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Background The molecular mechanisms of inflammatory pathophysiology after intracerebral hemorrhage (ICH) are not well established. We report mRNA-seq and miRNA-seq in the peripheral blood of ICH patients with three serial samples during the first week after the stroke. Methods Twenty-seven ICH patients were enrolled via 24/7 screening and peripheral blood sampled at < 24 h (baseline), 72 h (+/-12 h), and 7 days (+/- 2 days) from last known normal. mRNA-seq and miRNA-seq were assessed for differential expression (DE) between the time point comparisons. Pathways identified via enrichment analysis (STRING, Reactome, Ingenuity Pathway Analysis) were tested via paired t-test and principal component analysis (PCA). Correlations between miRNA/mRNA pairs were computed. Results For DE mRNA at 72 h vs. baseline, the main enriched pathways pertained to neuronal function and synaptic transmission; prominent neuron-related genes from these pathways are also implicated in platelet activation. For 7 days vs. baseline, neutrophil degranulation and ribosomal biogenesis were the most enriched pathways; PCA also suggested neutrophil degranulation was the pathway most significantly different at 7 days vs. baseline compared with 72 h vs. baseline ( p  = 0.02). miR-3613 and miR-3690 had decreased expression at 7 days and were the miRNA most correlated with DE genes from the neutrophil degranulation pathway. Conclusion Neutrophil degranulation was the prominent enriched pathway at 7 days after ICH and correlated with decreased miR-3613 and miR-3690. Further research is warranted of neutrophils in post-ICH inflammatory pathophysiology and functional validation studies of miR-3613 and miR-3690 as potential regulators of neutrophil degranulation after ICH.

Intracerebral hemorrhage (ICH) is a severe neurological disorder with no proven treatment. Inflammation after ICH contributes to clinical outcomes, but the relevant molecular mechanisms remain poorly understood. In studies of peripheral leukocyte counts and mRNA-sequencing (mRNA-seq), our group previously reported that monocytes and Interleukin-8 (IL-8) were important contributors to post-ICH inflammation. microRNA (miRNA) are powerful regulators of gene expression and promising therapeutic targets. We now report findings from an integrated analysis of miRNA-seq and mRNA-seq in peripheral blood mononuclear cells (PBMCs) from a swine ICH model. In 10 pigs, one PBMC sample was collected immediately prior to ICH induction and a second 6 h later; miRNA-seq and mRNA-seq were completed for each sample. An aggregate score calculation determined which miRNA regulated the differentially expressed mRNA. Networks of molecular interactions were generated for the combined miRNA/target mRNA. A total of 227 miRNA were identified, and 46 were differentially expressed after ICH (FDR < 0.05). The anti-inflammatory miR-181a was decreased post-ICH, and it was the most highly connected miRNA in the miRNA/mRNA bioinformatic network analysis. miR-181a has interconnected pathophysiology with IL-8 and monocytes; in prior studies, we found that IL-8 and monocytes contributed to post-ICH inflammation and ICH clinical outcome, respectively. miR-181a was a significant mediator of post-ICH inflammation and is promising for further study, including as a potential therapeutic target. This investigation also demonstrated feasible methodology for miRNA-seq/mRNA-seq analysis in swine that is innovative, and with unique challenges, compared with transcriptomics research in more established species.

Background The development of tools that could help emergency department clinicians recognize stroke during triage could reduce treatment delays and improve patient outcomes. Growing evidence suggests that stroke is associated with several changes in circulating cell counts. The aim of this study was to determine whether machine-learning can be used to identify stroke in the emergency department using data available from a routine complete blood count with differential. Methods Red blood cell, platelet, neutrophil, lymphocyte, monocyte, eosinophil, and basophil counts were assessed in admission blood samples collected from 160 stroke patients and 116 stroke mimics recruited from three geographically distinct clinical sites, and an ensemble artificial neural network model was developed and tested for its ability to discriminate between groups. Results Several modest but statistically significant differences were observed in cell counts between stroke patients and stroke mimics. The counts of no single cell population alone were adequate to discriminate between groups with high levels of accuracy; however, combined classification using the neural network model resulted in a dramatic and statistically significant improvement in diagnostic performance according to receiver-operating characteristic analysis. Furthermore, the neural network model displayed superior performance as a triage decision making tool compared to symptom-based tools such as the Cincinnati Prehospital Stroke Scale (CPSS) and the National Institutes of Health Stroke Scale (NIHSS) when assessed using decision curve analysis. Conclusions Our results suggest that algorithmic analysis of commonly collected hematology data using machine-learning could potentially be used to help emergency department clinicians make better-informed triage decisions in situations where advanced imaging techniques or neurological expertise are not immediately available, or even to electronically flag patients in which stroke should be considered as a diagnosis as part of an automated stroke alert system.

