencing, has not brought significant benefit for the patient in terms of early diagnosis. Therefore, we are currently searching for biomarkers, through "omics" technologies with sufficient diagnostic specificity and sensitivity, able to predict the clinical course of the disease and the patient response to therapy. Current progress in the use of systems biology technologies brings us hope that by using big data from clinical trials such biomarkers will be found.

Effectiveness of Superior Hypogastric Plexus Block for Pain Control Compared to Epidural Anesthesia in Women Requiring Uterine Artery Embolization for the Treatment of Uterine Fibroids - A Retrospective Evaluation.

PURPOSE: To assess the effectiveness of pain management with superior hypogastric plexus block (SHPB) compared to epidural anesthesia (EDA) in women requiring uterine artery embolization (UAE). MATERIALS AND METHODS: In this retrospective, single-center, non-randomized trial we included 79 women with symptomatic uterine fibroids who were scheduled for percutaneous, transcatheter UAE. According to their informed decision, the women were assigned to two different approaches of pain management including either SHPB or EDA. The effectiveness outcome measure was patient reported pain using a numeric rating scale ranging from 1 to 10. The pain score was assessed at UAE, 2 hours thereafter, and at subsequent intervals of 6 hours up to 36 hours after intervention. RESULTS: Treatment groups did not differ significantly regarding age, pain score for regular menstrual cramps, uterine fibroid size, location, and symptoms of uterine fibroids. During UAE and up to 6 hours thereafter, women who received SHPB experienced stronger pain than those who received EDA (mean pain score during UAE: 3.3 vs. 1.5, p < 0.001; at 2 hours: 4.4 vs. 2.8, p = 0.012; at 6 hours: 4.4 vs. 2.6, p = 0.021). The maximum pain level was 5.8 ±â€Š2.9 with SHPB and 4.5 ±â€Š2.9 with EDA (p = 0.086). Women with a history of severe menorrhagia tended to experience worse pain than those without (regression coefficient 2.5 [95 % confidence interval -0.3 to 5.3], p = 0.076). CONCLUSION: Among women who underwent UAE, pain management including SHPB resulted in stronger pain during and after the procedure than pain treatment including EDA. KEY POINTS: · Pain control with superior hypogastric plexus block was worse than epidural anesthesia.. · Peak of pain was at 12 hours after uterine artery embolization.. · Maximum pain was independent from uterine fibroid size or location.. CITATION FORMAT: · Malouhi A, Aschenbach R, Erbe A et al. Effectiveness of Superior Hypogastric Plexus Block for Pain Control Compared to Epidural Anesthesia in Women Requiring Uterine Artery Embolization for the Treatment of Uterine Fibroids - A Retrospective Evaluation. Fortschr Röntgenstr 2021; 193: 289 - 297.

Truncated human cytidylate-phosphate-deoxyguanylate-binding protein for improved nucleic acid amplification technique-based detection of bacterial species in human samples.

A trunk of human cytidylate-phosphate-deoxyguanylate-binding protein/CXXC finger protein 1 (CFP1), immobilized onto an aminohexyl-Sepharose column, can be used as a preanalytical tool for the selective enrichment of bacterial DNA from mixed solutions with high amounts of human background DNA for nucleic acid amplification technique-based detection of pathogens. The transcriptional activator protein exhibits a high affinity for nonmethylated CpG dinucleotide motifs, which are differentially distributed in prokaryotic and higher eukaryotic genomes. The feasibility of the affinity chromatography (AC) step was tested with DNA from severely septic patients. AC using 16S rRNA gene primers substantially increased PCR sensitivity. Approximately 90% of eukaryotic DNA was removed, which significantly increased the signal-to-noise ratio. Threshold cycle values revealed that sensitivity was elevated at least 10-fold. The change in the ratio of bacterial DNA to human DNA increased from 26% to 74% the likelihood of culture-independent PCR-based identification of bacterial presence. Compared to the results seen with blood culture (which is the clinical gold standard for systemic infections, exhibiting 28% positives), the combination of AC and PCR achieves a significant increase in sensitivity and contributes to shortening the time to results for the initiation of guided antibiotic therapy.

