BACKGROUND:Neutrophil granulocytes actively contribute to tissue damage after ischemic stroke. The membrane protein CD177 is detectable on variable neutrophil numbers in most individuals (CD177 wild-type [CD177WT] genotype), whereas ≈5% of the general population completely lack CD177 (CD177-deficient [CD177null] genotype). Despite its known relevance in vasculitis, the role of ischemic stroke remains unknown. METHODS:In 2 prospective cohorts of patients with first-ever ischemic stroke (PROSCIS-B [Prospective Cohort With Incident Stroke Berlin], NOFF-S [Neutrophils: Origin, Fate & Function Stroke]), we assessed the effect of CD177null and CD177WT status on stroke severity and outcome (National Institutes of Health Stroke Scale and modified Rankin Scale) over 1 year or 3 months poststroke, respectively. By flow cytometry, we stratified CD177 expression level as CD177neg, CD177dim, and CD177high. The predictive value of the CD177 state was evaluated by multivariable regression and discrimination analyses. RESULTS:In PROSCIS-B (n=579; mean age, 68.1 years; 38.5% women) and NOFF-S (n=236, 68.4 years, 36.9% women), similar rates of patients were CD177null (n=26 [4.5%] and n=10 [4.2%], respectively). Patients with CD177null had a higher probability of unfavorable stroke outcome (modified Rankin Scale score 3-6) than patients with CD177WT (n=8 of 21 [38.1%] versus 90 of 462 [19.5%] with follow-up, P=0.05, in PROSCIS-B; n=8 of 10 [80.0%] versus n=23 of 142 [16.2%] with follow-up, P<0.0001, in NOFF-S). This association remained when adjusted for age, sex, initial stroke severity defined by National Institutes of Health Stroke Scale score, stroke subtype defined by TOAST (Trial of ORG 10172 in Acute Stroke Treatment), and reperfusion treatment (risk ratio, 3.8 [95% CI, 2.0-7.1]; P<0.001, in NOFF-S). In NOFF-S, the proportion of CD177dim neutrophils at admission was negatively associated with stroke severity at admission, while that of CD177high neutrophils predicted a favorable clinical outcome after 3 months. CD177 expression level significantly improved the prediction of stroke outcome in addition to clinical adjustment variables in area under the curve, net reclassification improvement, and integrated discrimination improvement analyses (P=0.004, P=0.001, and P<0.001, respectively, for CD177high). CONCLUSIONS:CD177 expression at admission is an easy-to-measure biomarker for patient stratification. CD177 holds potential as a therapeutic target to modulate immune responses after stroke. REGISTRATION:URL: https://www.clinicaltrials.gov; Unique identifier: NCT01363856.

Abstract BACKGROUND Mesenchymal stromal cell–derived extracellular vesicles (MSC-EVs) exert neuroprotective effects in ischemic stroke largely through immunomodulatory mechanisms. Monocytes are first-line responders to MSC-EVs. Their contribution to MSC-EV-induced neuroprotection remains poorly understood. This study investigated the role of monocytes in shaping neuroprotective responses to MSC-EVs after ischemic stroke. METHODS Male C57BL/6J mice were exposed to transient middle cerebral artery occlusion (MCAO). Monocytes were depleted using pharmacological (clodronate liposomes), immunological (anti-CCR2), or genetic ( Mrp8-Cre +/– Nr4a1 fl/fl ) approaches removing total, CCR2 + , or Ly6C low monocytes, respectively. In additional cohorts, neutrophils and T cells were simultaneously depleted by anti-Ly6G or anti-CD4/CD8 antibodies. Small EVs from clonally expanded immortalized MSCs were administered intravenously. Neurological deficits, ischemic injury, and immune responses were analyzed up to 72 hours post-MCAO. Complementary ex vivo studies were performed, in which MSC-EVs were administered to monocyte-depleted or non-depleted peripheral blood mononuclear cells (PBMCs) obtained from acute ischemic stroke patients. RESULTS In ischemic mice with intact monocyte compartment, MSC-EVs reduced neurological deficits, infarct volume, neuronal injury, and brain leukocyte infiltrates. These protective effects were abolished in monocyte-depleted mice, particularly following CCR2 + monocyte depletion. Under these conditions, MSC-EV treatment exacerbated neurological deficits, ischemic injury, and leukocyte infiltration, accompanied by neutrophil and T cell expansion and overactivation. Depletion of neutrophils or T cells prevented the EV-induced worsening of stroke outcome in monocyte-deficient mice. Ly6C low monocytes played a crucial role in orchestrating immune responses to MSC-EVs. Their depletion abolished EV-induced neuroprotection. In stroke patient PBMCs, MSC-EVs induced phenotypic reprogramming of monocytes, whereas they promoted CD4 + and CD8 + T cell activation in the absence of monocytes. CONCLUSIONS Monocytes shape the immunomodulatory actions of MSC-EVs. In their absence, MSC-EVs trigger neutrophil and T cell overactivation that worsens stroke outcome. These findings highlight the importance of monocyte- and T cell-related potency assays for the clinical translation of MSC-EV therapies.

