Data_Sheet_1_Circulating Endothelial Cells as Promising Biomarkers in the Differential Diagnosis of Primary Angiitis of the Central Nervous System.docx

Background: Diagnosis of primary angiitis of the central nervous system (PACNS) and discrimination of PACNS from its mimics, e. g., reversible cerebral vasoconstriction syndrome (RCVS) or moyamoya disease (MMD) as non-inflammatory vasculopathies, still remain challenging. Circulating endothelial cells (CEC) are well-established markers for endothelial damage and potential biomarkers in PACNS. This study aimed to investigate if CECs may also help to distinguish an active PACNS from its important differentials (RCVS, MMD).Methods: CECs were assessed in 47 subjects. Twenty-seven patients with PACNS were included, seven with an active disease (aPACNS), 20 in remission (rPACNS). Seven patients with RCVS/MMD were analyzed. Thirteen healthy subjects served as controls (HC). CECs were measured by immunomagnetic isolation from peripheral venous blood. Mann-Whitney-U-Tests were applied for between-group comparisons. The Benjamini-Hochberg-procedure was applied to adjust for multiple comparisons.Results: In aPACNS, CECs were significantly elevated compared to HC (480 vs. 40 CEC/ml, p Conclusions: CECs may serve as biomarkers for diagnosis, treatment monitoring, and also for differential diagnosis of PACNS. CECs seem to be a marker of endothelial injury with higher levels in inflammatory than non-inflammatory vasculopathies. Larger patient samples are required to corroborate these findings.</p

Data_Sheet_2_Circulating Endothelial Cells as Promising Biomarkers in the Differential Diagnosis of Primary Angiitis of the Central Nervous System.docx

Background: Diagnosis of primary angiitis of the central nervous system (PACNS) and discrimination of PACNS from its mimics, e. g., reversible cerebral vasoconstriction syndrome (RCVS) or moyamoya disease (MMD) as non-inflammatory vasculopathies, still remain challenging. Circulating endothelial cells (CEC) are well-established markers for endothelial damage and potential biomarkers in PACNS. This study aimed to investigate if CECs may also help to distinguish an active PACNS from its important differentials (RCVS, MMD).Methods: CECs were assessed in 47 subjects. Twenty-seven patients with PACNS were included, seven with an active disease (aPACNS), 20 in remission (rPACNS). Seven patients with RCVS/MMD were analyzed. Thirteen healthy subjects served as controls (HC). CECs were measured by immunomagnetic isolation from peripheral venous blood. Mann-Whitney-U-Tests were applied for between-group comparisons. The Benjamini-Hochberg-procedure was applied to adjust for multiple comparisons.Results: In aPACNS, CECs were significantly elevated compared to HC (480 vs. 40 CEC/ml, p Conclusions: CECs may serve as biomarkers for diagnosis, treatment monitoring, and also for differential diagnosis of PACNS. CECs seem to be a marker of endothelial injury with higher levels in inflammatory than non-inflammatory vasculopathies. Larger patient samples are required to corroborate these findings.</p

Deficiency in Serine Protease Inhibitor Neuroserpin Exacerbates Ischemic Brain Injury by Increased Postischemic Inflammation

<div><p>The only approved pharmacological treatment for ischemic stroke is intravenous administration of plasminogen activator (tPA) to re-canalize the occluded cerebral vessel. Not only reperfusion but also tPA itself can induce an inflammatory response. Microglia are the innate immune cells of the central nervous system and the first immune cells to become activated in stroke. Neuroserpin, an endogenous inhibitor of tPA, is up-regulated following cerebral ischemia. To examine neuroserpin-dependent mechanisms of neuroprotection in stroke, we studied neuroserpin deficient (<i>Ns^−/−</i>) mice in an animal model of temporal focal ischemic stroke. Infarct size and neurological outcome were worse in neuroserpin deficient mice even though the fibrinolytic activity in the ischemic brain was increased. The increased infarct size was paralleled by a selective increase in proinflammatory microglia activation in <i>Ns^−/−</i> mice. Our results show excessive microglial activation in <i>Ns^−/−</i> mice mediated by an increased activity of tPA. This activation results in a worse outcome further underscoring the potential detrimental proinflammatory effects of tPA.</p></div

Total number of subtests with PR<15 (18 participants).

