The role of cortisol in immunosuppression in subarachnoid haemorrhage.

BACKGROUND: We sought to determine the extent to which cortisol suppressed innate and T cell-mediated cytokine production and whether it could be involved in reducing peripheral cytokine production following subarachnoid haemorrhage (SAH). METHODS: Whole blood from healthy controls, patients with SAH and healthy volunteers was stimulated with lipopolysaccharide (LPS), to stimulate innate immunity, or phytohaemagglutinin (PHA), to stimulate T cell-mediated immunity. Varying concentrations of cortisol were included, with or without the cortisol antagonist RU486. Concentration of interleukin-6 (IL-6), IL-1ß and tumour necrosis factor-alpha) TNFα were determined as a measure of innate immunity. IL-6, IL-17 (interferon gamma) IFNƔ and IL-17 were determined as an indicator of T cell-mediated immunity. RESULTS: Suppression of innate responses to LPS was apparent in whole blood from SAH patients, relative to healthy controls, and TNFα production was inversely correlated with plasma cortisol concentration. Cytokine production in whole blood from healthy volunteers was inhibited by cortisol concentrations from 0.33 µM, or 1 µM and above, and these responses were effectively reversed by the cortisol antagonist RU-486. In SAH patients, RU-486 reversed suppression of innate TNF-α and IL-6 responses, but not IL-1ß or T cell-mediated responses. CONCLUSION: These data suggest that cortisol may play a role in reducing innate, but not T cell-mediated immune responses in patients with injuries such as SAH and that cortisol antagonists could be effective in boosting early innate responses.

The Chemical History of a Bubble.

Acoustic cavitation (the growth, oscillation, and rapid collapse of bubbles in a liquid) occurs in all liquids irradiated with sufficient intensity of sound or ultrasound. The collapse of such bubbles creates local heating and provides a unique source of energy for driving chemical reactions. In addition to sonochemical bond scission and formation, cavitation also induces light emission in many liquids. This phenomenon of sonoluminescence (SL) has captured the imagination of many researchers since it was first observed 85 years ago. SL provides a direct probe of cavitation events and has provided most of our understanding of the conditions created inside collapsing bubbles. Spectroscopic analyses of SL from single acoustically levitated bubbles as well as from clouds of bubbles have revealed molecular, atomic, and ionic line and band emission riding atop an underlying continuum arising from radiative plasma processes. Application of spectrometric methods of pyrometry and plasma diagnostics to these spectra has permitted quantitative measurement of the intracavity conditions: relative peak intensities for temperature measurements, peak shifts and broadening for pressures, and peak asymmetries for plasma electron densities. The studies discussed herein have revealed that extraordinary conditions are generated inside the collapsing bubbles in ordinary room-temperature liquids: observable temperatures exceeding 15 000 K (i.e., three times the surface temperature of our sun), pressures of well over 1000 bar (more than the pressure at the bottom of the Mariana Trench), and heating and cooling rates in excess of 1012 K·s-1. Scientists from many disciplines, and even nonscientists, have been and continue to be intrigued by the consequences of dynamic bubbles in liquids. As chemists, we are fascinated by the high energy reactions and processes that occur during acoustic cavitation and by the use of SL as a spectroscopic probe of the events during cavitation. Within the chemical realm of SL and cavitation there are many interesting questions that are now answered but also many that remain to be explored, so we hope that this Account reveals to the reader some of the most fascinating of those curiosities as we explore the chemical history of a bubble. The high energy species produced inside collapsing bubbles also lead to secondary reactions from the high energy species created within the collapsing bubble diffusing into the bulk liquid and expanding the range of sonochemical reactions observed, especially in redox reactions relevant to nanomaterials synthesis. Bubbles near solid surfaces deform upon collapse, which lessens the internal heating within the bubble, as shown by SL studies, but introduces important mechanical consequences in terms of surface damage and increased surface reactivity. Our understanding of the conditions created during cavitation has informed the applications of ultrasound to a wide range of chemical applications, from nanomaterials to synthetically useful organic reactions to biomedical and pharmaceutical uses. Indeed, we echo Michael Faraday's observation concerning a candle flame, "There is not a law under which any part of this universe is governed which does not come into play and is touched upon in these phenomena." ( Faraday , M. The Chemical History of a Candle ; Harper & Brothers : New York , 1861 ).

SCIL-STROKE (Subcutaneous Interleukin-1 Receptor Antagonist in Ischemic Stroke): A Randomized Controlled Phase 2 Trial.