ObjectivesAcute mesenteric ischemia (AMI) is a rare disease with a relatively high mortality rate. We aimed to investigate the factors influencing in-hospital mortality in AMI patients and develop a nomogram for early risk stratification.MethodsClinical data of AMI patients hospitalized from January 2013 to October 2025 were retrospectively analyzed. Univariate and multivariate logistic regression identified independent pre-treatment risk factors, and a nomogram was constructed. Discrimination, calibration, and clinical utility were evaluated using receiver operating characteristic curve analysis, Hosmer-Lemeshow test, calibration curves, and decision curve analysis (DCA), with Bootstrap internal validation. Subgroup validation was performed by age, sex, and other subgroups. A further adjusted multivariate model examined associations of early clinical characteristics and treatments with mortality.ResultsAmong 235 patients, 44 died in hospital (18.7%). Advanced age, heart failure, higher white blood cell (WBC), and creatinine were independent predictors. The model showed good discrimination (Bootstrap area under the curve: 0.849, 95% CI: 0.780–0.909) and fit (Hosmer-Lemeshow: χ² = 10.111, P = 0.257), with good calibration and net benefit on DCA. Performance remained stable across subgroups. Further analysis that incorporated early clinical characteristics and treatment measures found that age, heart failure, WBC, aspartate aminotransferase, anticoagulation therapy, and vasopressor use were associated with mortality. Anticoagulation therapy was negatively correlated with mortality risk, whereas vasopressor use was positively correlated with mortality risk.ConclusionsThis study identified risk factors for in-hospital mortality in AMI patients and developed a predictive nomogram model. This aids in early high-risk patient identification, timely intervention, optimized treatment decisions, and improved patient outcomes.

Autophagy has crucial roles for ischemia/reperfusion (I/R) injury. To define the role of the autophagy hub protein p62/SQSTM1 in I/R injury, we conducted gain-of-function and loss-of-function experiments in a set of cell types, including two neuron-like cell lines, primary neurons, brain endothelial and astroglial-like cells, which we combined with mouse ischemic stroke studies. p62 levels post-I/R increased alongside intracellular ROS changes. p62 overexpression increased and p62 knockdown or pharmacological deactivation reduced I/R injury. Autophagic flux was p62-dependent, but oxygen-independent. Using p62 domain deletion mutants we identified p62's ZZ domain as key factor mediating autophagy and cell death. Death-promoting effects of p62 involved elevated ROS burden. At the same time, p62 activated a broad network of cytoprotective responses, which included NRF2-associated antioxidant signaling and inhibition of the pro-inflammatory NFκB pathway, which were bidirectionally linked with p62, and downregulation of the ER stress sensor BiP/GRP78 with consecutive activation of the UPR PERK branch. Our study establishes p62 as a master regulator of I/R injury, which offers itself as target for stroke therapies.

