Increased CX3CL1 in cerebrospinal fluid and ictal serum t-tau elevations in migraine: results from a cross-sectional exploratory case-control study.

BACKGROUND: To date, migraine is diagnosed exclusively based on clinical criteria, but fluid biomarkers are desirable to gain insight into pathophysiological processes and inform clinical management. We investigated the state-dependent profile of fluid biomarkers for neuroaxonal damage and microglial activation as two potentially relevant aspects in human migraine pathophysiology. METHODS: This exploratory study included serum and cerebrospinal fluid (CSF) samples of patients with migraine during the headache phase (ictally) (n = 23), between attacks (interictally) (n = 16), and age/sex-matched controls (n = 19). Total Tau (t-Tau) protein, glial fibrillary acidic protein (GFAP), ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), and neurofilament light chain (NfL) were measured with the Neurology 4-plex kit on a Single Molecule Array SR-X Analyzer (Simoa® SR-X, Quanterix Corp., Lexington, MA). Markers of microglial activation, C-X3-C motif chemokine ligand 1 (CX3CL1) and soluble triggering receptor expressed on myeloid cells 2 (sTREM2), were assessed using an immunoassay. RESULTS: Concentrations of CX3CL1 but not sTREM2 were significantly increased both ictally and interictally in CSF but not in serum in comparison to the control cohort (p = 0.039). ROC curve analysis provided an AUC of 0.699 (95% CI 0.563 to 0.813, p = 0.007). T-Tau in serum but not in CSF was significantly increased in samples from patients taken during the headache phase, but not interictally (effect size: η2 = 0.121, p = 0.038). ROC analysis of t-Tau protein in serum between ictal and interictal collected samples provided an AUC of 0.729 (95% CI 0.558 to 0.861, p = 0.006). The other determined biomarkers for axonal damage were not significantly different between the cohorts in either serum or CSF. DISCUSSION: CX3CL1 in CSF is a novel potential fluid biomarker of migraine that is unrelated to the headache status. Serum t-Tau is linked to the headache phase but not interictal migraine. These data need to be confirmed in a larger hypothesis-driven prospective study.

Effects of body mass index on the immune response within the first days after major stroke in humans.

INTRODUCTION: Immunological alterations associated with increased susceptibility to infection are an essential aspect of stroke pathophysiology. Several immunological functions of adipose tissue are altered by obesity and are accompanied by chronic immune activation. The purpose of this study was to examine immune function (monocytes, granulocytes, cytokines) as a function of body mass index (BMI: 1st group: 25; 2nd group: 25 BMI 30; 3rd group: 30) and changes in body weight post stroke. METHOD: Fat status was assessed using standardized weight measurements on days 1, 2, 3, 4, 5, and 7 after ischemic stroke in a cohort of 40 stroke patients and 16 control patients. Liver fat and visceral fat were assessed by MRI on day 1 or 2 [I] and on day 5 or 7 [II]. Leukocyte subpopulations in peripheral blood, cytokines, chemokines, and adipokine concentrations in sera were quantified. In a second cohort (stroke and control group, n = 17), multiple regression analysis was used to identify correlations between BMI and monocyte and granulocyte subpopulations. RESULTS: Weight and fat loss occurred from the day of admission to day 1 after stroke without further reduction in the postischemic course. No significant changes in liver or visceral fat were observed between MRI I and MRI II. BMI was inversely associated with IL-6 levels, while proinflammatory cytokines such as eotaxin, IFN-ß, IFN -γ and TNF-α were upregulated when BMI increased. The numbers of anti-inflammatory CD14+CD16+ monocytes and CD16+CD62L- granulocytes were reduced in patients with higher BMI values, while that of proinflammatory CD16dimCD62L+ granulocytes was increased. CONCLUSION: A small weight loss in stroke patients was detectable. The data demonstrate a positive correlation between BMI and a proinflammatory poststroke immune response. This provides a potential link to how obesity may affect the clinical outcome of stroke patients.

Serum Biomarkers of a Pro-Neuroinflammatory State May Define the Pre-Operative Risk for Postoperative Delirium in Spine Surgery.