Gene profiling in human blood leucocytes during recovery from septic shock.

OBJECTIVE: To assess blood leucocytes gene profiling during recovery phase of septic shock; to test the relation between encoding gene expression and protein level. STUDY DESIGN: Gene expression levels were studied at days 0, 1, 7 and 28 (D0, 1, 7 and 28) on a dedicated microarray of 340 genes involved in inflammatory processes. SETTINGS: 16-bed intensive care unit, Lariboisière University hospital. PATIENTS: Seventeen septic shock patients enrolled when at least one additional organ dysfunction occurred. MEASUREMENTS AND RESULTS: Changes over time were compared with D0 via the ratio Dx/D0. The time-related gene expression study showed significant changes in ten genes. Among them, S100A8 and S100A12 had a reduced expression over time compared with D0, whereas CD74's expression increased. The microarray results were validated by RT-qPCR for four genes. The S100A8 plasma levels decrease along recovery in parallel with the gene expression decrease. The CD74 gene expression evolution significantly correlated with HLA-DR monocyte expression. CONCLUSIONS: These results are the first description of variations in expression of key inflammatory genes in the course of the septic shock recovery period.

Immunotherapy of Sepsis: Blind Alley or Call for Personalized Assessment?

Sepsis is the most frequent cause of death in noncoronary intensive care units. In the past 10 years, progress has been made in the early identification of septic patients and their treatment. These improvements in support and therapy mean that mortality is gradually decreasing, however, the rate of death from sepsis remains unacceptably high. Immunotherapy is not currently part of the routine treatment of sepsis. Despite experimental successes, the administration of agents to block the effect of sepsis mediators failed to show evidence for improved outcome in a multitude of clinical trials. The following survey summarizes the current knowledge and results of clinical trials on the immunotherapy of sepsis and describes the limitations of our knowledge of the pathogenesis of sepsis. Administration of immunomodulatory drugs should be linked to the current immune status assessed by both clinical and molecular patterns. Thus, a careful daily review of the patient's immune status needs to be introduced into routine clinical practice giving the opportunity for effective and tailored use of immunomodulatory therapy.

Transcription in response to physical stress--clues to the molecular mechanisms of exercise-induced asthma.

To clarify stress-induced immunological reactions and molecular events during exercise and the potential relevance to exercise-induced bronchoconstriction, transcriptional responses to standardized physical stress were determined. Six healthy, young volunteers underwent an endurance exercise of 90% of their individual anaerobic threshold for 90 min. Time-dependent alterations in the expression pattern of leukocytes from healthy, trained subjects were analyzed by DNA microarrays before and 2 h and 6 h after exercise. Starting out from a large collection of cDNA library clones comprising more than 70,000 human expressed sequence tags, we selected, designed, and immobilized oligonucleotide probes (60-70mers) for transcripts of 5000 stress- and inflammation-relevant genes. Exercise-induced stress provoked changes in the expression of 433 gene activities 2 h and/or 6 h after exercise, which could be grouped into six clusters. The most prominent feature was an enhanced transcription of two genes, coding for 5-lipoxygenase (ALOX5) and ALOX5-activating protein. Moreover, enhanced levels of leukotriene B4 (LTB4) and LTC4 (P<0.05) were detected in plasma after exercise. Our data demonstrate that exercise alters the activities of a distinct number of genes. In particular, they possibly provide novel insights into the molecular mechanisms of exercise-induced bronchoconstriction and suggest that enhanced transcription of ALOX5 and its activating protein together with a present predisposition of the subject critically contribute to exercise-induced asthma.

A Transcriptomic Biomarker to Quantify Systemic Inflammation in Sepsis - A Prospective Multicenter Phase II Diagnostic Study.

