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After stroke and other brain injuries, there is a high incidence of respiratory complications such as pneumonia or acute lung injury. The molecular mechanisms that drive the brain-lung interaction post-stroke have not yet been elucidated. We performed transient middle cerebral artery occlusion (MCAO) and sham surgery on C57BL/6J mice and collected bronchoalveolar lavage fluid (BALF), serum, brain, and lung homogenate samples 24 h after surgery. A 92 proteins-panel developed by Olink Proteomics® was used to analyze the content in BALF and lung homogenates. MCAO animals had higher protein concentration levels in BALF than sham-controls, but these levels did not correlate with the infarct volume. No alteration in alveolar-capillary barrier permeability was observed. A total of 12 and 14 proteins were differentially expressed between the groups (FDR < 0.1) in BALF and lung tissue homogenates, respectively. Of those, HGF, TGF-α, and CCL2 were identified as the most relevant to this study. Their protein expression patterns were verified by ELISA. This study confirmed that post-stroke lung damage was not associated with increased lung permeability or cerebral ischemia severity. Furthermore, the dysregulation of HGF, TGF-α, and CCL2 in BALF and lung tissue after ischemia could play an important role in the molecular mechanisms underlying stroke-induced lung damage., This project received funding from Instituto de Salud Carlos III (ISCIII) [PI17/02130, PI21/00939], co-financed by the European Regional Development Fund (FEDER), from the Fundació La Marató de TV3 [201706]. The participating centers take part into RICORS-ICTUS network [RD21/0006/0024, RD21/0006/0007, RD21/0006/0015] from Instituto de Salud Carlos III (ISCIII) [PI17/02130, PI21/00939], co-financed by the European Regional Development Fund (FEDER).

ObjectiveTo validate a panel of blood biomarkers to differentiate between ischemic stroke (IS) and intracerebral hemorrhage (ICH) in patients with suspected stroke.MethodsPatients with suspected stroke admitted within 4.5 hours after onset were enrolled. Blood samples were collected at hospital admission. Glial fibrillary acid protein (GFAP), retinol binding protein 4 (RBP-4), N-terminal proB-type natriuretic peptide (NT-proBNP), and endostatin were measured by immunoassays. Cutoff points were obtained for 100% specificity for IS. A high-sensitivity assay to measure GFAP and rapid point-of-care tests (POCTs) to measure RBP-4 and NT-proBNP were used in subsets of patients. Biomarker panels were evaluated in another cohort of 62 stroke mimics.ResultsA total of 189 patients (154 IS and 35 ICH) were enrolled. Patients with IS had higher RBP-4, NT-proBNP, and endostatin and lower GFAP levels than patients with ICH. The best biomarker combination for the identification of IS was RBP-4+NT-proBNP, which was able to identify 29.7% of patients with IS with 100% specificity. In the subset of patients for whom GFAP was measured with the high-sensitivity assay, RBP-4, NT-proBNP, and GFAP identified 51.5% of patients with IS with 100% specificity. When stroke mimics were included, specificities were reduced to 98.4 and 96.8%, respectively. POCTs of RBP-4 and NT-proBNP showed results similar results to those of conventional ELISAs.ConclusionsA biomarker panel including RBP-4, NT-proBNP, and GFAP provided moderate but potentially useful sensitivity rates at 100% specificity for IS diagnosis. If confirmed in future studies, this strategy might allow prehospital treatment in selected patients.Classification of EvidenceThis study provides Class I evidence that a biomarker panel including RBP-4, NT-proBNP, and GFAP distinguishes IS from ICH with moderate accuracy.

Stroke remains a leading cause of death and disability worldwide. Despite continuous advances, the identification of key molecular signatures in the hyper-acute phase of ischemic stroke is still a primary interest for translational research on stroke diagnosis, prognosis, and treatment. Data integration from high-throughput -omics techniques has become crucial to unraveling key interactions among different molecular elements in complex biological contexts, such as ischemic stroke. Thus, we used advanced data integration methods for a multi-level joint analysis of transcriptomics and proteomics data sets obtained from mouse brains at 2 h after cerebral ischemia. By modeling net-like correlation structures, we identified an integrated network of genes and proteins that are differentially expressed at a very early stage after stroke. We validated 10 of these deregulated elements in acute stroke, and changes in their expression pattern over time after cerebral ischemia were described. Of these, CLDN20, GADD45G, RGS2, BAG5, and CTNND2 were next evaluated as blood biomarkers of cerebral ischemia in mice and human blood samples, which were obtained from stroke patients and patients presenting stroke-mimicking conditions. Our findings indicate that CTNND2 levels in blood might potentially be useful for distinguishing ischemic strokes from stroke-mimicking conditions in the hyper-acute phase of the disease. Furthermore, circulating GADD45G content within the first 6 h after stroke could also play a key role in predicting poor outcomes in stroke patients. For the first time, we have used an integrative biostatistical approach to elucidate key molecules in the initial stages of stroke pathophysiology and highlight new notable molecules that might be further considered as blood biomarkers of ischemic stroke.

The aim of the study was to determine markers of atrial dysfunction in patients with cryptogenic stroke to predict episodes of paroxysmal atrial fibrillation with high risk of embolization (HpAF). We classified patients included in the Crypto-AF study, Cryptogenic Stroke registry, to detect paroxysmal atrial fibrillation (pAF) with wearable Holter, according to the longest episode of pAF in three groups: without pAF detection, episodes of pAF shorter than 5 h, and episodes of pAF longer than 5 h (HpAF). Atrial dysfunction surrogates were evaluated: EKG pattern, Holter record and echocardiography parameters (left atria volume (LAVI), and peak atrial longitudinal and contraction strain (PALS and PACS). The level of N-terminal pro b-type natriuretic peptide (NT-proBNP) was determined. All patients were followed for 2 years to detect pAF and stroke recurrence. From 308 patients, 253 patients with high quality Holter analysis were selected. The distribution was No pAF 78.6% (n = 199), pAF 5 h 7.9% (n = 20), and HpAF 5 h 13.4% (n = 34). Age of the patients and combination of PALS and NT-proBNP independently predicted HpAF OR 1.07 (1.00; 1.15) and OR 3.05 (1.08; 8.60) respectively. The validity of PALS and NT-proBNP to detect patients at risk of HpAF was higher than the validity of age (AUC 0.82, sensitivity 78.95%, specificity 63%). Patients with PALS 25% and NT-proBNP 283 pg/ml had more detection of pAF during follow-up 35% vs. 5.1% OR 2.33 (1.05-5.13) (p 0.001). Multimodal assessment of atrial dysfunction with PALS and NT-proBNP improved the prediction of pAF episodes with high embolic risk in patients with cryptogenic stroke.

CCL23 is a chemokine implicated in inflammation and host defense responses. It has been recently associated with acquired brain damage and stroke outcomes. In this study, we reported the role of CCL23 in Alzheimer's disease (AD). We evaluated the levels of CCL23 in 659 individuals: cognitively normal, mild cognitive impaired (MCI), and AD patients. Two cross-sectional (study 1, n = 53; study 2, n = 200) and two longitudinal (study 3, n = 74; study 4, n = 332) studies were analyzed separately. CCL23 levels in the blood and/or cerebrospinal fluid (CSF) of each study were measured by immunoassays. Globally, our results suggest a predictive role of CCL23 protein levels both in the plasma in study 3 (hazard ratio (HR) = 2.5 (confidence interval (CI) 95% : 1.2-5.3), p = 0.02) and in the CSF in study 4 (HR = 3.05 (CI 95% : 1.02-5), p = 0.04) in cases of MCI that progress to AD. Moreover, we observed that the APOE ϵ4 allele was associated with higher levels of CCL23 in study 2 (470.33 pg/mL (interquartile range (IQR): 303.33-597.76) versus 377.94 pg/mL (IQR: 267.16-529.19), p = 0.01) (APOE genotypes were available in studies 2 and 4). Together, these findings support the role of CCL23 in neuroinflammation in the early stages of AD, suggesting that CCL23 might be a candidate blood biomarker for MCI to AD progression.