Extracellular vesicles (EVs) convey complex signals between cells that can be used to promote neuronal plasticity and neurological recovery in brain disease models. These EV signals are multimodal and context-dependent, making them unique therapeutic principles. This review analyzes how EVs released from various cell sources control neuronal metabolic function, neuronal survival and plasticity. Preferential sites of EV communication in the brain are interfaces between pre- and postsynaptic neurons at synapses, between astrocytes and neurons at plasma membranes or tripartite synapses, between oligodendrocytes and neurons at axons, between microglial cells/macrophages and neurons, and between cerebral microvascular cells and neurons. At each of these interfaces, EVs support mitochondrial function and cell metabolism under physiological conditions and orchestrate neuronal survival and plasticity in response to brain injury. In the injured brain, the promotion of neuronal survival and plasticity by EVs is tightly linked with EV actions on mitochondrial function, cell metabolism, oxidative stress and immune responses. Via the stabilization of cell metabolism and immune balance, neuronal plasticity responses are activated and functional neurological recovery is induced. As such, EV lay the ground for neuronal plasticity.

Small extracellular vesicles (sEVs) obtained from mesenchymal stromal cells (MSCs) have shown considerable promise as restorative stroke treatment. In a head-to-head comparison in mice exposed to transient proximal middle cerebral artery occlusion (MCAO), sEVs obtained from MSCs cultured under hypoxic conditions particularly potently enhanced long-term brain tissue survival, microvascular integrity, and angiogenesis. These observations suggest that hypoxic preconditioning might represent the strategy of choice for harvesting MSC-sEVs for clinical stroke trials. To test the efficacy of hypoxic MSCs in a second stroke model in an additional species, we now exposed 6–8-month-old Sprague–Dawley rats to permanent distal MCAO and intravenously administered vehicle, platelet sEVs, or sEVs obtained from hypoxic MSCs (1

Post-injury dysfunction of humoral immunity accounts for infections and poor outcomes in cardiovascular diseases. Among immunoglobulins (Ig), IgA, the most abundant mucosal antibody, is produced by plasma B cells in intestinal Peyer's patches (PP) and lamina propria. Here we show that patients with stroke and myocardial ischemia (MI) had strongly reduced IgA blood levels. This was phenocopied in experimental mouse models where decreased plasma and fecal IgA were accompanied by rapid loss of IgA-producing plasma cells in PP and lamina propria. Reduced plasma IgG was detectable in patients and experimental mice 3-10 d after injury. Stroke/MI triggered the release of neutrophil extracellular traps (NETs). Depletion of neutrophils, NET degradation or blockade of NET release inhibited the loss of IgA+ cells and circulating IgA in experimental stroke and MI and in patients with stroke. Our results unveil how tissue-injury-triggered systemic NET release disrupts physiological Ig secretion and how this can be inhibited in patients. Tuz et al. report that stroke and myocardial infarction induce the release of neutrophil extracellular traps (NETs), triggering the loss of B cells and a decrease in immunoglobulin A secretion, and that inhibition of NETs prevents the loss of immunoglobulin A in mice and in patients with stroke.

Sterile tissue injury after stroke causes lymphocyte contraction in lymphoid tissues and may decrease circulating IgA-levels. Intestinal Peyer’s patches (PP) harbor large numbers of IgA+ B cell precursors and plasma cells. Whether and how tissue injury triggers PP-B cell death, thereby mediating IgA-loss, is unknown. We found decreased circulating IgA levels in stroke and myocardial infarction patients. Experimental stroke and myocardial infarction in mice phenocopied the human situation. Decreased plasma and fecal IgA were accompanied by rapid and macroscopic shrinkage of PP caused by substantial losses of PP-resident IgA + precursors and plasma cells in mice. Tissue injury induced neutrophil activation endowed with the release of toxic neutrophil extracellular traps (NETs). Antibody-mediated or genetically-induced neutrophil loss, digestion of NETs, or inhibition of their release by the Gasdermin D blockade completely prevented lymphocyte loss and PP shrinkage. We also identified NETs in the plasma of stroke and myocardial infarction patients. Hence, tissue injury induces systemic NET-release, which might be targeted to maintain immune homeostasis at mucosal barriers.