Advances in spine surgery enable technically safe interventions in older patients with disabling spine disease, yet postoperative delirium (POD) poses a serious risk for postoperative recovery. This study investigates biomarkers of pro-neuroinflammatory states that may help objectively define the pre-operative risk for POD. This study enrolled patients aged ≥60 scheduled for elective spine surgery under general anesthesia. Biomarkers for a pro-neuroinflammatory state included S100 calcium-binding protein ß (S100ß), brain-derived neurotrophic factor (BDNF), Gasdermin D, and the soluble ectodomain of the triggering receptor expressed on myeloid cells 2 (sTREM2). Postoperative changes of Interleukin-6 (IL-6), Interleukin-1ß (IL-1ß), and C-reactive protein (CRP) were assessed as markers of systemic inflammation preoperatively, intraoperatively, and early postoperatively (up to 48 h). Patients with POD (n = 19, 75.7 ± 5.8 years) had higher pre-operative levels of sTREM2 (128.2 ± 69.4 pg/mL vs. 97.2 ± 52.0 pg/mL, p = 0.049) and Gasdermin D (2.9 ± 1.6 pg/mL vs. 2.1 ± 1.4 pg/mL, p = 0.29) than those without POD (n = 25, 75.6 ± 5.1 years). STREM2 was additionally a predictor for POD (OR = 1.01/(pg/mL) [1.00-1.03], p = 0.05), moderated by IL-6 (Wald-χ2 = 4.06, p = 0.04). Patients with POD additionally showed a significant increase in IL-6, IL-1ß, and S100ß levels on the first postoperative day. This study identified higher levels of sTREM2 and Gasdermin D as potential markers of a pro-neuroinflammatory state that predisposes to the development of POD. Future studies should confirm these results in a larger cohort and determine their potential as an objective biomarker to inform delirium prevention strategies.

Modulation of cytokine release from peripheral blood mononuclear cells from multiple sclerosis patients by coenzyme A and soraphen A.

By applying the acetyl-CoA-carboxylase inhibitors soraphen A (SorA) and coenzyme A (CoA) ex vivo, we aimed to reduce proinflammatory cytokine release by PBMCs and increase anti-inflammatory cytokine levels, thereby demonstrating a possible application of those pathways in future multiple sclerosis (MS) therapy. In a prospective exploratory monocentric study, we analysed cytokine production by PBMCs treated with SorA (10 or 50 nM) and CoA (600 µM). Thirty-one MS patients were compared to 18 healthy age-matched controls. We demonstrated the immunomodulatory potential of SorA and CoA in targeting the immune function of MS patients, with an overall reduction of cytokines except of IL-2, IL-6 and IL-10.

Duration of Surgery and Intraoperative Blood Pressure Management Are Modifiable Risk Factors for Postoperative Neurocognitive Disorders After Spine Surgery: Results of the Prospective CONFESS Study.

STUDY DESIGN: Prospective quasi-experimental observational study. OBJECTIVE: The objective of this study was to evaluate whether duration of surgery is a modifiable risk factor for postoperative delirium (POD) after spine surgery and explore further modifiable risk factors. In addition, we sought to investigate the association between POD and postoperative cognitive dysfunction and persistent neurocognitive disorders. SUMMARY OF BACKGROUND DATA: Advances in spine surgery enable technically safe interventions in elderly patients with disabling spine disease. The occurrence of POD and delayed neurocognitive complications ( e.g. postoperative cognitive dysfunction/persistent neurocognitive disorder) remain a concern since these contribute to inferior functional outcomes and long-term care dependency after spine surgery. MATERIALS AND METHODS: This prospective single-center study recruited patients aged 60 years or above and scheduled for elective spine surgery between February 2018 and March 2020. Functional (Barthel Index, BI) and cognitive outcomes [Consortium to Establish a Registry for Alzheimer's Disease (CERAD) test battery; telephone Montréal Cognitive Assessment] were assessed at baseline, three (V3), and 12 months postoperatively. The primary hypothesis was that the duration of surgery predicts POD. Multivariable predictive models of POD included surgical and anesthesiological parameters. RESULTS: Twenty-two percent of patients developed POD (n=22/99). In a multivariable model, duration of surgery [OR adj =1.61/h (95% CI, 1.20-2.30)], age [OR adj =1.22/yr (95% CI, 1.10-1.36)], and baseline deviations of intraoperative systolic blood pressure [25th percentile: OR adj =0.94/mm Hg (95% CI, 0.89-0.99); 90th percentile: OR adj =1.07/mm Hg (95% CI, 1.01-1.14)] were significantly associated with POD. Postoperative cognitive scores generally improved (V3, ΔCERAD total z -score: 0.22±0.63). However, this positive group effect was counteracted by POD [beta: -0.87 (95% CI, -1.31 to 0.42)], older age [beta: -0.03/yr (95% CI, -0.05 to 0.01)], and lack of functional improvement [ΔBI; beta: -0.04/point (95% CI, -0.06 to 0.02)]. Cognitive scores at twelve months remained inferior in the POD group, adjusted for baseline cognition/age. CONCLUSIONS: This study identified distinct neurocognitive effects after spine surgery, which are influenced by perioperative risk factors. Potential cognitive benefits are counteracted by POD, rendering its prevention critical in an aging population.

