Baillie JK, Walsh TS, Campbell L, Shankar-Hari M, Singer M on behalf of the A2B investigators

Numerous immune signaling mechanisms have been hypothesised to cause delirium[refs]. Alpha2 adrenoreceptor agonism modulates some of these immune signals, as detected in vitro by transcriptomics of quiescent[ref] and activated[ref] cells, cytokine measurements[ref] and assays of cellular function[ref]. in vivo data from humans under immune activation are not available. If, as suggested by the background evidence supporting the A2B trial, alpha2-adrenoreceptor agonists (a2aa) modulate the immune processes that lead to delirium, then the A2B trial constitutes a unique opportunity to infer a causative role for specific mediators in the pathogenesis of delirium.

Modifiable immune signals in peripheral blood cause delirium.

Detect inflammatory signals that are (a) modified by alpha agonism and (b) associated with reduction in delirium or (c) associated with reduction in duration of invasive ventilation n ctrl = 579 dex = 579 clon = 579

We will systematically review published studies of (a) delirium and (b) immune effects of a2aa on gene expression and protein production and release in humans and mice. We will perform a short series of in vitro experiments to provide additional gene expression data from dex and clonidine-treated immune cells from circulating populations from 3 volunteers (neutrophils, monocytes, T-lymphocytes, B-lymphocytes) under quiescent and stimulated (LPS) conditions at therapeutically-relevant doses. In this phase gene expression will be assayed using CAGE to provide the maximum regulatory signaling information. Signals will be systematically collapsed onto human gene names using publicly available annotation and orthology data, as in our previous work1. We will use a circular crossvalidation algorithm to perform a data-driven evaluation and collation of existing data. Associated cytokines will be systematically identified using annotated pathway data (KEGG, Reactome, Wikipathways). We anticipate that this will yeild a shortlist of 500-1000 genes and cytokines. From these we will select the subset of targets that have also been previously associated with delirium. We will obtain [samples] from all patients in the A2B study in participating units. Samples will be assayed by [assays] in the subgroup of patients with both (a) successful adherence to protocol and (b) evidence of delirium and (c) evidence of systemic inflammation. In order to detect the biological effect of treatment, samples will be obtained 48-72h after beginning treatment (see trial protocol). For each gene or cytokine in the shortlist of hypothesised a2aa-responsive causative factors in delirium, we will quantify evidence for causality by combining (a) statistical evidence for a difference between treatment and control groups and (b) statistical evidence for an association with outcome. After correction for multiple comparisons we will identify causative factors in delirium.

1. decompose transcriptome/cytokines to unique profiles 2. detect a2aa-responsive delirium-associated factors in training set 3. test significant associations in test set.

1. Schroder, K. et al. Conservation and divergence in Toll-like receptor 4-regulated gene expression in primary human versus mouse macrophages. Proc. Natl. Acad. Sci. U. S. A. 109, E944-953 (2012).