IntroductionCurrent guidelines recommend blood pressure (BP) lowering in patients after acute intracerebral haemorrhage (ICH) without guidance on initial choice of antihypertensive class. This study sought to determine if initial antihypertensive class differentially effects acute BP lowering in a large multiethnic ICH cohort.MethodsSubjects enrolled in the Ethnic/Racial Variations in ICH study between August 2010 and August 2017 with elevated admission BP and who received labetalol, nicardipine or hydralazine monotherapy as initial antihypertensive were analysed. Primary outcomes were systolic and diastolic BP changes from baseline to first BP measurement after initial antihypertensive treatment. Secondary outcomes included haematoma expansion (HE), hospital length of stay (LOS) and modified Rankin Score (mRS) up to 12 months after ICH. Exploratory outcomes assessed effects of race/ethnicity. Linear and logistic regression analyses, adjusted for relevant covariates, were performed to determine associations of antihypertensive class with outcomes.ResultsIn total, 1156 cases were used in analyses. Antihypertensive class was associated with diastolic BP change (p=0.003), but not systolic BP change (p=0.419). Initial dosing with nicardipine lowered acute diastolic BP than labetalol (least square mean difference (labetalol-nicardipine)=5.47 (2.37, 8.57), p<0.001). Initial antihypertensive class was also found to be associated with LOS (p=0.028), but not with HE (p=0.406), mortality (p=0.118), discharge disposition (p=0.083) or mRS score at discharge, 3, 6 and 12 months follow-up (p=0.262, 0.276, 0.152 and 0.36, respectively). Race/ethnicity variably affected multivariable models.ConclusionIn this large acute ICH cohort, initial antihypertensive class was associated with acute diastolic, but not systolic, BP-lowering suggesting differential effects of antihypertensive agents.Trial registration number NCT01202864.

Intracerebral hemorrhage (ICH) is a severe neurological disorder with no proven treatment. Our prior research identified a significant association with monocyte level and ICH mortality. To advance our understanding, we sought to identify gene expression after ICH using a swine model to test the hypothesis that ICH would induce peripheral blood mononuclear cell (PBMC) gene expression. In 10 pigs with ICH, two PBMC samples were drawn from each with the first immediately prior to ICH induction and the second six hours later. RNA-seq was performed with subsequent bioinformatics analysis using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Ingenuity® Pathway Analysis (IPA). There were 182 significantly upregulated and 153 significantly down-regulated differentially expressed genes (DEGs) after ICH. Consistent with findings in humans, significant GO and KEGG pathways were primarily related to inflammation and the immune response. Five genes, all upregulated post-ICH and known to be associated with monocyte activation, were repeatedly DEGs in the significant KEGG pathways: CD14, TLR4, CXCL8, IL-18, and CXCL2. In IPA, the majority of upregulated disease/function categories were related to inflammation and immune cell activation. TNF and LPS were the most significantly activated upstream regulators, and ERK was the most highly connected node in the top network. ICH induced changes in PBMC gene expression within 6 h of onset related to inflammation, the immune response, and, more specifically, monocyte activation. Further research is needed to determine if these changes affect outcomes and may represent new therapeutic targets.