Astrocytic responses are critical for the maintenance of neuronal networks in health and disease. In stroke, reactive astrocytes undergo functional changes potentially contributing to secondary neurodegeneration, but the mechanisms of astrocyte-mediated neurotoxicity remain elusive. Here, we investigated metabolic reprogramming in astrocytes following ischemia-reperfusion in vitro, explored their role in synaptic degeneration, and verified the key findings in a mouse model of stroke. Using indirect cocultures of primary mouse astrocytes and neurons, we demonstrate that transcription factor STAT3 controls metabolic switching in ischemic astrocytes promoting lactate-directed glycolysis and hindering mitochondrial function. Upregulation of astrocytic STAT3 signaling associated with nuclear translocation of pyruvate kinase isoform M2 and hypoxia response element activation. Reprogrammed thereby, the ischemic astrocytes induced mitochondrial respiration failure in neurons and triggered glutamatergic synapse loss, which was prevented by inhibiting astrocytic STAT3 signaling with Stattic. The rescuing effect of Stattic relied on the ability of astrocytes to utilize glycogen bodies as an alternative metabolic source supporting mitochondrial function. After focal cerebral ischemia in mice, astrocytic STAT3 activation was associated with secondary synaptic degeneration in the perilesional cortex. Inflammatory preconditioning with LPS increased astrocytic glycogen content, reduced synaptic degeneration, and promoted neuroprotection post stroke. Our data indicate the central role of STAT3 signaling and glycogen usage in reactive astrogliosis and suggest novel targets for restorative stroke therapy.

AbstractAcute myocardial infarction (AMI) is considered to be one of the most common cardiovascular complications. Recently, various research studies have shown that exosomes play a significant rolein the development and progression of cardiovascular diseases. However, there is still a lack of relevant research on the relationship between plasma exosomes and AMI. This retrospective study investigated the base date of patients with AMI(n = 20), stable angina pectoris (SAP, n = 20), and noncoronary heart disease (CON, n = 20). Proteomics was used to systematically screen the differential proteins of plasma exosomes in patients with clinical AMI, SAP, and CON. Then, the results were further verified by parallel reaction monitoring (PRM) and ELISA . Among the differential expression proteins, 5 proteins were quantified by PRM. Compared with the CON group, heparin cofactor 2 (SERPIND1), mannan-binding lectin serine protease 1 (MASP1), ficolin-2 (FCN2), and α1-Microglobulin/bikuninprecursor (AMBP)were upregulated in AMI and SAP, and they were more highly expressed in AMI than in SAP. Additionally, human leukocyte antigen (HLA-C) was found to be downregulated not only in exosomes, but also in plasma. The expression of four plasma exosomes biomarkersin AMI patients and stable angina pectoris (SAP) was higher than that in noncoronary heart disease (NCHD) patients, and HLA-C was found to be downregulated not only in exosomes, but also in plasma. The obtained results serve as anew candidate targetfor the detectionand therapyof AMI.