Predictors of post-stroke delirium incidence and duration: Results of a prospective observational study using high-frequency delirium screening.

BACKGROUND: Post-stroke delirium (PSD) is a modifiable predictor for worse outcome in stroke. Knowledge of its risk factors would facilitate clinical management of affected patients, but recently updated national guidelines consider available evidence insufficient. AIMS: The study aimed to establish risk factors for PSD incidence and duration using high-frequency screening. METHODS: We prospectively investigated patients with ischemic stroke admitted within 24 h. Patients were screened twice daily for the presence of PSD throughout the treatment period. Sociodemographic, treatment-related, and neuroimaging characteristics were evaluated as predictors of either PSD incidence (odds ratios (OR)) or duration (PSD days/unit of the predictor, b), using logistic and linear regression models, respectively. RESULTS: PSD occurred in 55/141 patients (age = 73.8 ± 10.4 years, 61 female, National Institutes of Health Stroke Scale (NIHSS) = 6.4 ± 6.5). Age (odds ratio (OR) = 1.06 (95% confidence interval (CI): 1.02-1.10), b = 0.08 (95% CI = 0.04-0.13)), and male gender (b = 0.99 (95% CI = 0.05-1.93)) were significant non-modifiable risk factors. In a multivariable model adjusted for age and gender, presence of pain (OR < sub > mvar = 1.75 (95% CI = 1.12-2.74)), urinary catheter (OR < sub > mvar = 3.16 (95% CI = 1.10-9.14)) and post-stroke infection (PSI; OR < sub > mvar = 4.43 (95% CI = 1.09-18.01)) were predictors of PSD incidence. PSD duration was impacted by presence of pain (b < sub > mvar = 0.49 (95% CI = 0.19-0.81)), urinary catheter (b < sub > mvar = 1.03 (95% CI = 0.01-2.07)), intravenous line (b < sub > mvar = 0.36 (95% CI = 0.16-0.57)), and PSI (b < sub > mvar = 1.60 (95% CI = 0.42-2.78)). PSD (OR = 3.53 (95% CI = 1.48-5.57)) and PSI (OR = 5.29 (95% CI = 2.92-7.66)) independently predicted inferior NIHSS at discharge. Insular and basal ganglia lesions increased the PSD risk about four- to eight-fold. DISCUSSION/CONCLUSION: This study identified modifiable risk factors, the management of which might reduce the negative impact PSD has on outcome.

Preterm ETs Are Significantly Reduced Compared with Adults and Partially Reduced Compared with Term Infants.

The release of DNA by cells during extracellular trap (ET) formation is a defense function of neutrophils and monocytes. Neutrophil ET (NET) formation in term infants is reduced compared to adults. Objective: The aim was to quantify NET and monocyte ET (MET) release and the respective key enzymes myeloperoxidase (MPO) and neutrophil elastase (NE) in preterm infants. In this prospective explorative study, ET induction was stimulated by N-formylmethionine-leucyl-phenylalanine (fMLP), phorbol 12-myristate 13-acetate (PMA), lipopolysaccharide (LPS), and lipoteichoic acid (LTA) in the cord blood of preterm infants (n = 55, 23-36 weeks) compared to term infants and adults. METs were quantified by microscopy, and NETs by microscopy and flow cytometry. We also determined the MPO levels within NETs and the intracellular concentrations of NE and MPO in neutrophils. The percentage of neutrophils releasing ET was significantly reduced for preterm infants compared to adults for all stimulants, and with a 68% further reduction for PMA compared to term infants (p = 0.0141). The NET area was not reduced except for when fMLP was administered. The amount of MPO in NET-producing cells was reduced in preterm infants compared to term infants. For preterm infants, but not term infants, the percentage of monocytes releasing ETs was significantly reduced compared to healthy adults for LTA and LPS stimulation. Conclusion: In preterm infants, ETs are measurable parts of the innate immune system, but are released in a reduced percentage of cells compared to adults.

Neurotrophin-3 attenuates human peripheral blood T cell and monocyte activation status and cytokine production post stroke.