Accurate stroke recognition during triage can streamline care and afford patients earlier access to life-saving interventions. However, the tools currently available to clinicians for prehospital and early in-hospital identification of stroke are limited. The peripheral immune system is intricately involved in stroke pathology and thus may be targetable for the development of immunodiagnostics. In this preliminary study, we sought to determine whether the circulating antibody pool is altered early in stroke, and whether such alterations could be leveraged for diagnosis. One hundred microliters of peripheral whole blood was sampled from 19 ischemic stroke patients, 17 hemorrhagic stroke patients, and 20 stroke mimics in the acute phase of care. A custom-fabricated high-density peptide array comprising 125,000 unique probes was used to assess the binding characteristics of blood-borne antibodies, and a random forest-based approach was used to select a parsimonious set of probes with an optimal ability to discriminate between groups. The coordinate antibody binding intensities of the top 17 probes identified in our analysis displayed an ability to differentiate the total pool of stroke patients from stroke mimics with 92% sensitivity and 90% specificity, as well as detect hemorrhage with 88% sensitivity and 87% specificity, as determined using a same-set cross-validation. These preliminary findings suggest that stroke-associated alterations in the circulating antibody pool may have clinical utility for diagnosis during triage, and that such a possibility warrants further investigation.

Objective ST2 is a member of the toll‐like receptor superfamily that can alter inflammatory signaling of helper T‐cells. We investigated whether soluble ST2 (sST2) could independently predict outcome and hemorrhagic transformation (HT) in the setting of stroke. Methods We measured sST2 in patients enrolled in the Specialized Program of Translational Research in Acute Stroke (SPOTRIAS) network biomarker study. 646 patients had plasma samples collected at the time of hospital admission and 210 patients had a second sample collected 48 h after stroke onset. Functional outcome was assessed using the modified Rankin Scale (mRS), with good and poor outcomes defined as mRS 0‐2 and 3‐6, respectively. HT was classified using ECASS criteria. The relationships between sST2, outcome, and HT were evaluated using multivariable logistic regression, Kaplan–Meier survival analysis and receiver operating characteristic curves. Results 646 patients were included in the analysis (mean age 69 years; 44% women), with a median NIHSS of 5 [IQR: 2–12]. The median sST2 level on hospital admission was 35.0 ng/mL [IQR: 25.7–49.8 ng/mL] and at 48 h it was 37.4 ng/mL [IQR 27.9–55.6 ng/mL]. sST2 was independently associated with poor outcome (OR: 2.77, 95% CI: 1.54–5.06; P = 0.003) and mortality (OR: 3.56, 95% CI: 1.58–8.38, P = 0.001) after multivariable adjustment. Plasma sST2 was also associated with hemorrhagic transformation after adjustment for traditional risk factors (OR: 5.58, 95% CI: 1.40–37.44, P = 0.039). Interpretation Soluble ST2 may serve as a prognostic biomarker for outcome and hemorrhagic transformation in patients with acute stroke. ST2 may link neuroinflammation and secondary injury after stroke.

Debate continues about the value of event adjudication in clinical trials and whether independent centralized assessments improve reliability and validity of study results in masked randomized trials compared with local, investigator-assessed end points. To assess the results of the adjudicated end point process in the Platelet-Oriented Inhibition in New TIA and Minor Ischemic Stroke (POINT) trial by comparing end points assessed by local site investigators with centrally adjudicated end points. This is an ad hoc secondary analysis of a randomized, double-blind clinical trial comparing safety and effectiveness of clopidogrel bisulphate plus aspirin vs placebo plus aspirin. Patients received either 600 mg of clopidogrel bisulphate on day 1, then 75 mg per day through day 90 plus 50 to 325 mg of aspirin per day, or the same range of dosages of placebo plus aspirin. Investigators reported all potential end points; independent masked adjudicators were randomly assigned to review using definitions specified in the study protocol. This was a multicenter study; 269 international sites in 10 countries enrolled from May 28, 2010, to December 19, 2017. The study enrolled 4881 patients 18 years or older with transient ischemic attack or minor acute ischemic stroke within 12 hours of symptom onset and followed for 90 days from randomization; last follow-up was completed in March 2018. Independent adjudicators external to the study and masked to study treatment assignment adjudicated 467 primary and secondary effectiveness outcomes and major and minor bleeding events, including the primary composite end point, which was the risk of a composite of major ischemic events at 90 days, defined as ischemic stroke, myocardial infarction, or death from an ischemic vascular event. The primary safety end point was major hemorrhage. All components of the primary and safety outcomes were adjudicated. In this secondary analysis of an international randomized clinical trial, a total of 269 sites worldwide randomized 4881 patients (median age, 65.0 years; interquartile range, 55-74 years); 55.0% were male. The primary results have been published previously. The hazard ratios for clopidogrel plus aspirin vs placebo plus aspirin for the primary composite end point were 0.75 (95% CI, 0.59-0.95) for adjudicator-assessed events and 0.76 (95% CI, 0.60-0.95) for investigator-assessed events. Agreement between adjudicator and investigator assessments was 90.7%. The hazard ratios for clopidogrel plus aspirin vs placebo plus aspirin for the primary safety end point were 2.32 (95% CI, 1.10-4.87) for adjudicator-assessed events and 2.58 (95% CI, 1.19-5.58) for investigator-assessed events, with an agreement rate of 77.5%. Independent end point adjudication did not substantially alter estimates of the primary treatment effectiveness in the POINT trial. ClinicalTrials.gov identifier: NCT00991029.