Background aims: Extracellular vesicles (EVs) derived from human mesenchymal stromal cells (MSCs) show immunomodulatory activity in different assays both in vitro and in vivo. In previous work, the authors com-pared the immunomodulatory potential of independent MSC-EV preparations in a multi-donor mixed lym-phocyte reaction (mdMLR) assay and an optimized steroid-refractory acute graft-versus-host disease (aGVHD) mouse model. The authors observed that only a proportion of the MSC-EV preparations showed immunomodulatory capabilities and demonstrated that only MSC-EV preparations with mdMLR immuno-modulating activities were able to suppress aGVHD symptoms in vivo and vice versa. Since the mdMLR assay is complex and depends on primary human cells of different donors, the authors sought to establish an assay that is much easier to standardize and fulfills the requirements for becoming qualified as a potency assay.Methods: The bona fide MSC antigen CD73 possesses ecto-5'-nucleotidase activity that cleaves pro-inflamma-tory extracellular adenosine monophosphate into anti-inflammatory adenosine and free phosphate. To test whether the ecto-5'-nucleotidase activity of the MSC-EV preparations reflected their immunomodulatory potential, the authors adopted an enzymatic assay that monitors the ecto-5'-nucleotidase activity of CD73 in a quantitative manner and compared the activity of well-characterized MSC-EV preparations containing or lacking mdMLR immunomodulatory activity.Results: The authors showed that the ecto-5'-nucleotidase activity of the MSC-EV preparations did not corre-late with their ability to modulate T-cell responses in the mdMLR assay and thus with their potency in improving disease symptomatology in the optimized mouse aGVHD model. Furthermore, the ecto-5'-nucleo-tidase activity was resistant to EV-destroying detergent treatment.Conclusions: Ecto-5'-nucleotidase activity neither reflects the potency of the authors' MSC-EV preparations nor provides any information about the integrity of the respective EVs. Thus, ecto-5'-nucleotidase enzyme activity is not indicative for the immunomodulatory potency of the authors' MSC-EV products. The develop-ment of appropriate potency assays for MSC-EV products remains challenging.(c) 2022 International Society for Cell & Gene Therapy. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Abstract Background The intravenous delivery of adult neural precursor cells (NPC) has shown promising results in enabling cerebroprotection, brain tissue remodeling, and neurological recovery in young, healthy stroke mice. However, the translation of cell-based therapies to clinical settings has encountered challenges. It remained unclear if adult NPCs could induce brain tissue remodeling and recovery in mice with hyperlipidemia, a prevalent vascular risk factor in stroke patients. Methods Male mice on a normal (regular) diet or on cholesterol-rich Western diet were exposed to 30 min intraluminal middle cerebral artery occlusion (MCAO). Vehicle or 106 NPCs were intravenously administered immediately after reperfusion, at 3 day and 7 day post-MCAO. Neurological recovery was evaluated using the Clark score, Rotarod and tight rope tests over up to 56 days. Histochemistry and light sheet microscopy were used to examine ischemic injury and brain tissue remodeling. Immunological responses in peripheral blood and brain were analyzed through flow cytometry. Results NPC administration reduced infarct volume, blood–brain barrier permeability and the brain infiltration of neutrophils, monocytes, T cells and NK cells in the acute stroke phase in both normolipidemic and hyperlipidemic mice, but increased brain hemorrhage formation and neutrophil, monocyte and CD4+ and CD8+ T cell counts and activation in the blood of hyperlipidemic mice. While neurological deficits in hyperlipidemic mice were reduced by NPCs at 3 day post-MCAO, NPCs did not improve neurological deficits at later timepoints. Besides, NPCs did not influence microglia/macrophage abundance and activation (assessed by morphology analysis), astroglial scar formation, microvascular length or branching point density (evaluated using light sheet microscopy), long-term neuronal survival or brain atrophy in hyperlipidemic mice. Conclusions Intravenously administered NPCs did not have persistent effects on post-ischemic neurological recovery and brain remodeling in hyperlipidemic mice. These findings highlight the necessity of rigorous investigations in vascular risk factor models to fully assess the long-term restorative effects of cell-based therapies. Without comprehensive studies in such models, the clinical potential of cell-based therapies cannot be definitely determined.

To investigate morphological predictors of neurological deterioration (ND) in patients with acute isolated pontine infarct. Acute isolated pontine infarct patients within 7 days after onset of stroke symptoms were included retrospectively and classified into ND and non-ND groups. Morphological phenotypes (paramedian pontine infarct [PPI], atypical PPI, small deep infarct, and other types), topographical location, and lesion size were evaluated on axial diffusion-weighted imaging. There were 210 eligible patients, of whom 62 patients had ND (29.5

Lymphocyte contraction (LC) in central immune organs is a concomitant of sterile tissue injury, for example after stroke. Intestinal Peyer’s patches (PP) harbor large numbers of B cells, but how sterile tissue injury leads to LC in PP has not been explored. We observed rapid and macroscopically evident shrinkage of PP after stroke and myocardial infarction. Light-sheet fluorescence microscopy and flow cytometry revealed a strong reduction in the number of PP‑resident B cells. Mechanistically, tissue injury triggered the activation of neutrophils that released B cell-toxic neutrophil extracellular traps (NETs) decorated with citrullinated histone-H3. Antibody-mediated or genetically induced neutrophil-loss, NETs-degradation or blockade of their generation completely reversed B cell loss and preserved the tissue architecture of PP. We also found NET-like elements in human post-stroke plasma. Hence, we propose that targeting NET-generation or -function counteracts post-injury B cell contraction in PP and thereby maintains immune homeostasis at mucosal barriers.