BACKGROUND AND PURPOSE: The proinflammatory cytokine IL-1 (interleukin-1) has a deleterious role in cerebral ischemia, which is attenuated by IL-1 receptor antagonist (IL-1Ra). IL-1 induces peripheral inflammatory mediators, such as interleukin-6, which are associated with worse prognosis after ischemic stroke. We investigated whether subcutaneous IL-1Ra reduces the peripheral inflammatory response in acute ischemic stroke. METHODS: SCIL-STROKE (Subcutaneous Interleukin-1 Receptor Antagonist in Ischemic Stroke) was a single-center, double-blind, randomized, placebo-controlled phase 2 trial of subcutaneous IL-1Ra (100 mg administered twice daily for 3 days) in patients presenting within 5 hours of ischemic stroke onset. Randomization was stratified for baseline National Institutes of Health Stroke Scale score and thrombolysis. Measurement of plasma interleukin-6 and other peripheral inflammatory markers was undertaken at 5 time points. The primary outcome was difference in concentration of log(interleukin-6) as area under the curve to day 3. Secondary outcomes included exploratory effect of IL-1Ra on 3-month outcome with the modified Rankin Scale. RESULTS: We recruited 80 patients (mean age, 72 years; median National Institutes of Health Stroke Scale, 12) of whom 73% received intravenous thrombolysis with alteplase. IL-1Ra significantly reduced plasma interleukin-6 (P<0.001) and plasma C-reactive protein (P<0.001). IL-1Ra was well tolerated with no safety concerns. Allocation to IL-1Ra was not associated with a favorable outcome on modified Rankin Scale: odds ratio (95% confidence interval)=0.67 (0.29-1.52), P=0.34. Exploratory mediation analysis suggested that IL-1Ra improved clinical outcome by reducing inflammation, but there was a statistically significant, alternative mechanism countering this benefit. CONCLUSIONS: IL-1Ra reduced plasma inflammatory markers which are known to be associated with worse clinical outcome in ischemic stroke. Subcutaneous IL-1Ra is safe and well tolerated. Further experimental studies are required to investigate efficacy and possible interactions of IL-1Ra with thrombolysis. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: ISRCTN74236229.

Reduction of inflammation after administration of interleukin-1 receptor antagonist following aneurysmal subarachnoid hemorrhage: results of the Subcutaneous Interleukin-1Ra in SAH (SCIL-SAH) study.

OBJECTIVE Aneurysmal subarachnoid hemorrhage (aSAH) is a devastating cerebrovascular event with long-term morbidity and mortality. Patients who survive the initial bleeding are likely to suffer further early brain injury arising from a plethora of pathological processes. These may result in a worsening of outcome or death in approximately 25% of patients and may contribute to longer-term cognitive dysfunction in survivors. Inflammation, mediated by the cytokine interleukin-1 (IL-1), is an important contributor to cerebral ischemia after diverse forms of brain injury, including aSAH. Its effects are attenuated by its naturally occurring antagonist, IL-1 receptor antagonist (IL-1Ra [anakinra]). The authors hypothesized that administration of additional subcutaneous IL-1Ra would reduce inflammation and associated plasma markers associated with poor outcome following aSAH. METHODS This was a randomized, open-label, single-blinded study of 100 mg subcutaneous IL-1Ra, administered twice daily in patients with aSAH, starting within 3 days of ictus and continuing until 21 days postictus or discharge from the neurosurgical center, whichever was earlier. Blood samples were taken at admission (baseline) and at Days 3-8, 14, and 21 postictus for measurement of inflammatory markers. The primary outcome was difference in plasma IL-6 measured as area under the curve between Days 3 and 8, corrected for baseline value. Secondary outcome measures included similar area under the curve analyses for other inflammatory markers, plasma pharmacokinetics for IL-1Ra, and clinical outcome at 6 months. RESULTS Interleukin-1Ra significantly reduced levels of IL-6 and C-reactive protein (p < 0.001). Fibrinogen levels were also reduced in the active arm of the study (p < 0.002). Subcutaneous IL-1Ra was safe, well tolerated, and had a predictable plasma pharmacokinetic profile. Although the study was not powered to investigate clinical effect, scores of the Glasgow Outcome Scale-extended at 6 months were better in the active group; however, this outcome did not reach statistical significance. CONCLUSIONS Subcutaneous IL-1Ra is safe and well tolerated in aSAH. It is effective in reducing peripheral inflammation. These data support a Phase III study investigating the effect of IL-1Ra on outcome following aSAH. Clinical trial registration no.: EudraCT: 2011-001855-35 ( www.clinicaltrialsregister.eu ).