Development of a dysregulated immune response discriminates sepsis from uncomplicated infection. Currently used biomarkers fail to describe simultaneously occurring pro- and anti-inflammatory responses potentially amenable to therapy. Marker candidates were screened by microarray and, after transfer to a platform allowing point-of-care testing, validated in a confirmation set of 246 medical and surgical patients. We identified up-regulated pathways reflecting innate effector mechanisms, while down-regulated pathways related to adaptive lymphocyte functions. A panel of markers composed of three up- (Toll-like receptor 5; Protectin; Clusterin) and 4 down-regulated transcripts (Fibrinogen-like 2; Interleukin-7 receptor; Major histocompatibility complex class II, DP alpha1; Carboxypeptidase, vitellogenic-like) described the magnitude of immune alterations. The created gene expression score was significantly greater in patients with definite as well as with possible/probable infection than with no infection (median (Q25/Q75): 80 (60/101)) and 81 (58/97 vs. 49 (27/66), AUC-ROC=0.812 (95%-CI 0.755-0.869), p<0.0001). Down-regulated lymphocyte markers were associated with prognosis with good sensitivity but limited specificity. Quantifying systemic inflammation by assessment of both pro- and anti-inflammatory innate and adaptive immune responses provides a novel option to identify patients-at-risk and may facilitate immune interventions in sepsis.

Genome-wide association study of survival from sepsis due to pneumonia: an observational cohort study.

BACKGROUND: Sepsis continues to be a major cause of death, disability, and health-care expenditure worldwide. Despite evidence suggesting that host genetics can influence sepsis outcomes, no specific loci have yet been convincingly replicated. The aim of this study was to identify genetic variants that influence sepsis survival. METHODS: We did a genome-wide association study in three independent cohorts of white adult patients admitted to intensive care units with sepsis, severe sepsis, or septic shock (as defined by the International Consensus Criteria) due to pneumonia or intra-abdominal infection (cohorts 1-3, n=2534 patients). The primary outcome was 28 day survival. Results for the cohort of patients with sepsis due to pneumonia were combined in a meta-analysis of 1553 patients from all three cohorts, of whom 359 died within 28 days of admission to the intensive-care unit. The most significantly associated single nucleotide polymorphisms (SNPs) were genotyped in a further 538 white patients with sepsis due to pneumonia (cohort 4), of whom 106 died. FINDINGS: In the genome-wide meta-analysis of three independent pneumonia cohorts (cohorts 1-3), common variants in the FER gene were strongly associated with survival (p=9·7 × 10(-8)). Further genotyping of the top associated SNP (rs4957796) in the additional cohort (cohort 4) resulted in a combined p value of 5·6 × 10(-8) (odds ratio 0·56, 95% CI 0·45-0·69). In a time-to-event analysis, each allele reduced the mortality over 28 days by 44% (hazard ratio for death 0·56, 95% CI 0·45-0·69; likelihood ratio test p=3·4 × 10(-9), after adjustment for age and stratification by cohort). Mortality was 9·5% in patients carrying the CC genotype, 15·2% in those carrying the TC genotype, and 25·3% in those carrying the TT genotype. No significant genetic associations were identified when patients with sepsis due to pneumonia and intra-abdominal infection were combined. INTERPRETATION: We have identified common variants in the FER gene that associate with a reduced risk of death from sepsis due to pneumonia. The FER gene and associated molecular pathways are potential novel targets for therapy or prevention and candidates for the development of biomarkers for risk stratification. FUNDING: European Commission and the Wellcome Trust.

Direct influence of mild hypothermia on cytokine expression and release in cultures of human peripheral blood mononuclear cells.

Hypothermia is associated with elevated frequency of infectious complications. Dysfunction of the immune response caused by hypothermia has been demonstrated in both clinical and animal studies, but it still remains unclear to what extent immunocompetent cells are directly influenced by hypothermia. To estimate the direct influence of mild hypothermia on cytokine expression and release by human peripheral blood mononuclear cells (PBMC), primary cultures of PBMC were incubated at 34 degrees C or 32 degrees C activated by lipopolysaccharide (LPS), phytohemagglutinin (PHA), or tumor necrosis factor-alpha (TNF-alpha). The cytokine gene expression was evaluated by RT-PCR. Release of interleukin-2 (IL-2), IL-6, IL-10, and TNF-alpha was measured by ELISA. Mild hyperthermia significantly impaired IL-2 gene expression in PHA-stimulated cultures of PBMC and decreased IL-2 release in all variants of cultures. Secretion of IL-6, IL-10, and TNF-alpha was decreased in hypothermic cultures of PBMC stimulated with the T lymphocyte activator PHA. Slight suppression of IL-10 secretion was observed also in TNF-alpha-stimulated hypothermic cultures of PBMC. TNF-alpha release increased slightly in mild hypothermia control cultures. Our data demonstrate that the direct influence of hypothermia on cytokine expression and release from PBMC is not uniform. Reduction of IL-2 production might play a crucial role in the impairment of immune response in hypothermia.