[Background and objective] AFRICAT is a prospective cohort study intending to develop an atrial fibrillation (AF) screening program through the combination of blood markers, rhythm detection devices, and long-term monitoring in our community. In particular, we aimed to validate the use of NT-proBNP, and identify new blood biomarkers associated with AF. Also, we aimed to compare AF detection using various wearables and long-term Holter monitoring., [Methods] 359 subjects aged 65–75 years with hypertension and diabetes were included in two phases: Phase I (n = 100) and Phase II (n = 259). AF diagnosis was performed by baseline 12-lead ECG, 4 weeks of Holter monitoring (NuuboTM), and/or medical history. An aptamer array including 1310 proteins was measured in the blood of 26 patients. Candidates were selected according to p-value, logFC and biological function to be tested in verification and validation phases. Several screening devices were tested and compared: AliveCor, Watch BP, MyDiagnostick and Fibricheck., [Results] AF was present in 34 subjects (9.47%). The aptamer array revealed 41 proteins with differential expression in AF individuals. TIMP-2 and ST-2 were the most promising candidates in the verification analysis, but none of them was further validated. NT-proBNP (log-transformed) (OR = 1.934; p, [Conclusion] The inclusion and monitoring of a cohort of primary care patients for AF detection, together with the testing of biomarkers and screening devices provided useful lessons about AF screening in our community. An AF screening strategy using rhythm detection devices and short monitoring periods among high-risk patients with high NT-proBNP levels could be feasible., This work was supported by Fundació Marató de TV3 in the research call “La Marató 2014: malalties del cor” [grant number: 201528-30-31-3]. EP received a predoctoral grant from Vall d’Hebron Institute of Research. Neurovascular Research Laboratory also takes part in the Span-ish stroke research network INVICTUS+ [RD16/0019]. This project is supported by AFFECT-EU, receiving funding from the European Union’s Horizon 2020 research and innovation pro-gramme under grant agreement N°847770.

[Background] Atrial fibrillation (AF) has been associated with an increased risk of silent brain infarcts (SBI) and cognitive impairment, even in patients with low embolic risk. We aimed to test the association between 11 blood-biomarkers representing different AF-related pathways, and SBI, white matter hyperintensities (WMH), and cognitive decline in patients with AF and low embolic risk., [Methods] The present study followed a cross-sectional design. 70 patients with a history of AF and CHADS2 score ≤1, and 10 controls with neither AF nor SBI were included. All patients underwent a 3T brain MRI. Cortical and large subcortical ischemic lesions were considered presumed embolic origin lesions. White matter hyperintensities (WMH) were measured according to the Fazekas scale. A subset of patients underwent cognitive evaluation with the MoCA test. Circulating proteins were measured under blind conditions in a laboratory at Roche Diagnostics, Germany., [Results] 45 patients presented SBI in the MRI, and 25 did not. Ang-2, FGF-23, and BMP-10 were increased in patients with SBI. Ang-2 was elevated only in patients with embolic infarcts, whereas FGF-23 and BMP-10 tended to be elevated in patients with both types of infarcts. Ang-2 (OR = 1.56 [0.94-2.59], p = 0.087), and BMP-10 (OR = 4.83 [0.99–23.60], p = 0.052) were the biomarkers that showed the highest association with SBI when entered in a multivariable logistic regression model corrected by age. No biomarker was found associated with WMH or mild cognitive impairment., [Conclusions] BMP-10, and Ang-2 were increased in patients with SBI. Its usefulness to detect SBI in AF patients should be further explored., A Junta de Andalucía grant (PIN-0144-2016) supported partially the study. The Fundación Cajasol contributed to the study. Neurovascular Research Groups at Seville and Barcelona are part of the Spanish Neurovascular Disease Research Network (RICORS-ICTUS, RD21/0006/0007).

We aimed to discover blood biomarkers associated with longitudinal changes in white matter hyperintensities (WMH). This study was divided into a discovery phase and a replication phase. Subjects in both studies were patients with hypertension, aged 50–70, who underwent two magnetic resonance imaging (MRI) sessions and blood extractions over a 4-year follow-up period. In the discovery phase, we screened 1305 proteins in 12 subjects with WMH progression and in 12 matched control subjects. We found that 41 proteins were differentially expressed: 13 were upregulated and 28 were downregulated. We subsequently selected three biomarkers for replication in baseline and follow-up samples in 80 subjects with WMH progression and in 80 control subjects. The selected protein candidates for the replication were MMP9 (matrix metalloproteinase-9), which was higher in cases, MET (hepatocyte growth factor receptor) and ASAH2 (neutral ceramidase), which were both lower in cases of WMH progression. Baseline biomarker concentrations did not predict WMH progression. In contrast, patients with WMH progression presented a steeper decline in MET over time. Furthermore, cases showed higher MMP9 and lower ASAH2 levels than controls at the follow-up. These results indicate that MMP9, MET, and ASAH2 are potentially associated with the progression of WMH, and could therefore be interesting candidates to validate in future studies., Funds were obtained from the Instituto de Salud Carlos III (Grant Numbers: PI14/01535, PI17/02222, ICI14/307, PI19/00217, CP15/00010, and JR15/00032), incorporation of scientists and technicians to research groups (PERIS, SLT006/17/00266) and the AGAUR (FI_DGR 2017, Grant Number 2017_FI_B 00064), with the support of the Secretary of Universities and Research (Department of Economy and Knowledge, Generalitat de Catalunya), and was cofinanced by the European Regional Development Fund. The neurovascular research laboratory receives funds from the Spanish research stroke network (RD/16/0019/0021).

An accurate etiological classification is key to optimize secondary prevention after ischemic stroke, but the cause remains undetermined in one third of patients. Several studies pointed out the usefulness of circulating gene expression markers to discriminate cardioembolic (CE) strokes, mainly due to atrial fibrillation (AF), while only exploring them in small cohorts. A systematic review of studies analyzing high-throughput gene expression in blood samples to discriminate CE strokes was performed. Significantly dysregulated genes were considered as candidates, and a selection of them was validated by RT-qPCR in 100 patients with defined CE or atherothrombotic (LAA) stroke etiology. Longitudinal performance was evaluated in 12 patients at three time points. Their usefulness as biomarkers for AF was tested in 120 cryptogenic strokes and 100 individuals at high-risk for stroke. Three published studies plus three unpublished datasets were considered for candidate selection. Sixty-seven genes were found dysregulated in CE strokes. CREM, PELI1, and ZAK were verified to be up-regulated in CE vs LAA (p = 0.010, p = 0.003, p, Neurovascular Research Laboratory acknowledges funding for this project by PI15/00354 and PI18/804 grant from Fondo de Investigaciones Sanitarias and takes part in the Spanish stroke research network INVICTUS+ (RD16/0019/0021) of the Instituto de Salud Carlos III (co-financed by the European Regional Development Fund, FEDER). Moreover, it has been partially financed by 201528-30-31-3 grant from Fundació la Marató de TV3. A.B is supported by a Juan Rodés research contract JR16/00008 from the Instituto de Salud Carlos III.