Do model polymer therapeutics sufficiently diffuse through articular cartilage to be a viable therapeutic route?

The ability of a polymer therapeutic to access the appropriate subcellular location is crucial to its efficacy and is defined to a large part by the many and complex cellular biological and biochemical barriers such that a construct must traverse. It is shown here that model dextrin conjugates are able to pass through a cartilaginous extracellular matrix into chondrocytes, with little perturbation of the matrix structure, indicating that targeting of potential therapeutics through a cartilaginous extracellular matrix should be proven possible. Rapid chondrocytic targeting of drugs which require intra cellularisation for their activity and uniform extracellular concentrations of drugs with an extracellular target, is thus enabled though polymer conjugation.

Study design and population pharmacokinetic analysis of a phase II dose-ranging study of interleukin-1 receptor antagonist.

Interleukin-1 receptor antagonist, a naturally-occurring antagonist to the pro-inflammatory cytokine Interleukin-1, is already in clinical use. In experimental models of stroke, Interleukin-1 receptor antagonist in cerebrospinal fluid has been associated with cerebral neuroprotection and in a phase I clinical trial in patients with subarachnoid haemorrhage it crosses the blood-cerebrospinal fluid barrier. The aims of the current work were to design a dose-ranging clinical study in patients and to analyse the plasma and cerebrospinal fluid data obtained using a population pharmacokinetic modelling approach. The study was designed using prior information: a published population pharmacokinetic model and associated parameter estimates. Simulations were carried out to identify combinations of intravenous bolus and 4 h infusion doses that could achieve a concentration of 100 ng/ml in cerebrospinal fluid within approximately 30 min. The most informative time points for plasma and cerebrospinal fluid were obtained prospectively; optimisation identified five sampling time points that were included in the 15 time points in the present study design. All plasma and cerebrospinal fluid concentration data from previous and current studies were combined for updated analysis. The result of the simulations showed that a dosage regimen of 500 mg intravenous bolus and 10 mg/kg/h could achieve the target concentration, however four other regimens that represent a stepwise increase in maximum concentration were also selected. Analysis of the updated data showed improvement in parameter accuracy and predictive performance of the model; the percentage relative standard errors for fixed and random-effects parameters were <15 and 35% respectively. A dose-ranging study was successfully designed using modelling and simulation.

Diagnosis of Stroke-Associated Pneumonia: Recommendations From the Pneumonia in Stroke Consensus Group.

BACKGROUND AND PURPOSE: Lower respiratory tract infections frequently complicate stroke and adversely affect outcome. There is currently no agreed terminology or gold-standard diagnostic criteria for the spectrum of lower respiratory tract infections complicating stroke, which has implications for clinical practice and research. The aim of this consensus was to propose standardized terminology and operational diagnostic criteria for lower respiratory tract infections complicating acute stroke. METHODS: Systematic literature searches of multiple electronic databases were undertaken. An evidence review and 2 rounds of consensus consultation were completed before a final consensus meeting in September 2014, held in Manchester, United Kingdom. Consensus was defined a priori as ≥75% agreement between the consensus group members. RESULTS: Consensus was reached for the following: (1) stroke-associated pneumonia (SAP) is the recommended terminology for the spectrum of lower respiratory tract infections within the first 7 days after stroke onset; (2) modified Centers for Disease Control and Prevention (CDC) criteria are proposed for SAP as follows-probable SAP: CDC criteria met, but typical chest x-ray changes absent even after repeat or serial chest x-ray; definite SAP: CDC criteria met, including typical chest x-ray changes; (3) there is limited evidence for a diagnostic role of white blood cell count or C-reactive protein in SAP; and (4) there is insufficient evidence for the use of other biomarkers (eg, procalcitonin). CONCLUSIONS: Consensus operational criteria for the terminology and diagnosis of SAP are proposed based on the CDC criteria. These require prospective evaluation in patients with stroke to determine their reliability, validity, impact on clinician behaviors (including antibiotic prescribing), and clinical outcomes.

How is pneumonia diagnosed in clinical stroke research? A systematic review and meta-analysis.