BACKGROUND AND PURPOSE: Stroke therapy still lacks successful measures to improve post stroke recovery. Neurotrophin-3 (NT-3) is one promising candidate which has proven therapeutic benefit in motor recovery in acute experimental stroke. Post stroke, the immune system has opposing pathophysiological roles: pro-inflammatory cascades and immune cell infiltration into the brain exacerbate cell death while the peripheral immune response has only limited capabilities to fight infections during the acute and subacute phase. With time, anti-inflammatory mechanisms are supposed to support recovery of the ischemic damage within the brain parenchyma. However, interestingly, NT-3 can improve recovery in chronic neurological injury when combined with the pro-inflammatory stimulus lipopolysaccharide (LPS). AIM: We elucidated the impact of NT-3 on human monocyte and T cell activation as well as cytokine production ex vivo after stroke. In addition, we investigated the age-dependent availability of the high affinity NT-3 receptor TrkC upon LPS stimulation. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from acute stroke patients and controls and incubated with different dosages of NT-3 (10 and 100 ng/mL) and with or without LPS or anti-CD3/CD28 for 48 h. Total TrkC expression and cell activation (CD25, CD69 and HLA-DR) were assessed by FACS staining. IFN-γ, TNF-α, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A, IL-17F, IL-21 and IL-22 were quantified by cytometric bead array. RESULTS: Most monocytes and only a small proportion of T cells expressed TrkC in blood from humans without stroke. Activation of cells from young humans (without strokes) using anti-CD3/CD28 or LPS partially reduced the proportion of monocytes expressing TrkC whilst they increased the proportion of T cells expressing TrkC. In contrast, activation of cells from elderly humans (without strokes) did not affect the proportion of monocytes expressing TrkC and only anti-CD3/CD28 led to an increase in the proportion of CD4+ T cells expressing TrkC. In blood from stroke patients or controls, NT-3 treatment reduced the percentage of monocytes and CD4+ and CD8+ T cells that were activated and reduced all cytokines investigated besides IL-21. CONCLUSIONS: NT-3 attenuated immune responses in cells from stroke patients and controls. The mechanism whereby human immune cells respond to NT-3 may be via TrkC receptors whose levels are regulated by stimulation. Further work is required to determine whether the induction of sensorimotor recovery in rodents by NT-3 after CNS injury is caused by this attenuation of the immune response.

Impact of Gestational and Postmenstrual Age on Excretion of Fetal Zone Steroids in Preterm Infants Determined by Gas Chromatography-Mass Spectrometry.

CONTEXT: Fetal zone steroids (FZSs) are excreted in high concentrations in preterm infants. Experimental data suggest protective effects of FZSs in models of neonatal disease. OBJECTIVE: We aimed to characterize the postnatal FZS metabolome of well preterm and term infants. METHODS: Twenty-four-hour urinary FZS excretion rates were determined in early preterm (<30 weeks' gestation), preterm (30-36 weeks), and term (>37 weeks) infants. Pregnenolone and 17-OH-pregnenolone metabolites (n = 5), and dehydroepiandrosterone sulfate and metabolites (n = 12) were measured by gas chromatography mass spectrometry. Postnatal concentrations of FZSs were compared with already published prenatal concentrations in amniotic fluid. RESULTS: Excretion rates of total FZSs and most of the single metabolites were highest in early preterm infants. In this group, excretion rates approach those of term infants at term equivalent postmenstrual age. Preterm infants of 30-36 weeks had more than half lower median excretion rates of FZSs than early preterm infants at the same time of postmenstrual age. Postnatal concentrations of FZSs were partly more than 100-fold higher in all gestational age groups than prenatal concentrations in amniotic fluid at midgestation. CONCLUSION: The excretion rates of FZSs as a proxy of the involution of the fetal zone of the most immature preterm infants approached those of term infants at term equivalent. In contrast, the fetal zone in more mature preterm infants undergoes more rapid involution. These data in exclusively well neonates can serve as a basis to investigate the effects of illness on the FZS metabolome in future studies.

The neuroendocrine effects of dehydroepiandrosterone and 17ß-estradiol in the in vitro preterm hyperoxia infant model.