Platelet and leukocyte activation correlate with the severity of septic organ dysfunction.

This study was conducted to investigate the extent of platelet-leukocyte adhesion and platelet, monocyte, and neutrophil activation in septic patients and to analyze whether these variables correlate with the severity of sepsis. Forty-seven patients consecutively admitted to the operative ICU of a University Medical Centre and 12 control patients prior to elective surgery were included in this prospective cohort study. Patients were evaluated daily for sepsis criteria and sepsis-associated organ failure assessment (SOFA) score was used to describe the extent of sepsis-associated organ failure. Indicators for cell activation (CD62P on platelets and CD11b on neutrophils and monocytes) and binding of platelets to neutrophils and monocytes were analyzed by flow cytometry. CD62P was increased on platelets from patients with sepsis compared with patients who did not have sepsis. Patients with sepsis also had higher CD11b expression on neutrophils and monocytes. Statistical analyses revealed a positive correlation between platelet CD62P expression and severity of sepsis, as well as a positive correlation between the SOFA score and CD11b on monocytes. No correlation was found between the SOFA score and CD11b on neutrophils. Higher values for platelet-neutrophil adhesion were observed in patients with uncomplicated sepsis compared either with controls or to patients with septic shock. An inverse relation between severity of sepsis and extent of platelet-neutrophil adhesion was also obvious from correlation analysis. The results indicate that flow cytometry can be used to measure these parameters of cell activation in sepsis and that activation of platelets and monocytes as well as adhesion of platelets to neutrophils does play a role in the development of organ dysfunction.

Expression profiling: toward an application in sepsis diagnostics.

Sepsis is a common and serious health problem whereby improvements in diagnosis are crucial in increasing survival rates. To test whether profiling transcription is applicable to sepsis diagnosis, we analyzed whole blood using a microarray containing probes for 340 genes relevant to inflammation. The patient's gene expression pattern was highly homogenous, resulting in 69% of differentially expressed genes. With a positive predictive value of 98%, a list of 50 differentially expressed genes was compiled, and randomly chosen transcripts were confirmed by PCR. Here, we present the first evidence that microarrays can identify typical gene expression profiles in the blood of patients with severe sepsis. Regardless of the heterogeneity of the patients, we observed a striking correlation between the conventional diagnostic classification and our approach. The unity of responses suggests that the principle of this multiparameter approach can be adapted to early stage sepsis diagnosis.

Expression and regulation of CCL18 in synovial fluid neutrophils of patients with rheumatoid arthritis.

Rheumatoid arthritis (RA) is characterized by the recruitment of leukocytes and the accumulation of inflammatory mediators within the synovial compartment. Release of the chemokine CCL18 has been widely attributed to antigen-presenting cells, including macrophages and dendritic cells. This study investigates the production of CCL18 in polymorphonuclear neutrophils (PMN), the predominant cell type recruited into synovial fluid (SF). Microarray analysis, semiquantitative and quantitative reverse transcriptase polymerase chain reaction identified SF PMN from patients with RA as a novel source for CCL18 in diseased joints. Highly upregulated expression of other chemokine genes was observed for CCL3, CXCL8 and CXCL10, whereas CCL21 was downregulated. The chemokine receptor genes were differentially expressed, with upregulation of CXCR4, CCRL2 and CCR5 and downregulation of CXCR1 and CXCR2. In cell culture experiments, expression of CCL18 mRNA in blood PMN was induced by tumor necrosis factor alpha, whereas synthesis of CCL18 protein required additional stimulation with a combination of IL-10 and vitamin D3. In comparison, recruited SF PMN from patients with RA were sensitized for CCL18 production, because IL-10 alone was sufficient to induce CCL18 release. These results suggest a release of the T cell-attracting CCL18 by PMN when recruited to diseased joints. However, its production is tightly regulated at the levels of mRNA expression and protein synthesis.