BACKGROUND AND PURPOSE: Diagnosis of pneumonia complicating stroke is challenging, and there are currently no consensus diagnostic criteria. As a first step in developing such consensus-based diagnostic criteria, we undertook a systematic review to identify the existing diagnostic approaches to pneumonia in recent clinical stroke research to establish the variation in diagnosis and terminology. METHODS: Studies of ischemic stroke, intracerebral hemorrhage, or both, which reported occurrence of pneumonia from January 2009 to March 2014, were considered and independently screened for inclusion by 2 reviewers after multiple searches using electronic databases. The primary analysis was to identify existing diagnostic approaches for pneumonia. Secondary analyses explored potential reasons for any heterogeneity where standard criteria for pneumonia had been applied. RESULTS: Sixty-four studies (56% ischemic stroke, 6% intracerebral hemorrhage, 38% both) of 639 953 patients were included. Six studies (9%) reported no information on the diagnostic approach, whereas 12 (19%) used unspecified clinician-reported diagnosis or initiation of antibiotics. The majority used objective diagnostic criteria: 20 studies (31%) used respiratory or other published standard criteria; 26 studies (41%) used previously unpublished ad hoc criteria. The overall occurrence of pneumonia was 14.3% (95% confidence interval 13.2%-15.4%; I(2)=98.9%). Occurrence was highest in studies applying standard criteria (19.1%; 95% confidence interval 15.1%-23.4%; I(2)=98.5%). The substantial heterogeneity observed was not explained by stratifying for other potential confounders. CONCLUSIONS: We found considerable variation in terminology and the diagnostic approach to pneumonia. Our review supports the need for consensus development of operational diagnostic criteria for pneumonia complicating stroke.

The effect of intravenous interleukin-1 receptor antagonist on inflammatory mediators in cerebrospinal fluid after subarachnoid haemorrhage: a phase II randomised controlled trial.

BACKGROUND: Interleukin-1 (IL-1) is a key mediator of ischaemic brain injury induced by stroke and subarachnoid haemorrhage (SAH). IL-1 receptor antagonist (IL-1Ra) limits brain injury in experimental stroke and reduces plasma inflammatory mediators associated with poor outcome in ischaemic stroke patients. Intravenous (IV) IL-1Ra crosses the blood-brain barrier (BBB) in patients with SAH, to achieve cerebrospinal fluid (CSF) concentrations that are neuroprotective in rats. METHODS: A small phase II, double-blind, randomised controlled study was carried out across two UK neurosurgical centres with the aim of recruiting 32 patients. Adult patients with aneurysmal SAH, requiring external ventricular drainage (EVD) within 72 hours of ictus, were eligible. Patients were randomised to receive IL-1Ra (500 mg bolus, then a 10 mg/kg/hr infusion for 24 hours) or placebo. Serial samples of CSF and plasma were taken and analysed for inflammatory mediators, with change in CSF IL-6 between 6 and 24 hours as the primary outcome measure. RESULTS: Six patients received IL-1Ra and seven received placebo. Concentrations of IL-6 in CSF and plasma were reduced by one standard deviation in the IL-1Ra group compared to the placebo group, between 6 and 24 hours, as predicted by the power calculation. This did not reach statistical significance (P = 0.08 and P = 0.06, respectively), since recruitment did not reach the target figure of 32. No adverse or serious adverse events reported were attributable to IL-1Ra. CONCLUSIONS: IL-1Ra appears safe in SAH patients. The concentration of IL-6 was lowered to the degree expected, in both CSF and plasma for patients treated with IL-1Ra.

C-reactive protein predicts hematoma growth in intracerebral hemorrhage.

BACKGROUND AND PURPOSE: Early hematoma growth (EHG) occurs in about one third of patients with spontaneous intracerebral hemorrhage. The main aim of this study was to investigate the potential of plasma C-reactive protein (CRP) for predicting EHG after acute spontaneous intracerebral hemorrhage. METHODS: Plasma CRP was measured within 6 hours of onset (median, 120 minutes) in 399 patients with primary or vitamin K antagonist-associated spontaneous intracerebral hemorrhage and without recent infection. Computed tomography brain scans were performed at baseline and repeated within 24 hours (median, 22 hours). The primary outcome was EHG, defined as absolute growth>12.5 cm3 or relative growth>33%. Secondary outcomes included early neurological worsening (ENW) using the Glasgow Coma Scale and 30-day mortality. Multivariable regression analyses were used to evaluate associations of CRP concentration and outcomes. Kaplan-Meier analysis was used for survival. RESULTS: EHG occurred in 25.8%, ENW in 19.3%, and mortality was 31.8% at 30 days. Thirty-day mortality was significantly higher in patients with ENW (hazard ratio, 3.21; 95% confidence interval, 2.00-5.17; P<0.0001) and in patients with EHG (hazard ratio, 2.13; 95% confidence interval, 1.42-3.18; P<0.0001, log-rank test). Median CRP was 12 mg/L (interquartile range, 10-17) in the EHG group and 7 mg/L (interquartile range, 4-12.1) in those without EHG (P<0.0001). In multivariable analyses, plasma CRP>10 mg/L independently predicted EHG (odds ratio, 4.71; 95% confidence interval, 2.75-8.06; P<0.0001) and ENW (odds ratio, 2.70; 95% confidence interval, 1.50-4.84; P=0.0009). CONCLUSIONS: CRP>10 mg/L is independently predictive of EHG and ENW, both of which are associated with increased mortality. Inflammation may be important in contributing to EHG and warrants further investigation.