[Background and purpose] B‐type natriuretic peptide (BNP) and N‐terminal pro‐brain natriuretic peptide (NT‐proBNP) are well‐known surrogates of atrial fibrillation (AF) detection but studies usually present data on either BNP or NT‐proBNP. The aim was to determine and directly compare the validity of the two biomarkers as a tool to predict AF and guide prolonged cardiac monitoring in cryptogenic stroke patients., [Methods] Non‐lacunar acute ischaemic stroke (, [Results] From 320 patients evaluated, 218 were included in the analysis. AF was detected in 50 patients (22.9%). NT‐proBNP (P < 0.001) and BNP (P < 0.001) levels were higher in subjects with AF and their levels correlated (r = 0.495, P < 0.001). BNP showed an increased area under the curve (0.720 vs. 0.669; P = 0.0218) and a better predictive capacity (IDI = 3.63%, 95% confidence interval 1.36%–5.91%) compared to NT‐proBNP. BNP performed better than NT‐proBNP in a specific model (IDI = 3.7%, 95% confidence interval 0.87%–6.5%), whilst both biomarkers performed similarly in the case of a sensitive model., [Conclusions] Both BNP and NT‐proBNP were increased in cryptogenic stroke patients with AF detection. Interestingly, BNP outperforms NT‐proBNP, especially in terms of specificity., This work was supported by a Research Grant (grant number PI15/02265) from the Carlos III Institute of Health, Ministry of Economy and Competitiveness (Spain), within the National Research Program Scientific and Innovation Research National Plan 2013–2016, reference co‐funded with European Regional Development Fund. The Neurovascular Research Laboratory acknowledges funding for this project by PI18/00804 from Fondo de Investigaciones Sanitarias. The Spanish stroke research network INVICTUS+ (RD16/0019/00021) of the Instituto de Salud Carlos III (co‐financed by the European Regional Development Fund, FEDER) is also acknowledged. EP has received a predoctoral grant from Vall D'Hebron Institute of Research.

Background: Early cerebral hypoperfusion and ischemia occur after subarachnoid hemorrhage (SAH) and influence clinical prognosis. Pathophysiological mechanisms possibly involve inflammatory mediators. TNF-α has been associated with complications and prognosis after SAH. We investigated the relation of perfusion parameters and ischemic lesions, with levels of TNF-α main receptor, TNF-R1, after SAH, and their association with prognosis. Methods: We included consecutive SAH patients admitted within the first 72 h of SAH onset. Blood samples were simultaneously collected from a peripheral vein and from the parent artery of the aneurysm. Levels of TNF-R1 were measured using ELISA (R&D Systems Inc., USA). CT perfusion and MRI studies were performed in the first 72 h. Correlation and logistic regression analysis were used to identify outcome predictors. Results: We analyzed 41 patients. Increased levels of TNF-R1 correlated with increased T (arterial: r = −0.37, p = 0.01) and prolonged MTT (arterial: r = 0.355, p = 0.012; venous: r = 0.306, p = 0.026). Increased levels of both arterial and venous TNF-R1 were associated with increased number of lesions on DWI (p = 0.006). In multivariate analysis, venous TNFR1 levels > 1742.2 pg/mL (OR 1.78; 95%CI 1.18–2.67; p = 0.006) and DWI lesions (OR 14.01; 95%CI 1.19–165.3; p = 0.036) were both independent predictors of poor outcome (mRS ≥ 3) at 6 months. Conclusion: Increased levels of TNF-R1 in arterial and venous blood correlate with worse cerebral perfusion and with increased burden of acute ischemic lesions in the first 72 h after SAH. Venous levels of TNF-R1 and DWI lesions were associated with poor outcome at 6 months. These results highlight the pathophysiological role of TNF-α pathways in SAH and suggest a possible role of combined imaging and laboratorial markers in determining prognosis in acute SAH., Dr Fragata was supported by Sociedade Portuguesa de AVC/Tecnifar. Dr Bustamante is supported by a Juan Rodes research contract (JR16/00008) from Instituto de Salud Carlos III.

Introduction and Purpose: Our aim was to review the characteristics of transient ischemic attack (TIA) and minor ischemic stroke patients monitored for atrial fibrillation (AF) epidoses detection within the first 4weeks after stroke to assess AF predictors. Materials and Methods: TIA and minor ischemic stroke patients (nihss≤ 5) were selected from CRYPTO-AF database. CRYPTO-AF is a prospective multicentre registry of patients with cryptogenic stroke older of 55 year-old. Monitoring started within the first 72 hours from stroke symptoms onset and was prolonged for 4 weeks. Clinical, cardiographic and blood test parameters of patients included were reviewed. Fisher exact and Mann Whitney tests were used to analyze categorical and continuous data. Results: In our cohort of 152 transient and minor ischemic stroke patients, 55.9% were men, mean age 73.18±10.24 and median NIHSS score was 2.31(0-5). A total of 30 patients (20.3%) were diagnosed with AF in the first month of monitoring, 6 patients (5.7%) within the first 3 days, 14 patients (13.3%) between 3 days and to 2 weeks of monitoring, and 14 partientes (12.4%) between the second and the fourth week. In these transient and minor ischemic stroke patients, age (p< 0.031), left atrial volume index (p< 0.023), the appearance of isolated extrasystoles during monitoring (p< 0.021), Type B natriuretic peptide (p< 0.011) and the longitudinal strain (p< 0.019) appeared as independet precitors of AF. However in the multivariate analysis adjusted for the above variables, only left atrial strain (OR 0.89, 95% CI: 0.797-0.991, p< 0.034) independently predicts AF detection. Conclusions: In our serie, only the left atrial strain appeared as a indepent predictor of AF. Given the known pathophysiology of TIA and minor ischemic stroke, the description of AF predictors would help to identify those patients who would benefit from completing a longer monitoring. More studies are needed to identify these predictors.

[Background and purpose]: Covert paroxysmal atrial fibrillation (pAF) is the most frequent cause of cardiac embolism. Our goal was to discover parameters associated with early pAF detection with intensive cardiac monitoring., [Method]: Crypto‐AF was a multicentre prospective study (four Comprehensive Stroke Centres) to detect pAF in non‐lacunar cryptogenic stroke continuously monitored within the first 28 days. Stroke severity, infarct pattern, large vessel occlusion (LVO) at baseline, electrocardiography analysis, supraventricular extrasystolia in the Holter examination, left atrial volume index and brain natriuretic peptide level were assessed. The percentage of pAF detection and pAF episodes lasting more than 5 h were registered., [Results]: Out of 296 patients, 264 patients completed the monitoring period with 23.1% (61/264) of pAF detection. Patients with pAF were older [odds ratio (OR) 1.04, 95% confidence interval (CI) 1.01–1.08], they had more haemorrhagic infarction (OR 4.03, 95% CI 1.44–11.22), they were more likely to have LVO (OR 4.29, 95% CI 2.31–7.97) (P < 0.0001), they had a larger left atrial volume index (OR 1.03, 95% CI 1.01–1.1) (P = 0.0002) and they had a higher level of brain natriuretic peptide (OR 1.01, 95% CI 1.0–1.1). Age and LVO were independently associated with pAF detection (OR 1.06, 95% CI 1.00–1.16, and OR 4.58, 95% CI 2.27– 21.38, respectively). Patients with LVO had higher cumulative incidence of pAF (log rank P < 0.001) and more percentage of pAF > 5 h [29.6% (21/71) vs. 8.3% (12/144); OR 4.62, 95% CI 2.11–10.08; P < 0.001]. In a mean follow‐up of 26.82 months (SD 10.15) the stroke recurrence rate was 4.6% (12/260)., [Conclusions]: Large vessel occlusion in cryptogenic stroke emerged as an independent marker of pAF., This work was supported by a research grant (grant number PI15/02265) from the Carlos III Institute of Health, Ministry of Economy and Competitiveness (Spain), within the National Research Program Scientific and Innovation Research National Plan 2013–2016, reference co‐funded with the European Regional Development Fund.