Preterm birth causes neurological deficits. Previously, we demonstrated that fetal zone steroids reduce hyperoxia-mediated cell death in vitro. In immature oligodendrocytes (OLN-93 cells), dehydroepiandrosterone + 17ß-estradiol co-treatment had synergistic beneficial effects while signals were transduced through different receptors. In immature astrocytes (C6 cells), both hormones compete for the same receptor and no synergistic effects were observed. 17ß-estradiol and progesterone drastically decrease while fetal zone steroids, mainly dehydroepiandrosterone, remain persistently high within preterm infants until term. Substitution of 17ß-estradiol and progesterone does not improve neurological outcomes. We investigated the influence of dehydroepiandrosterone, 17ß-estradiol or dehydroepiandrosterone + 17ß-estradiol treatment in C6 or OLN-93 cells on steroid receptor availability and activation of intracellular signaling molecules in hyperoxic cell culture. We sought explanations of the observed synergistic effect in preliminary study. In C6 cells, the generated signaling of dehydroepiandrosterone + 17ß-estradiol treatment has no synergistic effects. The combined effect on this particular pathway does not potentiate cell survival. In OLN-93 cells, we observed significant differences in the early generated signaling of 17ß-estradiol + dehydroepiandrosterone treatment to either 17ß-estradiol dehydroepiandrosterone alone but never to both at the same time. The latter finding needs, therefore, further investigation to explain synergistic effects. Nevertheless, we add insight into the receptor and signaling cascade alterations induced by 17ß-estradiol, dehydroepiandrosterone or 17ß-estradiol + dehydroepiandrosterone treatment of C6 and OLN-93 cells in hyperoxia.

Stroke-Induced Modulation of Myeloid-Derived Suppressor Cells (MDSCs) and IL-10-Producing Regulatory Monocytes.

Background: Stroke patients are at risk of acquiring secondary infections due to stroke-induced immune suppression (SIIS). Immunosuppressive cells comprise myeloid-derived suppressor cells (MDSCs) and immunosuppressive interleukin 10 (IL-10)-producing monocytes. MDSCs represent a small but heterogeneous population of monocytic, polymorphonuclear (or granulocytic), and early progenitor cells ("early" MDSC), which can expand extensively in pathophysiological conditions. MDSCs have been shown to exert strong immune-suppressive effects. The role of IL-10-producing immunosuppressive monocytes after stroke has not been investigated, but monocytes are impaired in oxidative burst and downregulate human leukocyte antigen-DR isotype (HLA-DR) on the cell surface. Objectives: The objective of this work was to investigate the regulation and function of MDSCs as well as the immunosuppressive IL-10-producing monocytes in experimental and human stroke. Methods: This longitudinal, monocentric, non-interventional prospective explorative study used multicolor flow cytometry to identify MDSC subpopulations and IL-10 expression in monocytes in the peripheral blood of 19 healthy controls and 27 patients on days 1, 3, and 5 post-stroke. Quantification of intracellular STAT3p and Arginase-1 by geometric mean fluorescence intensity was used to assess the functionality of MDSCs. In experimental stroke induced by electrocoagulation in middle-aged mice, monocytic (CD11b+Ly6G-Ly6Chigh) and polymorphonuclear (CD11b+Ly6G+Ly6Clow) MDSCs in the spleen were analyzed by flow cytometry. Results: Compared to the controls, stroke patients showed a relative increase in monocytic MDSCs (percentage of CD11b+ cells) in whole blood without evidence for an altered function. The other MDSC subgroups did not differ from the control. Also, in experimental stroke, monocytic, and in addition, polymorphonuclear MDSCs were increased. The numbers of IL-10-positive monocytes did not differ between the patients and controls. However, we provide a new insight into monocytic function post-stroke since we can report that a differential regulation of HLA-DR and PD-L1 was found depending on the IL-10 production of monocytes. IL-10-positive monocytes are more activated post-stroke, as indicated by their increased HLA-DR expression. Conclusions: MDSC and IL-10+ monocytes can induce immunosuppression within days after stroke.

Corrigendum: Clinical Improvement Following Stroke Promptly Reverses Post-stroke Cellular Immune Alterations.

[This corrects the article DOI: 10.3389/fneur.2019.00414.].

Impact of Storage Conditions on the Breast Milk Peptidome.

Human donor milk (HDM) provides appropriate nutrition and offers protective functions in preterm infants. The aim of the study is to examine the impact of different storage conditions on the stability of the human breast milk peptidome. HDM was directly frozen at -80 °C or stored at -20 °C (120 h), 4 °C (6 h), or room temperature (RT for 6 or 24 h). The milk peptidome was profiled by mass spectrometry after peptide collection by ultrafiltration. Profiling of the peptidome covered 3587 peptides corresponding to 212 proteins. The variance of the peptidome increased with storage temperature and time and varied for different peptides. The highest impact was observed when samples were stored at RT. Smaller but significant effects were still observed in samples stored at 4 °C, while samples showed highest similarity to those immediately frozen at -80 °C when stored at -20 °C. Peptide structures after storage at RT for 24 h point to the increased activity of thrombin and other proteases cleaving proteins at lysine/arginine. The results point to an ongoing protein degradation/peptide production by milk-derived proteases. They underline the need for immediate freezing of HDM at -20 °C or -80 °C to prevent degradation of peptides and enable reproducible investigation of prospectively collected samples.