Background: Blood biomarkers for ischemic and hemorrhagic stroke diagnosis remain elusive. Recent investigations suggested that apolipoproteins (Apo), matrix metalloproteinases (MMP), and paraoxonase-1 may be associated with stroke. We hypothesized that Apo A-I, Apo C-I, Apo C-III, MMP-3, MMP-9, and paraoxonase-1 are differentially expressed in ischemic stroke, hemorrhagic stroke, and controls. Methods: In a single center prospective observational study, consecutive stroke cases were enrolled if blood samples were obtainable within 12 hours of symptom onset. Age (+/– five years), race, and sex matched controls were recruited. Multiplex assays were used to measure protein levels. The Wilcoxon Signed Ranks Test and Mann-Whitney U Test were used to compare biomarker values between ischemic stroke patients and controls, hemorrhagic stroke patients and controls, and ischemic and hemorrhagic stroke patients. The 95% confidence intervals (CI) for the difference of two medians were calculated. Gel filtration chromatography was used to characterize HDL fractions in a subset of five case-control pairs. Results: Fourteen ischemic stroke case-control pairs and 23 intracerebral hemorrhage (ICH) case-control pairs were enrolled. Median Apo A-I levels were lower in ischemic stroke versus controls (140mg/dL vs 175mg/dL, difference 35mg/dL, 95% CI –54 to –16) , and in ischemic stroke versus ICH (140mg/dL vs 180mg/dL, difference 40mg/dL, 95% CI -57 to -23). Median paraoxonase-1 was lower in ischemic stroke compared to both ICH and matched controls. Median Apo C-I was slightly lower in ischemic stroke compared to ICH cases. There were no differences between groups for MMP-3, MMP-9, and Apo C-III. A specific HDL fraction was identified for male ICH cases and female ICH controls. Conclusion: Apo A-I and paraoxonase-1 levels may be clinically useful for ischemic stroke diagnosis and for differentiating ischemic and hemorrhagic stroke. Further evaluation of HDL fractions in stroke patients and controls, and the associated protein composition, is warranted and could lead to identification of novel biomarkers

Like most complex phenotypes, exceptional longevity is thought to reflect a combined influence of environmental (e.g., lifestyle choices, where we live) and genetic factors. To explore the genetic contribution, we undertook a genome-wide association study of exceptional longevity in 801 centenarians (median age at death 104 years) and 914 genetically matched healthy controls. Using these data, we built a genetic model that includes 281 single nucleotide polymorphisms (SNPs) and discriminated between cases and controls of the discovery set with 89% sensitivity and specificity, and with 58% specificity and 60% sensitivity in an independent cohort of 341 controls and 253 genetically matched nonagenarians and centenarians (median age 100 years). Consistent with the hypothesis that the genetic contribution is largest with the oldest ages, the sensitivity of the model increased in the independent cohort with older and older ages (71% to classify subjects with an age at death>102 and 85% to classify subjects with an age at death>105). For further validation, we applied the model to an additional, unmatched 60 centenarians (median age 107 years) resulting in 78% sensitivity, and 2863 unmatched controls with 61% specificity. The 281 SNPs include the SNP rs2075650 in TOMM40/APOE that reached irrefutable genome wide significance (posterior probability of association = 1) and replicated in the independent cohort. Removal of this SNP from the model reduced the accuracy by only 1%. Further in-silico analysis suggests that 90% of centenarians can be grouped into clusters characterized by different \"genetic signatures\" of varying predictive values for exceptional longevity. The correlation between 3 signatures and 3 different life spans was replicated in the combined replication sets. The different signatures may help dissect this complex phenotype into sub-phenotypes of exceptional longevity.