To investigate the topography and etiologies of acute cerebellar infarcts (ACIs) that presented as isolated acute vestibular syndromes (AVSs). ACI was ascertained on magnetic resonance diffusion-weighted imaging combined with apparent diffusion coefficient sequence and was categorized into the simple (territory and small infarct) and the complicated (concomitant infarcts in territories of posterior circulation besides cerebellum). Infarct topography and etiologies were compared between ACI patients with isolated AVS and non-isolated AVS (general and/or local neurological symptoms and/or signs with or without AVS). We enrolled 129 ACI patients, and 53 patients (53/129, 41.1%) had isolated AVS. In isolated AVS, the infarct lesions could be territory infarcts, small infarcts involving cortical, subcortical, and areas directly related to vestibular structures, and the primary etiologies were of large artery atherosclerosis and small vessel disease. Compared with the patients with non-isolated AVS, those with isolated AVS had more prevalence of small vessel disease (OR 6.30, 2.16–18.39; p = 0.001) and more probability of small infarcts (OR 6.04, 95%CI 2.31–15.76; p < 0.0001). In isolated AVS patients, the small infarct located more frequently in cerebellar hemispheres than the areas directly related to vestibular structures (27/35 vs 8/35), and the territory infarct located more frequently in the area supplied by posterior inferior cerebellar artery than the other areas (9/13 vs 4/13). Our study found that ACI could be presented as isolated AVS, which occurred more frequently in patients with small hemisphere infarct or infarct in the territory supplied by posterior inferior cerebellar artery.

Background and aims: The sedentary behavior in people's daily life has continued to increase in recent years, causing many studies to focus on its relationship with diseases. Several studies have shown that sedentary behavior is an independent risk factor for cardiovascular disease and metabolic disease. Therefore, we performed a meta-analysis to assess the association between sedentary behavior and the risk of stroke.Methods and results: Two independent investigators searched for prospective cohort studies on the association between sedentary behavior and stroke risk, published before February 2022. We pooled adjusted effect size and performed the dose-response analysis by random-effect model. Seven studies with 677,614 participants and 15,135 stroke events during a median follow-up of 12.2 years were included. The pooled hazard ratio (HR) of stroke was 1.16 (95% confidence interval [CI]: 1.09-1.24) with no significant heterogeneity (I2 = 0.0%, p for heterogeneity = 0.983). In dose-response analysis, a nonlinear association between sedentary behavior and stroke risk was discovered. Stroke risk began to increase when sedentary time exceeded 3.7 h/d (HR, 1.01; 95% CI, 0.97-1.05). And when reached 11 h/d, a significantly increased risk of stroke was observed (HR, 1.21; 95% CI 1.12-1.31).Conclusion: A nonlinear association was found in the dose-response analysis, with increased risk only when sedentary time exceeded a certain level. Further research is needed to explain the biological mechanisms by which sedentary time above a certain threshold significantly increases (c) 2022 Published by Elsevier B.V. on behalf of The Italian Diabetes Society, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition and the Department of Clinical Medicine and Surgery, Federico II University.

Ischemic stroke is a highly prevalent condition that frequently results in life-long disability and death. Considerable efforts have been made to establish treatments that prevent secondary ischemic damage and promote stroke recovery. Until now, the recanalization of occluded blood vessels via thrombolysis and thrombectomy, although highly potent, remains the only treatment in humans that enhances stroke outcome. Small extracellular vesicles are non-replicating, nano-sized (70–150 nm) lipid bilayer-enclosed vesicles, which have shown remarkable biological activities in various physiological and pathophysiological contexts. When administered post-stroke, mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) induce neuroprotection, promote brain remodeling and plasticity, and enhance neurological recovery in rodents and non-human primates via mechanisms that involve immunomodulation and anti-inflammation. In this review, experimental studies on the therapeutic actions of MSC-EVs in animal stroke models are summarized and perspectives for clinical translation are outlined.