Early and accurate diagnosis of stroke improves the probability of positive outcome. The objective of this study was to identify a pattern of gene expression in peripheral blood that could potentially be optimised to expedite the diagnosis of acute ischaemic stroke (AIS). A discovery cohort was recruited consisting of 39 AIS patients and 24 neurologically asymptomatic controls. Peripheral blood was sampled at emergency department admission, and genome-wide expression profiling was performed via microarray. A machine-learning technique known as genetic algorithm k-nearest neighbours (GA/kNN) was then used to identify a pattern of gene expression that could optimally discriminate between groups. This pattern of expression was then assessed via qRT-PCR in an independent validation cohort, where it was evaluated for its ability to discriminate between an additional 39 AIS patients and 30 neurologically asymptomatic controls, as well as 20 acute stroke mimics. GA/kNN identified 10 genes (ANTXR2, STK3, PDK4, CD163, MAL, GRAP, ID3, CTSZ, KIF1B and PLXDC2) whose coordinate pattern of expression was able to identify 98.4% of discovery cohort subjects correctly (97.4% sensitive, 100% specific). In the validation cohort, the expression levels of the same 10 genes were able to identify 95.6% of subjects correctly when comparing AIS patients to asymptomatic controls (92.3% sensitive, 100% specific), and 94.9% of subjects correctly when comparing AIS patients with stroke mimics (97.4% sensitive, 90.0% specific). The transcriptional pattern identified in this study shows strong diagnostic potential, and warrants further evaluation to determine its true clinical efficacy.
These findings suggest that assessment of peripheral blood cfDNA levels may be useful for the identification of ischaemic stroke in the acute care setting, and provide associative evidence that cfDNA is a potential activator of the peripheral innate immune system in response to cerebral ischaemia.
Our group recently identified 16 genes whose peripheral blood expression levels are differentially regulated in acute ischemic stroke. The purpose of this study was to determine whether the early expression levels of any of these 16 genes are predictive for post-stroke blood brain barrier (BBB) disruption. Transcriptional expression levels of candidate genes were measured in peripheral blood sampled from ischemic stroke patients at emergency department admission, and BBB permeability was assessed at 24 hour follow up via perfusion-weighted imaging. Early heightened expression levels of AKAP7, a gene encoding a protein kinase A-binding scaffolding molecule, were significantly associated with BBB disruption 24 hours post-hospital admission. We then determined that AKAP7 is predominantly expressed by lymphocytes in peripheral blood, and strongly co-expressed with ITGA3, a gene encoding the adhesion molecule integrin alpha 3. Subsequent in vitro experiments revealed that heightened expression of AKAP7 and ITGA3 in primary human lymphocytes is associated with a highly adherent phenotype. Collectively, our results suggest that AKAP7 expression levels may have clinical utility as a prognostic biomarker for post-stroke BBB complications, and are likely elevated early in patients who later develop post-stroke BBB disruption due to the presence of an invasive lymphocyte population in the peripheral blood.
CD163 is a scavenger receptor expressed on innate immune cell populations which can be shed from the plasma membrane via the metalloprotease ADAM17 to generate a soluble peptide with lympho-inhibitory properties. The purpose of this study was to investigate CD163 as a possible effector of stroke-induced adaptive immune system suppression. Liquid biopsies were collected from ischemic stroke patients (n = 39), neurologically asymptomatic controls (n = 20), and stroke mimics (n = 20) within 24 hours of symptom onset. Peripheral blood ADAM17 activity and soluble CD163 levels were elevated in stroke patients relative to non-stroke control groups, and negatively associated with post-stroke lymphocyte counts. Subsequent in vitro experiments suggested that this stroke-induced elevation in circulating soluble CD163 likely originates from activated monocytic cells, as serum from stroke patients stimulated ADAM17-dependant CD163 shedding from healthy donor-derived monocytes. Additional in vitro experiments demonstrated that stroke-induced elevations in circulating soluble CD163 can elicit direct suppressive effects on the adaptive immune system, as serum from stroke patients inhibited the proliferation of healthy donor-derived lymphocytes, an effect which was attenuated following serum CD163 depletion. Collectively, these observations provide novel evidence that the innate immune system employs protective mechanisms aimed at mitigating the risk of post-stroke autoimmune complications driven by adaptive immune system overactivation, and that CD163 is key mediator of this phenomenon.
CD163 is a scavenger receptor expressed on innate immune cell populations which can be shed from the plasma membrane via the metalloprotease ADAM17 to generate a soluble peptide with lympho-inhibitory properties. The purpose of this study was to investigate CD163 as a possible effector of stroke-induced adaptive immune system suppression. Liquid biopsies were collected from ischemic stroke patients (n=39), neurologically asymptomatic controls (n=20), and stroke mimics (n=20) within 24 hours of symptom onset. Peripheral blood ADAM17 activity and soluble CD163 levels were elevated in stroke patients relative to non-stroke control groups, and negatively associated with post-stroke lymphocyte counts. Subsequent in vitro experiments suggested that this strokeinduced elevation in circulating soluble CD163 likely originates from activated monocytic cells, as serum from stroke patients stimulated ADAM17-dependant CD163 shedding from healthy donor-derived monocytes.Additional in vitro experiments demonstrated that stroke-induced elevations in circulating soluble CD163 can elicit direct suppressive effects on the adaptive immune system, as serum from stroke patients inhibited the proliferation of healthy donor-derived lymphocytes, an effect which was attenuated following serum CD163 depletion. Collectively, these observations provide novel evidence that the innate immune system employs protective mechanisms aimed at mitigating the risk of post-stroke autoimmune complications driven by adaptive immune system overactivation, and that CD163 is key mediator of this phenomenon. PREPRINT
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