Patients’ outcome prediction after ischemic stroke is still challenging. Aquaporin-4 (AQP4) is a water channel that is up-regulated in the brain after the ischemic event, but its presence in bloodstream of stroke patients has not been previously studied. The aim of this pilot study was to investigate circulating AQP4 levels after stroke and its correlation with infarct growth and neurological outcome. AQP4 level was determined by ELISA in serum from 42 t-PA-treated ischemic stroke patients at admission (before t-PA) and 13 healthy subjects. To assess infarct growth, serial brain diffusion-weighted magnetic resonance images were performed at hospital admission and 1–3 days after. Neurological improvement was defined as a ≥4-point decrease in NIHSS score compared to baseline score. Despite stroke patients and healthy controls had similar baseline circulating AQP4 levels, among strokes AQP4 level negatively correlated with NIHSS score at admission (R= −0.34, p = 0.029) and with infarct growth after 1–3 days of stroke onset (R=−0.36; p = 0.018). Furthermore, baseline AQP4 level was higher in those stroke patients showing a neurological improvement 48 h after stroke onset (p = 0.030) and at hospital discharge (p = 0.037). Baseline AQP4 levels also resulted to be an independent predictor of good neurological outcome at both studied time points (ORadj: 14.33[1.82–112.92], p = 0.012 at 48 h; ORadj: 4.86[0.98–24.12], p = 0.053 at discharge) in logistic regression analysis, adjusted by age, sex, baseline NIHSS and significant variables in the univariate analysis. Overall, we have explored circulating AQP4 levels, and our data suggest that AQP4 could be used as a biomarker of neurological recovery in the acute-subacute phase of ischemic stroke., Neurovascular Research Laboratory acknowledges funding for this project by PI15/00354 and PI18/00804 grants from Fondo de Investigaciones Sanitarias of the Instituto de Salud Carlos III (co-financed by the European Regional Development Fund, FEDER). Neurovascular Research Laboratory also takes part into the Spanish stroke research network INVICTUS+ (RD16/0019). L. Ramiro is supported by a predoctoral fellowship grant from the Instituto de Salud Carlos III (IFI17/00012). In addition, we acknowledge Miriam Echevarría from the Instituto de Biomedicina de Sevilla-IBiS by interesting conversations and advice on the role of Aquaporins in neurological diseases.

Immunoassays and immunosensors are extensively described diagnostic tools. However, they often entail time-consuming multi-step procedures that are difficult to perform outside centralized diagnostic laboratories, such as at point-of-care settings, where extremely simple assay paths are required. In this work, a commercial polymeric enzyme, polyHRP, has been used as a signal amplifier for detection of matrix metalloproteinase 9 (MMP-9). Contrary to previous reports, signal enhancement has been exploited here to reduce the number and duration of the steps of a classical multistep enzyme-linked immunosorbent assay (ELISA) while retaining its original sensitivity. The two assay formats produced, a simplified ELISA and an electrochemical immunosensor, could be carried out in about 1 h and 45 min, respectively, but detected MMP-9 with linear range, limit of detection (LOD), limit of quantification (LOQ), sensitivity, and variability comparable to the original ELISA that took >5 h, and quantitated successfully MMP-9 in plasma samples and uterine aspirates from patients. These results demonstrate that the implementation of signal amplifiers, such as polyHRP, allows the optimization of simplified immunoassays, maintaining the high performance obtained with longer multi-step paths. Furthermore, the strategy reported is extremely simple and potentially implemented elsewhere by other researchers with minimal technical requirements.

BACKGROUND CCL23 role in the inflammatory response after acute brain injuries remains elusive. Here, we evaluated whether CCL23 blood levels associate with acquired cerebral lesions and determined CCL23 predictive capacity for assessing stroke prognosis. We used preclinical models to study the CCL23 homologous chemokines in rodents, CCL9 and CCL6. METHODS Baseline CCL23 blood levels were determined on 245 individuals, including ischaemic strokes (IS), stroke mimics and controls. Temporal profile of circulating CCL23 was explored from baseline to 24 h in 20 of the IS. In an independent cohort of 120 IS with a 3-month follow-up, CCL23 blood levels were included in logistic regression models to predict IS outcome. CCL9/CCL6 cerebral expression was evaluated in rodent models of brain damage. Both chemokines were also profiled in circulation and histologically located on brain following ischaemia. RESULTS Baseline CCL23 blood levels did not discriminate IS, but permitted an accurate discrimination of patients presenting acute brain lesions (P = 0.003). IS exhibited a continuous increase from baseline to 24 h in circulating CCL23 (P

ObjectiveWe aimed to analyze ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) in relation to arterial recanalization in patients treated with IV tissue plasminogen activator (tPA) and in relation to futile recanalization in patients treated with mechanical thrombectomy.MethodsAcute ischemic stroke patients (n = 108) with documented arterial occlusions treated with IV-tPA were selected. ADAMTS13 activity was measured by ELISA in samples collected before treatment. Recanalization was assessed at 2 hours by transcranial Doppler. In 78 consecutive patients treated with endovascular thrombectomy, ADAMTS13 antigen was measured by ELISA and futile recanalization was defined as complete recanalization plus modified Rankin Scale score >2 at 3 months. Independent predictors of recanalization and futile recanalization were determined by logistic regression, adjusted by age, NIH Stroke Scale score, and time from stroke onset.ResultsPatients who achieved tPA-induced recanalization had higher baseline ADAMTS13 activity (78.1% [68%–88%] vs 70.1% [61%–79%], p = 0.021). In logistic regression analysis, ADAMTS13 activity >75% was an independent predictor of recanalization (odds ratio = 6.76 [1.52–30.02], p = 0.012), together with absence of early ischemic signs and Oxfordshire Community Stroke Project classification. Regarding endovascular therapies, a reduced ADAMTS13 concentration (p = 0.034), together with age and diabetes mellitus. The addition of ADAMTS13 to clinical predictors of tPA-induced recanalization and futile recanalization improved discrimination and reclassification (integrated discrimination improvement = 10.06% and 28.4%, net reclassification improvement = 61.0% and 107.4%, respectively).ConclusionsA reduced ADAMTS13 was associated with poor response to recanalization therapies. If confirmed in future prospective studies, a panel of blood biomarkers including ADAMTS13 might be a useful tool to guide reperfusion therapies.

We describe the feasibility of monitoring with a Textile Wearable Holter (TWH) in patients included in Crypto AF registry.We monitored cryptogenic stroke patients from stroke onset (3days) continuously during 28days. We employed a TWH composed by a garment and a recorder. We compared two garments (Lead and Vest) to assess rate of undiagnosed Atrial Fibrillation (AF) detection, monitoring compliance, comfortability (1 to 5 points), skin lesions, and time analyzed. We describe the timing of AF detection in three periods (0-3, 4-15 and 16-28days).The rate of undiagnosed AF detection with TWH was 21.9% (32 out of 146 patients who completed the monitoring). Global time compliance was 90% of the time expected (583/644h). The level of comfortability was 4 points (IQR 3-5). We detected reversible skin lesions in 5.47% (8/146). The comfortability was similar but time compliance (in hours) was longer in Vest group 591 (IQR [521-639]) vs. Lead 566 (IQR [397-620]) (p=0.025). Also, time analyzed was more prolonged in Vest group 497 (IQR [419-557]) vs. Lead (336h (IQR [140-520]) (p=0.001)). The incidence of AF increases from 5.6% (at 3days) to 17.5% (at 15th day) and up to 20.9% (at 28th day). The percentage of AF episodes detected only in each period was 12.5% (0-3days); 21.7% (4-15days) and 19% (16-28days).28days Holter monitoring from the acute phase of the stroke was feasible with TWH. Following our protocol, only five patients were needed to screen to detected one case of AF.

Introduction: One of the main limitations in biomarker research for stroke-associated pneumonia (SAP) is the lack of a gold-standard for clinical diagnosis. We aimed to evaluate the diagnostic accuracy of candidate biomarkers related to the features on thoracic high-resolution computed tomography (HRCT). Methods: Patients with ischemic stroke with baseline NIHSS>10 were enrolled between March-2016 and August-2017. Blood samples were collected at baseline, 24 and 48 hours after stroke onset, to determine C-reactive protein (CRP), serum amyloid protein A (SAA), mid-regional proadrenomedullin (MR-proADM), and soluble receptor of urokinase-type plasminogen activator (suPAR). Biomarkers were measured by immunoassays. Pneumonia was diagnosed as the presence of a new consolidation, infectious nodules or ground-glass pattern, in a thoracic HRCT performed 5-7 days after stroke. Results: From 46 included patients, thoracic HRCT was performed in 41 patients. Five (12.5%) were diagnosed as pneumonia and 3 (7.3%) with acute bronchitis. No blood marker was associated with pneumonia on thoracic HRCT at baseline. At 24 hours, a cut-off of MR-proADM>1.023 nmol/L had 80% sensitivity and 72.2% specificity for pneumonia. At 48 hours, both SAA>25.15 mg/dL (60% sensitivity, 85.7% specificity) and suPAR>3.14 ng/mL (80% sensitivity, 57.1% specificity) were associated with infections. The combination of SAA>25.15 mg/dL and suPAR>3.14 ng/mL at 48 hours had 80% sensitivity and 95.8% specificity when both biomarkers were above the cut-off, and 60% sensitivity and 94.1% specificity when one of them was above the cut-off. Conclusions: An objective diagnostic test such as thoracic HRCT could be of interest for future research in SAP. The evaluated biomarkers will require further validation in larger samples. However, these biomarkers represent promising tools to be evaluated in future projects on SAP early diagnosis and guidance on antibiotic therapy.