Spermine and spermidine modulate T-cell function in older adults with and without cognitive decline ex vivo.

The global increase in neurodegenerative disorders is one of the most crucial public health issues. Oral polyamine intake was shown to improve memory performance which is thought to be mediated at least in part via increased autophagy induced in brain cells. In Alzheimer's Disease, T-cells were identified as important mediators of disease pathology. Since autophagy is a central regulator of cell activation and cytokine production, we investigated the influence of polyamines on T-cell activation, autophagy, and the release of Th1/Th2 cytokines from blood samples of patients (n=22) with cognitive impairment or dementia in comparison to healthy controls (n=12) ex vivo. We found that spermine downregulated all investigated cytokines in a dose-dependent manner. Spermidine led to an upregulation of some cytokines for lower dosages, while high dosages downregulated all cytokines apart from upregulated IL-17A. Autophagy and T-cell activation increased in a dose-dependent manner by incubation with either polyamine. Although effects in patients were seen in lower concentrations, alterations were similar to controls.We provide novel evidence that supplementation of polyamines alters the function of T-cells. Given their important role in dementia, these data indicate a possible mechanism by which polyamines would help to prevent structural and cognitive decline in aging.

Immune Alterations Following Neurological Disorders: A Comparison of Stroke and Seizures.

Background: Granulocytes and monocytes are the first cells to invade the brain post stroke and are also being discussed as important cells in early neuroinflammation after seizures. We aimed at understanding disease specific and common pathways of brain-immune-endocrine-interactions and compared immune alterations induced by stroke and seizures. Therefore, we compared granulocytic and monocytic subtypes between diseases and investigated inflammatory mediators. We additionally investigated if seizure type determines immunologic alterations. Material and Methods: We included 31 patients with acute seizures, 17 with acute stroke and two control cohorts. Immune cells were characterized by flow cytometry from blood samples obtained on admission to the hospital and the following morning. (i) Monocytes subpopulations were defined as classical (CD14++CD16-), (ii) intermediate (CD14++CD16+), and (iii) non-classical monocytes (CD14dimCD16+), while granulocyte subsets were characterized as (i) "classical granulocytes" (CD16++CD62L+), (ii) pro-inflammatory (CD16dimCD62L+), and (iii) anti-inflammatory granulocytes (CD16++CD62L-). Stroke patient's blood was additionally drawn on days 3 and 5. Cerebrospinal fluid mitochondrial DNA was quantified by real-time PCR. Plasma High-Mobility-Group-Protein-B1, metanephrine, and normetanephrine were measured by ELISA. Results: HLA-DR expression on monocytes and their subpopulations (classical, intermediate, and non-classical monocytes) was reduced after stroke or seizures. Expression of CD32 was increased on monocytes and subtypes in epilepsy patients, partly similar to stroke. CD32 and CD11b regulation on granulocytes and subpopulations (classical, anti-inflammatory, pro-inflammatory granulocytes) was more pronounced after stroke compared to seizures. On admission, normetanephrine was upregulated in seizures, arguing for the sympathetic nervous system as inducer of immune alterations similar to stroke. Compared to partial seizures, immunologic changes were more pronounced in generalized tonic-clonic seizures. Conclusion: Seizures lead to immune alterations within the immediate postictal period similar but not identical to stroke. The type of seizures determines the extent of immune alterations.

Functional analysis of granulocyte and monocyte subpopulations in neonates.

