Andriy Ruryk

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.

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.

Addressable bipartite molecular hook (ABMH): immobilized hairpin probes with sensitivity below 50 fM.

Sensitivity and specificity of nucleic acid binding probes immobilized on solid supports are essential features of microarrays. Whereas conventional biochips apply nonquenched linear probes (cDNA, oligonucleotides), hairpin structures containing a fluorophore-quencher system comprise important prerequisites required for ideal transcriptional probes. We describe here the generation of addressable bipartite molecular hook (ABMH) probes and the characterization of their performance analyzing biological and clinical samples, also in comparison to linear oligonucleotide arrays. ABMH can be immobilized subsequent to reaction with the target sequence or the reaction carried out directly with the immobilized probe; target sequences are recognized with excellent sensitivity, specificity, and a detection limit below 50 fM. Due to excellent sensitivity and specificity, ABMH represent ideal candidates for the nonamplified microarray-based detection of low abundance nucleic acids, e.g., required in diagnostic assays.