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103029#pone-0103029-t005" target="_blank">Table 5</a> shows the total number of subtests with poor performance (PR<15) for each neuropsychological domain for both time points- 3months and 1 year after acute disease. Only patients who accomplished the assessment at both time points were included (18 of 20 patients, as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103029#pone-0103029-t004" target="_blank">table 4</a>).</p

Laboratory data.

<p>CRP – C-reactive protein;</p><p>LDH – Lactate dehydrogenase,</p><p><i>p</i> – p Value,</p><p>*- statistically significant.</p

Characteristics of the study patients.

<p>*- statistically significant.</p

Activation of microglia is increased in the absence of neuroserpin.

<p>(<b>A</b>) Absolute numbers of brain microglia in the ischemic hemisphere of wild type, and <i>Ns^−/−</i> mice 3 days after MCAO. Cell counts were determined by flow cytometry analysis of the CNS-infiltrating cells using TrueCount tubes. Brain microglia cells were identified as CD11b⁺ CD45^intermediate. Representative dot plots show CD11b⁺ CD45^high and CD11b⁺ CD45^intermediate-gated populations identifying macrophages and microglia respectively. The graphs show means±SD of 9–12 animals per group analyzed three days after MCAO in three or four independent experiments. <i>t</i> test was used to assess statistical significance. (<b>B</b>) Immunohistochemical analysis of absolute numbers of Iba-1 positive brain microglia/macrophages in the ischemic hemisphere of wild type, and <i>Ns^−/−</i> mice 3 days after MCAO. The graphs show means±SD of 3 animals per group. <i>t</i> test was used to assess statistical significance. (<b>C</b>) Immunohistochemical analysis of the activation state (resting, bushy and amoeboid) of Iba-1 positive microglia in the ipsilesional hippocampus and penumbra area and contralesional hippocampus area 3 days following 1 h MCAO. The graphs show means±SD of 3 animals per group and the statistical analysis was assessed using one-way ANOVA with Bonferroni post hoc test (scale bar = 20 µm).</p

Deficiency in neuroserpin is detrimental in stroke.

<p>(<b>A</b>) TTC staining for evaluation of infarct volume at day three (left panel) and (<b>B</b>) neurological scores at days one and three (right panel) of wt and <i>Ns^−/−</i> mice after MCAO. Data are represented as means±SD of 10 wt and eight <i>Ns^−/−</i> animals. <i>t</i> test was used to assess statistical significance for infarct sizes and Mann-Whitney U test for neurological scores. (<b>C</b>) Survival rate of wt (n = 13) and <i>Ns^−/−</i> mice (n = 13). Survival was analyzed by the <i>χ</i>² test (survival rate). (<b>D</b>) In all mice subjected to MCAO, regional cerebral blood flow (rCBF) was measured with Laser Doppler. The decrease in rCBF of approximately 90% was similar between <i>Ns^−/−</i> and wt mice. Ten minutes after reperfusion rCBF was reconstituted to at least 60% of baseline levels and was unaltered between <i>Ns^−/−</i> and wt animals. (<b>E</b>) Accumulation of fibrin(ogen) in the infarcted and in the contralesional hemispheres of wt (n = 3) and <i>Ns^−/−</i> mice (n = 3). Fibrin(ogen) formation was analyzed by immunoblotting following fixation with 4% PFA (upper panel) or w/o 4% PFA-fixation (lower panel) using a rabbit polyclonal fibrin/fibrinogen-specific antibody 24 h following ischemia. Asterisks indicate additional bands representing fibrin degradation. <i>t</i> test was used to assess statistical significance.</p

Neuropsychological domains and tests.

<p>Neuropsychological domains and tests.</p

Number of subtests with PR<15.

<p>n.c. =  not controlled.</p><p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103029#pone-0103029-t004" target="_blank">Table 4</a> depicts the number of subtests with poor performance (≤15 PR) for each neuropsychological domain and every single patient for both time points: 1. evaluation - 3months and 2. evaluation - 1 year after acute disease. In the second evaluation only selected tests with under average performance (≤15 PR) in the first assessment were repeated. All not-repeated subtests with scores >15 PR in the first testing are considered as <i>0</i> in the second one.</p