Hypertension is one of the principal risk factors for cerebral small vessel disease progression and cognitive impairment. We aimed to investigate how changes in cerebral small vessel disease lesions relate to cognitive decline and incident mild cognitive impairment in hypertensive patients. Data were obtained from the ISSYS cohort (Investigating Silent Strokes in Hypertensives: a Magnetic Resonance Imaging Study)—a longitudinal population-based study on hypertensive patients aged 50 to 70 years without dementia and stroke at baseline. Patients underwent a brain magnetic resonance imaging, a cognitive screening test, and cognitive diagnosis (normal aging or mild cognitive impairment) at baseline and follow-up. We evaluated incident lacunar infarcts and cerebral microbleeds. Changes in the periventricular and deep white matter hyperintensities (WMH) were qualitatively defined as none, minor, or marked. We followed up 345 patients (median age, 65 [61–68]; 55.4% men) for 3.95 (3.83–4.34) years. Incident mild cognitive impairment was diagnosed in 9.1% of the sample. Considering the progression of cerebral small vessel disease, the prevalence of incident infarcts was 6.1% and that of incident cerebral microbleeds was 5.5%; progression of periventricular WMH was 22% and that of deep WMH was 48%. Patients with marked progression of periventricular WMH showed a significant decrease in global cognition compared with patients without progression (adjusted mean [SE], −0.519 [0.176] versus 0.057 [0.044], respectively; P value=0.004) and a higher risk of incident mild cognitive impairment (OR, 6.184; 95% CI, 1.506–25.370; P value=0.011). Therefore, our results indicate that hypertensive patients with progression of periventricular WMH have higher odds of cognitive impairment, even in the early stages of cognitive decline.

© 2019 Springer Science+Business Media, LLC, part of Springer Nature and Neurocritical Care Society, Background: There is increasing evidence for the role of inflammation in clinical outcome after subarachnoid hemorrhage (SAH). Specifically, the TNF-alfa(α) pathway seems to be relevant after SAH. Although the TNF-α main receptor, TNF-R1 is associated with aneurysm growth and rupture, its relation to prognosis is unknown. We sought to compare TNF-R1 levels in peripheral venous blood and arterial blood closer to the ruptured aneurysm to study the association of TNF-R1 blood levels with poor prognosis (modified Rankin Scale > 2 at discharge, 3 and 6 months) and complications (hydrocephalus or delayed cerebral ischemia/DCI) following SAH. Methods: We included consecutive SAH patients admitted in the first 72 h of symptoms. Blood samples were simultaneously collected from a peripheral vein and from the main parent artery of the aneurysm. Levels of TNF-R1 were measured using enzyme-linked immunosorbent assays. Results: We analyzed 58 patients. Arterial and venous levels of TNF-R1 were correlated (R = 0.706, p < 0.001). In multivariate regression analysis, venous TNF-R1 was an independent predictor of poor outcome at 6 months after adjusting by age and sex [odds ratio (OR) 11.63; 95% CI 2.09-64.7, p = 0.005] and after adjusting by Glasgow Coma Scale and Fisher scales (OR 8.74; 95% CI 1.45-52.7, p = 0.018). There was no association of TNF-R1 with DCI. A cut-off for arterial TNF-R1 of 1523.7 pg/mL had 75% sensitivity/66% specificity for the prediction of hydrocephalus. Conclusion: Levels of venous TNF-R1 are associated with poor outcome in SAH. A specific association was found between levels of arterial TNF-R1 and hydrocephalus. These results are consistent with the role of TNF-α pathway in SAH and need to be validated in larger cohorts., Dr Fragata was supported by Sociedade Portuguesa de AVC/Tecnifar. Dr Bustamante is supported by a Juan Rodes research contract (JR16/00008) from Instituto de Salud Carlos III.

Objective: To study the association of N-terminal pro-brain natriuretic peptide (NT-proBNP) with several brain MRI markers of brain vascular disease in a sample of participants free of stroke and dementia. Methods: NT-proBNP plasma level was determined by means of a sandwich immunoassay method in a cohort study comprising 278 hypertensive patients. The presence of silent brain infarcts, brain microbleeds, enlarged perivascular spaces, and white matter hyperintensity volumes was assessed by brain MRI. We performed univariate and multivariate analyses to determine whether NT-proBNP was independently associated with these imaging markers, individually or combined. Results: Median age was 63 years, and 41.4% were women. NT-proBNP remained independently associated with silent brain infarcts (odds ratio [OR] per 1-SD increase in NT-proBNP 2.11, 95% confidence interval [CI] 1.44–3.10), brain microbleeds (OR 1.79, 95% CI 1.15–2.78), basal ganglia enlarged perivascular spaces (OR 1.55, 95% CI 1.12–2.15), and white matter hyperintensity volumes (β 1.60, 95% CI 0.47–2.74), even after controlling for vascular risk factors, cardiovascular risk, atrial fibrillation, previous heart disease, duration of hypertension, and preventive treatments. A score combining several imaging markers was also related to NT-proBNP levels (common OR per 1-SD increase 1.74, 95% CI 1.21–2.50). Conclusions: NT-proBNP is independently associated with silent cerebrovascular lesions and could be a surrogate marker of vascular brain damage in hypertension.

International audience; Aim: We report the study of a familial rare disease with recurrent venous thromboembolic events that remained undiagnosed for many years using standard coagulation and hemostasis techniques. Methods: Exome sequencing was performed in three familial cases with venous thromboembolic disease and one familial control using NimbleGen exome array. Clot lysis experiments were performed to analyze the reasons of the altered fibrinolytic activity caused by the mutation found. Results: We found a mutation that consists of a R458C substitution on the fibrinogen alpha chain (FGA) gene confirmed in 13 new familial subjects that causes a rare subtype of dysfibrinogenemia characterized by venous thromboembolic events. The mutation was already reported to be associated with a fibrinogen variant called fibrinogen Bordeaux. Clot-lysis experiments showed a decreased and slower fibrinolytic activity in carriers of this mutation as compared to normal subjects, thus demonstrating an impaired fibrinolysis of fibrinogen Bordeaux. Conclusions: The exome sequencing and clot-lysis experiments might be powerful tools to diagnose idiopathic thrombophilias after an unsuccessful set of biochemical laboratory tests. Fibrinogen Bor-deaux is associated with impaired fibrinolysis in this family with idiopathic thrombophilia.

We aimed to study whether worsening in markers of kidney function parallels the progression in cerebral small vessel disease (cSVD) and cognitive decline.Data from the ISSYS (Investigating Silent Strokes in Hypertensives Study), a longitudinal population-based study in hypertensives aged 50-70 and dementia and stroke-free at baseline. At both visits, patients underwent a brain MRI, a cognitive diagnosis (normal aging or mild cognitive impairment, [MCI]) and urine and blood sampling collection. We assessed the incidence of infarcts and cerebral microbleeds, and the progression of white matter hyperintensities at periventricular (PVH) and deep areas. We determined changes in albumin-creatinine ratio and estimated glomerular filtration rate (eGFR). These changes were dichotomized into microalbuminuria at follow-up -either in subjects with or without baseline microalbuminuria- and significant decline in eGFR -lowest quintile of eGFR change (-10.57 mL/min/1.73m360 patients were followed-up for 4 years. 80 (23%) patients presented microalbuminuria at follow-up and 68 (20.1%) experienced a significant eGFR decline. Considering cSVD change, we found a relationship between microalbuminuria at follow-up and progression in PVH (β = 0.31, P-value = .01). Regarding cognitive decline, presence of microalbuminuria at follow-up related to a steeper decrease in memory function (β = -0.36, P-value.01). Moreover, patients with significant decline in eGFR were at higher risk of incident MCI (OR = 3.54, P-value = .02). These associations were independent of progression of cSVD.The worsening in markers of kidney function paralleled the decrease in cognition and the progression of cSVD, and this may be explained by common shared underlying risk factors.

