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.

Role of tumor necrosis factor-α in the human systemic endotoxin-induced transcriptome.

TNFα has been implicated in the pathogenesis of various inflammatory diseases. Different strategies to inhibit TNFα in patients with sepsis and chronic inflammatory conditions have shown contrasting outcomes. Although TNFα inhibitors are widely used in clinical practice, the impact of TNFα antagonism on white blood cell gene expression profiles during acute inflammation in humans in vivo has not been assessed. We here leveraged the established model of human endotoxemia to examine the effect of the TNFα antagonist, etanercept, on the genome-wide transcriptional responses in circulating leukocytes induced by intravenous LPS administration in male subjects. Etanercept pre-treatment resulted in a markedly dampened transcriptional response to LPS. Gene co-expression network analysis revealed this LPS-induced transcriptome can be categorized as TNFα responsive and non-responsive modules. Highly significant TNFα responsive modules include NF-kB signaling, antiviral responses and T-cell mediated responses. Within these TNFα responsive modules we delineate fundamental genes involved in epigenetic modifications, transcriptional initiation and elongation. Thus, we provide comprehensive information about molecular pathways that might be targeted by therapeutic interventions that seek to inhibit TNFα activity during human inflammatory diseases.

In vivo biodistribution and pharmacokinetics of 18F-labelled Spiegelmers: a new class of oligonucleotidic radiopharmaceuticals.

PURPOSE: Single-stranded mirror-image oligonucleotides (Spiegelmers) are highly resistant to nuclease degradation and are capable of tightly and specifically binding to protein targets. Here we explored the potential of Spiegelmers as in vivo imaging probes for positron emission tomography (PET). METHODS: We investigated the biodistribution and pharmacokinetics of [18F]-L-DNA and [18F]-L-RNA Spiegelmers by dynamic quantitative whole-body PET imaging after intravenous administration in non-human primates. Their metabolic profile was explored in primates and rats, and ex vivo autoradiography of [(125)I]-L-RNA was performed in rat kidneys, the major organ for Spiegelmer uptake. RESULTS: Both [18F]-L-DNA and [18F]-L-RNA Spiegelmers were metabolically stable in plasma during 2 h after injection. No evidence of non-specific binding was found with either type of Spiegelmer in any tissue. CONCLUSION: The biodistribution and metabolic profiles of [18F]-L-DNA and [18F]-L-RNA Spiegelmers highlight their potential as radiotracers for in vivo imaging applications.

Inhibition of ghrelin action in vitro and in vivo by an RNA-Spiegelmer.

Employing in vitro selection techniques, we have generated biostable RNA-based compounds, so-called Spiegelmers, that specifically bind n-octanoyl ghrelin, the recently discovered endogenous ligand for the type 1a growth hormone secretagogue (GHS) receptor. Ghrelin is a potent stimulant of growth hormone release, food intake, and adiposity. We demonstrate that our lead compound, L-NOX-B11, binds ghrelin with low-nanomolar affinity and inhibits ghrelin-mediated GHS-receptor activation in cell culture with an IC(50) of 5 nM. l-NOX-B11 is highly specific for the bioactive, n-octanoylated form of ghrelin. Like the GHS receptor, it does not recognize the inactive unmodified peptide and requires only the N-terminal five amino acids for the interaction. The i.v. administration of polyethylene glycol modified l-NOX-B11 efficiently suppresses ghrelin-induced growth hormone release in rats. These results demonstrate that the neutralization of circulating bioactive ghrelin leads to inhibition of ghrelin's secretory effects in the CNS.

In vivo properties of an anti-GnRH Spiegelmer: an example of an oligonucleotide-based therapeutic substance class.

Spiegelmers are high-affinity l-enantiomeric oligonucleotide ligands that display high resistance to enzymatic degradation compared with d-oligonucleotides. The target binding properties of Spiegelmers can be designed by an in vitro-selection process starting from a random pool of oligonucleotides. Applying this method, a Spiegelmer with high affinity (K(D) = 20 nM) for the peptide hormone, gonadotropin-releasing hormone (GnRH) was isolated. The Spiegelmer acts as an antagonist to GnRH in Chinese hamster ovary cells stably expressing the human GnRH receptor, and its activity is unchanged by linking to 40-kDa polyethylene glycol. In a castrated rat model the Spiegelmer further demonstrated strong GnRH antagonist activity, which is more pronounced and persists longer with the polyethylene glycol-linked derivative. Furthermore, in rabbits the anti-GnRH Spiegelmer was shown to have a very low, possibly negligible immunogenic potential. These studies suggest that Spiegelmers could be of substantial interest in the development of new pharmaceutical approaches against GnRH and other targets.