BACKGROUND: Neonate immune cell functions lack full protection against pathogens. This could be either defect or protective mechanism against overshooting proinflammatory immune responses. We here analysed the function of classical, pro- and anti-inflammatory monocytes and granulocytes from neonates in comparison with adults to investigate if suppressed functions of subpopulations are causative for the unique neonatal immune status. Therefore, reactive oxygen species (ROS) and surface activation markers were quantified in subpopulations. METHODS: In a prospective, longitudinal study granulocyte and monocyte subpopulations were analysed in healthy term infants (> 37 week; n = 13) in comparison with healthy young adults (n = 11). Percentage (%) of cells expressing surface marker (HLA-DR, CD11b, CD62L, CD32, Toll-Like-Receptor-2) and expression per cell, determined by mean fluorescence intensity (MFI), were measured by flow cytometry. ROS production was induced by fMLP, PMA and E. coli in term neonates (> 37 week; n = 13). RESULTS: Classical granulocytes were down- and proinflammatory granulocytes upregulated in neonates compared with adults. Percentage of TLR-2 expressing granulocytes was increased in neonates. Granulocytic ROS production depended on stimulation. The percentage of anti-inflammatory monocytes was increased, while classical monocytes were reduced in neonates. HLA-DR (%, MFI) showed reduction for all monocyte subpopulations, while CD32, CD11b, CD62L and TLR-2 were differently regulated in comparison with adults. CONCLUSIONS: Differentially regulated granulocyte and monocyte subpopulations indicate a unique state of neonatal immunity to fight infections and prevent dysregulation. Further studies are needed to investigate the role of reduced granulocytic ROS formation and reduced monocytic HLA-DR in active disease.

Clinical Improvement Following Stroke Promptly Reverses Post-stroke Cellular Immune Alterations.

Background and Purpose: Stroke induces immediate profound alterations of the peripheral immune system rendering patients more susceptible to post-stroke infections. The precise mechanisms maintaining stroke-induced immune alterations (SIIA) remain unknown. High-Mobility-Group-Protein B1 (HMGB-1) is elevated for at least 7 days post-stroke and has been suggested to mediate SIIA. Patients with rapid clinical recovery of neurological deficits rarely develop severe infections. We therefore investigated whether rapid neurological recovery (either spontaneous or secondary to neurovascular recanalization therapy) alters the course of SIIA. National Institutes of Health Stroke Scale (NIHSS) served as surrogate marker for neurological improvement. Methods: Fluorescence-activated cell sorting was used to define leukocyte subpopulations. C-reactive protein (CRP), procalcitonin (PCT), HMGB-1, GM-CSF; IFN-ß, IFN-γ, IL-1ß, IL-1RA, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-17, IL-17F, IL-18, TNF-α, MIF, IL-8, MCP-1, MCP-4, MIP-3α, MIP-3ß, Eotaxin, soluble IL-6 receptor, E-selectin, and P-selectin were analyzed by ELISA or Multiplex Assays. Serum miRNA expression changes were analyzed by qPCR. Results: Cellular parameters were similar in the improved and non-improved cohort on admission. In patients with rapid clinical recovery absolute and relative leukocyte, neutrophil, and lymphocyte numbers normalized promptly overnight. In contrast, HMGB-1 serum levels did not differ between the two groups. Nine miRNA were found to be differentially expressed between improved and non-improved patients. Conclusions: SIIA are detectable on admission of acute stroke patients. While it was assumed that post-stroke immunosuppression is rapidly reversed with improvement this is the first data set that shows that improvement actually is associated with a rapid reversal of SIIA demonstrating that SIIA require a constant signal to persist. The observation that HMGB-1 serum concentrations were similar in improved and non-improved cohorts argues against a role for this pro-inflammatory mediator in the maintenance of SIIA. Serum miRNA observed to be regulated in stroke in other publications was counter regulated with improvement in our cohort.

Intrathecal inflammation in young stroke.

OBJECTIVES: Correct identification of inflammatory etiologies of stroke is of outmost importance as they require treatment of the underlying disease. Aim of this study was to determine the prevalence of inflammatory changes in cerebrospinal fluid (CSF) observed in young cryptogenic stroke patients. MATERIALS AND METHODS: Of 6476 records of patients diagnosed with ischemic stroke, 278 had confirmed ischemia in brain imaging and received lumbar puncture. A total of 122 were classified as young stroke (≤55 years), and 156 were classified as older stroke patients; lumbar puncture in this cohort was indicated due to atypical clinical presentation. RESULTS: An infectious etiology was detected in 2.5% of young stroke patients (n = 3: vasculitis due to opportunistic infection, vasculitis due to neuroborreliosis, secondary vasospasm after viral meningitis) and in 1.9% (n = 3) in the older stroke cohort (vasculitis due to neurotuberculosis, septic embolic ischemia, vasculitis post-haemophilus influenza meningoencephalitis). Isolated vasculitis was evident in one patient of the older stroke cohort (0.6%). Non-specific alterations in CSF included increased cell count in 10% in young and in 9.3% in the older stroke cohort. Intrathecal Ig synthesis was present in 3.4% of the younger and in 4% of the older stroke cohort. CONCLUSIONS: The prevalence of an infectious etiology in young stroke is modest but slightly higher in comparison with older stroke patients. As brain imaging is not always sufficient for suspecting vasculitis, we recommend implementation of lumbar puncture in young cryptogenic stroke patients. If an infectious disease is present in ischemic stroke, it is of high therapeutic relevance.