Introduction: Despite the usefulness of mechanical thrombectomy (MT), around 50% of the patients had poor outcome after achievement of complete recanalization. We aimed to test blood biomarkers to predict futile recanalization (FR) in patients treated with MT. Methods: Consecutive stroke patients were enrolled from August-2012 to August-2014. Blood samples were collected at admission. Biomarkers were selected among pathophysiological pathways related to FR: inflammation (VCAM-1), thrombosis (ADAMTS13), lipid metabolism (ApoC-III). FR was defined as complete recanalization after endovascular procedure (TICI 2b-3) and poor functional outcome (mRS>2) at three months. Comparisons were performed between patients with futile and successful recanalization. Independent predictors of FR were determined by logistic regression. The additional predictive value of blood biomarkers was evaluated by integrated discrimination improvement (IDI) and net reclassification improvement (NRI) indexes, considering thresholds of 25-75% risk categories for the later. Results: From 87 patients treated with MT, 73 were included. Complete recanalization was achieved in 58 patients, being futile in 33 patients (57%). Patients with FR had a trend towards lower levels of both ADAMTS13 and ApoC-III. The rate of FR when both biomarkers were under the 1 st quartile was 100%. Both biomarkers were independent predictors of FR in logistic regression analysis [ADAMTS13; OR=8.01 (1.20-53.5), p=0.032. ApoC-III; OR=6.94 (1.32-36.62), p=0.022], together with previous mRS [OR=5.75 (1.73-19.05), p=0.004] and diabetes [OR=7.6 (1.26-45.1), p=0.032]. The addition of both biomarkers improved discrimination [IDI=13.8% (4.7-22.9), p=0.003], and reclassification [60.2% (31.4-89.1), p Conclusions: Pre-treatment levels of ADAMTS13 and ApoC-III are predictors of FR. These blood markers might be used to guide reperfusion therapies.

Introduction: Traditional predictors of paroxysmal AF (PAF) including left atria size, CHADSVASC score or rate of atria premature beats are inaccurate to predict PAF in elderly stroke patients. We aimed to evaluate the yield of atrial dysfunction markers such as NT-ProBNP and Left atrial deformability in acute elderly cryptogenic stroke patients to predict PAF. Methods: We prospectively evaluated cryptogenic stroke patients Results: Out of 150 cryptogenic stroke patients, one hundred and ten patients fulfilled 4 weeks of Holter monitoring. Median age was 75 years (69-83).The percentage of PAF detection was 24.5%. Age, sex, hypertension, diabetes, CHADSVASC score, NIHSSS, left atria volume, left ventricle ejection fraction and rate of atria premature beats were comparable between patients with and without PAF detection. Admission NT-ProBNP levels were higher (346.9 pg/ml [175-685]) in whom PAF was detected compared to those who did not (203.5 pg/ml [76.75-453.95] (p=0.028). Conversely, LAS was lower 18.66% [13.98-26.71] in PAF compared to no PAF detected patients 27.43% [21.28-36-56] (p=0.003). Both markers were inversely correlated (Spearman Rho -0.31) (p=0.007). The AUC of NT-ProBNP was 0.65 (0-53-0.76) with cut-off point of NT-ProBNP> 102 pg/ml (sensitivity 96%, specificity 33%) and LAS was 0.73 (0.59-0.86) with cut-off point of LAS Conclusions: The combination of both NT-ProBNP and LAS markedly improve the predictive accuracy for PAF detection in elderly cryptogenic stroke patients.

Background and Purpose— Stroke diagnosis could be challenging in the acute phase. We aimed to develop a blood-based diagnostic tool to differentiate between real strokes and stroke mimics and between ischemic and hemorrhagic strokes in the hyperacute phase. Methods— The Stroke-Chip was a prospective, observational, multicenter study, conducted at 6 Stroke Centers in Catalonia. Consecutive patients with suspected stroke were enrolled within the first 6 hours after symptom onset, and blood samples were drawn immediately after admission. A 21-biomarker panel selected among previous results and from the literature was measured by immunoassays. Outcomes were differentiation between real strokes and stroke mimics and between ischemic and hemorrhagic strokes. Predictive models were developed by combining biomarkers and clinical variables in logistic regression models. Accuracy was evaluated with receiver operating characteristic curves. Results— From August 2012 to December 2013, 1308 patients were included (71.9% ischemic, 14.8% stroke mimics, and 13.3% hemorrhagic). For stroke versus stroke mimics comparison, no biomarker resulted included in the logistic regression model, but it was only integrated by clinical variables, with a predictive accuracy of 80.8%. For ischemic versus hemorrhagic strokes comparison, NT-proBNP (N-Terminal Pro-B-Type Natriuretic Peptide) >4.9 (odds ratio, 2.40; 95% confidence interval, 1.55–3.71; P 4.7 (odds ratio, 2.02; 95% confidence interval, 1.19–3.45; P =0.010), together with age, sex, blood pressure, stroke severity, atrial fibrillation, and hypertension, were included in the model. Predictive accuracy was 80.6%. Conclusions— The studied biomarkers were not sufficient for an accurate differential diagnosis of stroke in the hyperacute setting. Additional discovery of new biomarkers and improvement on laboratory techniques seem necessary for achieving a molecular diagnosis of stroke.

We aimed to evaluate the usefulness of sepsis biomarkers to predict stroke-associated infections. Soluble triggering receptor expressed on myeloid cells-1 (sTREM-1), mid-regional pro-adrenomedullin (MR-proADM), presepsin (sCD14), and soluble urokinase-type plasminogen activator receptor (suPAR), were explored in 125 blood samples collected at different time-points. At baseline, MR-proADM was an independent predictor of infection [>0.94pg/mL, OR=3.63 (1.16-11.33), p=0.026], as well as suPAR at 24h [>2185.8pg/mL, OR=5.81 (1.05-32.26), p=0.044]. Both MR-proADM and suPAR were raised in patients with infections throughout the first week after stroke. These results are especially relevant for MR-proADM given its early elevation, which would allow early preventive interventions.

Chemokines act mainly in guiding leukocyte migration along the endothelium. Apart from angiogenesis or neuronal survival, chemokines are involved in damage and repair of brain tissue after ischemic stroke. We studied the presence of chemokines directly in neurons and brain blood vessels that were obtained by means of laser microdissection from human ischemic brains. Using multiple ELISA Searchlight® array we evaluated nine chemokines (CCL1−5, CCL11, CCL17, CCL22, and CXCL8) in microdissected samples. We found higher levels of CCL1 and CCL2 in neurons than in vessels; CCL5 and CCL22 were decreased in the infarcted areas. The same ELISA array was performed in plasma samples from stroke patients. We explored the temporal profile of circulating chemokines from admission to 90 days after the cerebrovascular event, and found that only CCL22 showed significant changes along time and that these changes negatively correlated with neurological severity. When neurological outcome was assessed in the hyperacute phase of stroke no associations were found. From our study, we can conclude that these chemokines do not perform a clear role of outcome biomarkers. Further studies are necessary to assess which mechanisms underlie the association of chemokines with the neurological state at distinct time points since the differences found here could be reflecting the dual role of chemokines in neuroinflammation.