Platelet-derived microvesicles induce differential gene expression in monocytic cells: a DNA microarray study.

Platelet-derived microvesicles (PMV) that are shed from the plasma membrane of activated platelets, expose various platelet-type antigens on their surface and are able to adhere to other blood cells and endothelial cells. There are several clinical conditions with markedly increased numbers of PMV, e.g. acute coronary syndrome, thrombotic microangiopathy and sepsis. To prove whether PMV may contribute to an inflammatory response we used DNA microarray technology to study the effect of PMV on gene expression in the prototypic monocytic cell line MonoMac 6 (MM6). PMV were generated by activating human platelets in plasma with collagen and subsequent removal of platelets and plasma by repeated centrifugation. MM6 were incubated for 2 h with PMV in a ratio corresponding to 75 platelets/cell, or saline as control. After RNA isolation, reverse transcription and fluorescence labelling, cDNA was hybridized on a medium density microarray comprising 5308 probes addressing 4868 transcripts of 4730 human genes relevant to inflammation, immune response and related processes. The formation of PMV-MM6 conjugates was associated with significant variations in gene expression, i.e. 93 genes were found to be differentially expressed (P < 0.001; q < 0.087). Among them, 47 genes with annotated transcripts and proteins were identified. Using Ingenuity Pathway Analysis, 37 of the differentially expressed genes were identified as parts of networks associated with functional pathways including cell-to-cell signalling, cellular growth and proliferation, regulation of gene expression and lipid metabolism. For sphingosine kinase-1 the increased expression could be confirmed exemplarily not only by RT-PCR but also on the enzyme activity level. The data indicate that PMV signal differential expression of inflammation-relevant genes in monocytic cells and may represent a novel link between hemostasis and inflammation.

Plasma platelet-activating factor acetylhydrolase activity in critically ill patients.

OBJECTIVE: Platelet-activating factor (PAF) is a potent proinflammatory mediator in systemic inflammation and sepsis and is inactivated by the enzyme PAF-acetylhydrolase (PAF-AH). Recently, a large phase III clinical trial using recombinant PAF-AH to treat patients with severe sepsis was performed but failed to reduce 28-day mortality rate. To get more information on the activity of PAF-AH in sepsis, we repeatedly measured its activity in plasma in critically ill patients compared with healthy controls. DESIGN: Retrospective cohort study. SETTING: Intensive care unit. PATIENTS: Two hundred thirty-one patients who were admitted to an operative intensive care unit within 1 yr were enrolled and evaluated daily for American College of Chest Physicians/Society of Critical Care Medicine criteria. PAF-AH activity was measured as the release of [H]-acetate from [H]-acetyl-PAF. INTERVENTIONS: Analysis of plasma samples. MEASUREMENTS AND MAIN RESULTS: At the day of admission, PAF-AH activity of patients was below controls but markedly increased over time. Higher activities were seen in patients with severe sepsis or septic shock compared with those without organ failure. With respect to the clinical outcome, lower values were found in nonsurvivors only as long as they had not developed organ failure. In severe sepsis/septic shock, values of nonsurvivors exceeded those of survivors. PAF-AH activity was positively correlated with plasma levels of inflammatory mediators such as neopterine and tumor necrosis factor-alpha but not with acute phase reactants such as C-reactive protein, interleukin-6, or PCT. In addition, parenteral nutrition with lipid emulsions was seemingly associated with low PAF-AH activity compared with enteral nutrition. CONCLUSION: The data indicate severity- and time-dependent changes in PAF-AH activity and may help to explain the failure of recombinant PAF-AH treatment strategies that were not based on activity measurements.