Eva Möller

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.

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.

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.

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.

Genexpressionsdiagnostik zur Erkennung früher postoperativer Risiken

Septic syndroms and the associated perturbation of immune homeo — stasis represent a major health problem in intensive care settings. Aim of our development is a diagnostic tool for monitoring the post-operative host response to assess the risk of early infectious complications. The diagnostic application of an established RNA-biomarker panel from whole blood for septic patients, measured on a qPCR platform, will be extended towards immunosuppressed post-transplant patients.