Despite being ischemic stroke a leading cause of death and functional disability, there are no other accurate tools to predict outcome of patients beyond clinical variables such as age and stroke severity. In this scenario, defining protein changes associated with acute ischemic brain damage might help to identify new biomarker candidates for stroke prognosis. By means of mass spectrometry-based proteomics, we identified 51 proteins which levels were altered in the infarcted area of the human brain after stroke. Among 8 selected protein candidates, circulating levels of gelsolin, dihydropyrimidinase-related protein 2 and cystatin A were independent predictors of poor outcome. Logistic regression models including these innovative biomarkers significantly improved the predictive value with respect to the only use of clinical variables in both discrimination and reclassification analyses. Our results indicate that early blood determination of these three biomarkers might predict outcome of patients and might help in decision-making processes related to ischemic stroke management.Circulating levels of gelsolin, dihydopyrimidinase-related protein 2 and cystatin A, proteins found altered in human brain after cerebral ischemia, demonstrate potential usefulness as biomarkers for long-term stroke prognosis.

Background: Intracranial atherosclerotic disease (ICAD) is an important cause of ischemic stroke (IS) and endothelial dysfunction plays a critical role in its onset and progression. Endothelial progenitor cells (EPCs) and endothelial production of angiogenic growth factors (AGFs) may play an essential role in this process. This study investigated the association of EPCs and AGFs with ICAD severity. Methods: A total of 42 patients who had experienced a transient ischemic attack (TIA) or IS attributable to symptomatic ICAD were included. Clinical and neurosonological evaluations were conducted between 2.4 and 8.7 years after the initial cerebrovascular event. Severe ICAD was defined as the presence of at least 1 severe intracranial stenosis, and extensive ICAD as 3 or more intracranial stenoses. Blood samples were obtained to determine EPC levels using flow cytometry (CD34+KDR+ cells), and the plasma levels of several growth factors were assessed with a protein array (Searchlight®). Twenty-two individuals without cerebrovascular disease and with normal ultrasonographic examination were also included. Results: No difference in the count of circulating EPCs was found between patients and controls, and a moderate increase in the number of EPCs/ml was noted in patients with extensive ICAD (p = 0.05). Patients presented decreased levels of fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF-BB) compared with controls (p = 0.002, p = 0.079 and p = 0.061, respectively). Higher levels of FGF, VEGF and PDGF-BB were found in patients with severe ICAD (p = 0.007, p = 0.07 and p = 0.07, respectively), but there was no correlation between any AGFs and EPCs. Conclusions: Symptomatic ICAD patients have decreased levels of AGFs with no correlation to the number of circulating EPCs, while patients with severe ICAD have higher levels of EPCs, FGF, VEGF and PDGF-BBs. This suggests that reduced EPC and proangiogenic factor production capacity is implicated in ICAD pathogenesis, while the more severe forms of chronic brain hypoperfusion in ICAD patients might stimulate EPC mobilization and AGF production.

Infection is an independent risk factor for adverse outcome in stroke patients. The risk of developing an infection in this setting is partly related to a stroke-induced immunodepression, in which a shift to a predominant Th2 (immunosuppressive) phenotype has been postulated to play a major role. Our aim was to study whether clinical variables or changes in plasma cytokine expression can predict poststroke infections. Patients and Methods: Medical records of 92 stroke patients were reviewed, and the baseline concentration of cytokines from the Th1/Th2 system was determined. Clinical and serological predictors of incident infections and their prognostic significance were sought by means of univariate and multivariate analysis, and two predictive models for developing an infection were constructed by combining independent predictors (strictly clinical in one, and both clinical and serological in the other) for this outcome. The improvement conferred by the addition of immunological markers to the clinical model was assessed by comparing their respective ROC curves and by improvement (Net Reclassification Index and Integrated Discriminator Improvement) analysis. Results: Nineteen patients (20.7% of the study sample) developed an infection. Ongoing antiplatelet therapy at symptom onset (OR 0.02, 95% CI 0.001-0.23, p = 0.001), diabetes mellitus (OR 9.96, 95% CI 1.32-75.29, p = 0.03), IL-13 level 8.4 pg/ml (OR 60.17, 95% CI 1.78-2037.23, p = 0.02) were independently associated with the development of infections during hospital admission. The combined regression model predicted infection with an accuracy of 93.4%, an improvement in the predictive capacity of 17% (p < 0.001). Infection was associated with a worse neurological status at hospital discharge (median NIHSS score 11 (6-18) vs. 4 (1-11.5), p = 0.014). Conclusions: This study shows that bloodstream biomarkers are useful to improve the accuracy of clinical prognostic models for infection in the acute phase of stroke. The clinical predictors of infection in the acute phase of stroke are relatively well established in the medical literature, but further research to identify the optimal combination of biomarkers (possibly inflammatory and stress markers) to be included in a clinically useful model is needed. Such a model could be subsequently used in clinical trials to assess the effect of prophylactic and/or early antibiotic therapy in this setting, a currently controversial issue in this field.

Aim: Despite the benefits of recombinant t-PA (rt-PA) for stroke patients some of them suffer from adverse hemorrhagic transformations (HTs) following treatment. Our objective is to study the transcriptomics of HTs patients. Methods: We studied by microarrays 11 blood samples from patients with stroke that had received rt-PA of whom six of them had suffered a HT. For replication step RNA was collected from 14 new subjects (seven with HT, seven without) and then analyzed by real-time PCR. Four proteins were measured by ELISA in 72 new subjects to analyze their role as potential protein biomarkers. Results: The microarray analysis revealed that 14 genes were altered among the HT patients. The replication study confirmed these results for six genes. Two of them (BCL2 and OLFM4) are associated with apoptosis, whereas the other four (LTF, LCN2 [also known as NGAL], CEACAM8 and CRISP3) are involved in the regulation of neutrophil processes. Conclusion: Our data revealed that genes related to apoptosis and neutrophil regulation pathways could be associated with HTs after rt-PA. Original submitted 7 September 2012; Revision submitted 23 January 2013

Purpose The importance of troponin elevation at stroke presentation remains uncertain. We aimed to assess whether baseline ultrasensitive Troponin I (hs-TnI) predicts cardiac complications and outcome in acute stroke patients. Method Stroke patients admitted within 6 h were consecutively enrolled from May 2013 to March 2014. Blood samples were taken at admission to determine hs-TnI by chemiluminescent microparticle immunoassay. hs-TnI > 34.2 pg/ml (male) and >15.6 pg/ml (female) were considered elevated. Complications during in-hospital stay and outcome at 90 days were prospectively recorded. Independent predictors of cardiac complications (heart failure and acute coronary syndrome) and mortality were determined by logistic regression. The additional predictive value of hs-TnI was evaluated by integrated discrimination improvement index. A subanalysis was performed after excluding patients with previous cardiac diseases. Findings From 174 patients, 39(22%) had elevated hs-TnI, having these patients higher incidence of cardiac complications (57% versus 19%, p = 0.004). hs-TnI was an independent predictor of cardiac complications (OR = 16.1 (1.7–150.3)) together with diastolic blood pressure (OR = 0.92 (0.86–0.99)). Addition of hs-TnI to clinical variables significantly improved discrimination (IDI = 15.2% (7.8–22.7)). Subanalysis in patients without previous cardiac diseases showed similar results. Elevated hs-TnI was independently associated with 90 days mortality (OR = 3.6 (1.3–9.4)), but addition of hs-TnI to clinical data did not result in an increased discrimination. Discussion The present study confers hs-TnI a 2b level of evidence as a diagnostic tool to predict cardiac complications in stroke. Absence of serial hs-TnI measurements and limited sample size are the main weaknesses of the study. Conclusion Patients with elevated baseline hs-TnI showed a higher frequency of cardiac complications and a higher mortality. Measurement of hs-TnI in acute stroke might be useful to identify patients at a high risk of cardiac complications and death.