Siponimod (BAF312) Treatment Reduces Brain Infiltration but Not Lesion Volume in Middle-Aged Mice in Experimental Stroke.

Background and Purpose- The contribution of neuroinflammation and, in particular, the infiltration of the brain by lymphocytes is increasingly recognized as a substantial pathophysiological mechanism after stroke. The interaction of lymphocytes with endothelial cells and platelets, termed thromboinflammation, fosters microvascular dysfunction and secondary infarct growth. Siponimod is an S1PR (sphingosine-1-phosphate receptor) modulator, which blocks the egress of lymphocytes from lymphoid organs and has demonstrated beneficial effects in multiple sclerosis treatment. We investigated the effect of treatment with siponimod on stroke outcome in a mouse model of cerebral ischemia. Methods- Transient middle cerebral artery occlusion was induced in middle-aged wild-type mice. Animals were either treated with siponimod (3 mg/kg; intraperitoneal) or vehicle for 6 days. Stroke outcome was assessed by magnetic resonance imaging (spleen volume: prestroke, day 3, and day 7; infarct volume: days 1, 3, and 7) and behavioral tests (prestroke, day 2, and day 6). Immune cells of the peripheral blood and brain-infiltrating cells ipsilateral and contralateral were analyzed by VETScan and by flow cytometry. Results- Siponimod significantly induced lymphopenia on day 7 after transient middle cerebral artery occlusion and reduced T-lymphocyte accumulation in the central nervous system. No effect was detected for lesion size. Conclusions- For siponimod administered at 3 mg/kg in transient middle cerebral artery occlusion mouse model, our findings do not provide preclinical evidence for the use of S1PR1/5 modulators as neuroprotectant in stroke therapy.

Ischaemic stroke and the recanalization drug tissue plasminogen activator interfere with antibacterial phagocyte function.

BACKGROUND: Stroke induces immune alterations such as impaired oxidative burst and reduced release of neutrophil extracellular traps (NETs). We hypothesised that key enzymes of these defence mechanisms may be altered in ischaemic stroke. Therefore, we analysed the intra- and extracellular amounts of myeloperoxidase (MPO) and neutrophil elastase (NE) in patient sera and granulocytes and monocytes. Because the autonomous nervous system is thought to mediate stroke-induced immune alterations, we also studied the influence of stress hormones and acetylcholine on MPO and NE. Rapid recanalization by recombinant tissue plasminogen activator (r-tPA) is the only available treatment for ischaemic stroke besides thrombectomy, and its influence on antibacterial defence mechanisms of granulocytes and monocytes were addressed here. METHODS: Ex vivo: Intracellular and serum MPO and NE were measured on days 0, 1, 3 and 5 post-stroke by either flow cytometry or enzyme-linked immunosorbent assay (ELISA) and compared to controls. In vitro: Blood from healthy donors was incubated with catecholamines, dexamethasone and acetylcholine, and the percentage of NET-producing cells and the area covered by NETs were quantified immunohistochemically. Intra- and extracellular MPO and NE were quantified by flow cytometry or ELISA. Blood samples from healthy donors were incubated with r-tPA, and oxidative burst, phagocytosis, NETosis, cytokine release, MPO and NE were quantified by flow cytometry, ELISA and microscopy. RESULTS: MPO was reduced in granulocytes but increased in sera obtained from stroke patients compared to controls. NE was not altered intracellularly but was elevated in patient sera. The percentage of NET-producing neutrophils was decreased by stress hormones and increased by acetylcholine. Neither intracellular MPO nor NE was altered by hormone treatment; however, adrenaline and acetylcholine induced NE release. r-tPA led to reduced phagocytosis and oxidative burst in granulocytes and monocytes in vitro. NETosis, MPO release and cytokines were not altered, whereas NE release was enhanced by r-tPA. CONCLUSIONS: Intracellular reduction of MPO might be responsible for reduced NETosis in stroke patients. The impact of enhanced MPO and NE serum levels in stroke patients should be addressed in future studies. r-tPA impaired antibacterial defence function in vitro. Therefore, patients who undergo unsuccessful recanalization therapy might be at higher risk for infection, which should be analysed in future investigations. Immune alterations due to r-tPA effects in stroke patients should also be investigated.