The risk of recurrent stroke is likely related to etiology. Therefore it is important to identify which patients are at highest early risk. We evaluated whether selected blood biomarkers may aid in the diagnosis of stroke etiology. We studied consecutive non-lacunar stroke patients between November 2006 and January 2007, and selected undetermined origin strokes. Blood samples were drawn at arrival to test brain natriuretic peptide (BNP), D-dimer, CK-MB, myoglobin, and troponin. Second harmonic transthoracic echocardiography (SHTTE) and ECG-24 h monitoring were also performed within the first 24 h. We evaluated 294 patients with ischemic stroke; 89 had an initial undetermined origin. After a cardiological work-up, 49 were diagnosed as embolic including atrial fibrillation (4), severe aortic arch atheromatosis (24), severe wall abnormalities (12), valve disease (3), dilated cardiomyopathy (1), and patent foramen (5). Higher levels of CK-MB, BNP, and myoglobin were found in patients with embolic source in SHTTE, but only CK-MB >1.5 ng/ml and BNP >64 pg/ml remained as independent predictors: BNP (OR 8.86; CI 95 % 2.79-28.09), CK-MB (OR 6.28; CI 95 % 1.66-23.69). BNP showed specificity of 75 %, sensitivity of 63.4 %, and positive predictive value (PPV) of 75.6 %. CK-MB had specificity of 85 %, sensitivity of 47.9 %, and PPV of 79.3 %. Measuring both biomarkers improves the finding of embolic source, increasing specificity to 95 % and PPV to 88.2 %. High-level CK-MB and BNP during the acute phase of ischemic stroke are associated with an embolic source. Measurement of both biomarkers may improve the diagnosis, guiding the need to perform a heart exploration.

Background Lp-PLA2 is a novel biomarker in cardiovascular diseases due to its ability to predict first-ever and recurrent stroke. Little information is known regarding its influence on early outcome after stroke. Objectives We aimed to investigate Lp-PLA2 in t-PA-treated stroke patients and to study its relationship with early outcome. Methods Lp-PLA2 mass and activity were measured in 135 healthy controls and also in stroke patients treated with t-PA at baseline ( n =99) and serially thereafter ( n =34) by means of the PLAC test at an automated Olympus analyzer and by a colorimetric activity method (diaDexus). NIHSS scores and TCD recordings were also obtained serially. Outcome was defined according to early neurological status, the presence of arterial recanalization and functional outcome at third month. Results Lp-PLA2 mass was increased as compared to controls, whereas Lp-PLA2 activity was significantly decreased at baseline as compared with controls and with 1 and 24h determinations. Lp-PLA2 mass and activity were not related with early (48h) neurological status. Regarding recanalization, higher mass and activity were found among patients who did not achieve complete recanalization by the end of t-PA treatment ( p =0.029 for mass, p =0.044 for activity). Lp-PLA2 mass and the existence of a proximal occlusion at baseline were the most powerful predictors for persistent occlusions (OR for proximal occlusion 6.8. p =0.036, OR for Lp-PLA2 mass 7.2 per standard deviation increase, p =0.008). Conclusions Significant changes in Lp-PLA2 concentrations occur early after stroke onset. Lp-PLA2 mass may add relevant information regarding early arterial recanalization in intravenous t-PA-treated stroke patients.

Background: Lipoprotein-associated phospholipase A2 (Lp-PLA2) has emerged as a novel biomarker in cardiovascular diseases due to its ability to predict stroke in population-based studies. We aimed to investigate Lp-PLA2 levels in transient ischemic attack (TIA) patients and to study their relationship with stroke recurrence. Methods: Lp-PLA2 mass and activity were measured by means of the PLAC test with an automated Olympus analyzer and by a colorimetric activity method (diaDexus) in 166 TIA patients and 144 healthy controls. Vascular risk factors and stroke etiology were assessed. Outcome was defined as the presence of recurrent stroke/TIA within 7 and 30 days after the index TIA. Multivariate analyses were performed to identify potential predictors of recurrence. Results: Both Lp-PLA2 mass and activity (p < 0.05) were higher in TIA than in controls. Several risk factors or previous treatments were associated with Lp-PLA2 mass and activity level. During follow-up, 20 strokes/TIA (12%) occurred within the first 30 days and the presence of a large-artery atherosclerosis etiology of stroke (HR 3.28, p = 0.011), together with the past medical history of hyperlipidemia (HR 3.68, p = 0.008) and Lp-PLA2 activity of >207 nmol/ml/min (HR 2.7, p = 0.042) were all significant predictors for recurrent stroke/TIA. Conclusions: Lp-PLA2 activity might add significant prognostic information in the early evaluation of TIA patients.

Background: Vascular adhesion protein-1 (VAP-1) is a cell surface and circulating enzyme that belongs to the semicarbazide-sensitive amine oxidase (SSAO) family, which oxidatively deaminates primary amines and is implicated in leukocyte extravasation. Our aim was to investigate the alteration of soluble VAP-1/SSAO activity in plasma samples after acute intracerebral hemorrhage (ICH) and its presence in human ICH brain tissue. Methods: VAP-1/SSAO activity was determined in plasma of 66 ICH patients and 58 healthy controls. In addition, we assessed the expression of VAP-1/SSAO in postmortem brain tissue from hemorrhagic stroke patients by Western blot and immunohistochemistry. Results: We observed significantly higher levels of plasma VAP-1/SSAO activity in patients with ICH compared to matched elderly controls (p = 0.001). Plasma VAP-1/SSAO activity Conclusions: Our data demonstrate that plasma VAP-1/SSAO activity is increased in ICH and predicts neurological outcome, suggesting a possible contribution of the soluble protein in secondary brain damage. Furthermore, anti-VAP-1/SSAO strategies might be a promising approach to prevent neurological worsening following ICH.

Cerebral amyloid angiopathy (CAA) is one of the main causes of intracerebral hemorrhage (ICH) in the elderly. Matrix metalloproteinases (MMPs) have been implicated in blood-brain barrier disruption and ICH pathogenesis. In this study, we determined the levels MMP-2 and MMP-9 in plasma and their brain expression in CAA-associated hemorrhagic stroke. Although MMP-2 and MMP-9 plasma levels did not differ among patients and controls, their brain expression was increased in perihematoma areas of CAA-related hemorrhagic strokes compared with contralateral areas and nonhemorrhagic brains. In addition, MMP-2 reactivity was found in β-amyloid (Aβ)-damaged vessels located far from the acute ICH and in chronic microbleeds. MMP-2 expression was associated to endothelial cells, histiocytes and reactive astrocytes, whereas MMP-9 expression was restricted to inflammatory cells. In summary, MMP-2 expression within and around Aβ-compromised vessels might contribute to the vasculature fatal fate, triggering an eventual bleeding.

Background and purpose Circulating lipoprotein-associated phospholipase A 2 (Lp-PLA 2 ) has emerged as a novel biomarker for cardiovascular diseases. Our aim was to determine Lp-PLA 2 mass and activity in a selected cohort of first-ever transient ischemic attack (TIA) or ischemic stroke patients with intracranial atherosclerotic disease (ICAD) and to investigate its relationship with the presence of classical vascular risk factors, response to secondary prevention treatments and risk of recurrent vascular events. Methods Lp-PLA 2 mass and activity were measured 3 months after TIA or stroke by means of the PLAC test and CAM-assay (diaDexus, Inc.) respectively in 75 patients. Classic vascular risk factors, preventive treatments and clinical characteristics at the time of the index event were recorded. Follow-up transcranial Doppler ultrasonography (TCD) was performed and the presence of a new vascular event was assessed every 6 months. Results Several preventive treatments (statins and clopidogrel) were significantly associated with lower Lp-PLA 2 mass and activity. During follow-up (median time 23 months), eighteen patients (24%) suffered a new vascular event. Baseline factors associated with new vascular events were: history of coronary artery disease, number of intracranial stenoses detected by TCD and also Lp-PLA 2 activity, which was the only independent predictor for new vascular events (hazard ratio 2.89; 95% CI 1.029 to 8.096; p =0.044) after multivariate analysis (Cox regression). Conclusions Lp-PLA 2 activity might be a useful tool to identify intracranial large-artery occlusive disease patients at higher risk of suffering new vascular events.