Overcoming matrix matching problems in multiplex cytokine assays.

Failure to match assay matrices with samples in immunoassays can result in incorrect sample values being reported. For multiplex assays this presents particular problems, due to the need to find a matrix suitable for all the analytes. Here, we describe strategies adopted to overcome matrix problems identified in establishing a cytokine multiplex assay in human plasma. Standard analytes were diluted in plasma samples to identify representative plasma for assay development. Horse sera were screened to evaluate potential interference before using to adjust a matrix to match plasma samples. Suitability of the matrix match was confirmed by evaluating recovery of known concentrations of analytes from plasma. Individual plasmas modified the assay signal for some analytes to a variable extent, particularly for IL-1α and IL-1ß. Addition of horse serum to assay buffer improved matching to plasma samples, although endogenous MCP-1 activity was apparent in one sample. Matching of plasma and assay matrices allowed recoveries within 10% to 20% of the expected values, unless the samples contained atypical interfering activity. Attention to choice of samples and diluent used for assay development is particularly important for measurement of sample analytes in cytokine multiplex assays.

Inflammation as a predictor for delayed cerebral ischemia after aneurysmal subarachnoid haemorrhage.

BACKGROUND: The mechanism of development of delayed cerebral ischemia (DCI) after aneurysmal subarachnoid hemorrhage (SAH) is poorly understood. Inflammatory processes are implicated in the development of ischemic stroke and may also predispose to the development of DCI following SAH. The objective of this study was to test whether concentrations of circulating inflammatory markers (C-reactive protein (CRP), interleukin-6 (IL-6) and interleukin 1 receptor antagonist (IL-1Ra)) were predictive for DCI following SAH. Secondary analyses considered white cell count (WCC) and erythrocyte sedimentation rate (ESR). METHODS: This was a single-center case-control study nested within a prospective cohort. Plasma inflammatory markers were measured in patients up to 15 days after SAH (initial, peak, average, final and rate of change to final). Cases were defined as those developing DCI. Inflammatory markers were compared between cases and randomly selected matched controls. RESULTS: Among the 179 participants there were 46 cases of DCI (26%). In primary analyses the rate of change of IL-6 was associated with DCI (OR 2.3 (95% CI 1.1 to 5.0); p=0.03). The final value and rate of change of WCC were associated with DCI (OR 1.2 (95% CI 1.0 to 1.3) and OR 1.3 (95% CI 1.0 to 1.6), respectively). High values of ESR were associated with DCI (OR 2.4 (95% CI 1.3 to 4.6) initial; OR 2.3 (95% CI 1.3 to 4.2) average; OR 2.1 (95% CI 1.1 to 3.9) peak; and OR 2.0 (95% CI 1.2 to 3.3) final value). CONCLUSIONS: Leucocytosis and change in IL-6 prior to DCI reflect impending cerebral ischemia. The time-independent association of ESR with DCI after SAH may identify this as a risk factor. These data suggest that systemic inflammatory mechanisms may increase the susceptibility to the development of DCI after SAH.

Cerebrospinal fluid and plasma cytokines after subarachnoid haemorrhage: CSF interleukin-6 may be an early marker of infection.

BACKGROUND: Cytokines and cytokine receptor concentrations increase in plasma and cerebrospinal fluid (CSF) of patients following subarachnoid haemorrhage (SAH). The relationship between plasma and CSF cytokines, and factors affecting this, are not clear. METHODS: To help define the relationship, paired plasma and cerebrospinal fluid (CSF) samples were collected from patients subject to ventriculostomy. Concentrations of key inflammatory cytokines, interleukin (IL)-1ß, IL-1 receptor antagonist (IL-1Ra), IL-1 receptor 2, IL-6, IL-8, IL-10, tumour necrosis factor (TNF)-α, and TNF receptors (TNF-R) 1 and 2 were determined by immunoassay of CSF and plasma from 21 patients, where samples were available at three or more time points. RESULTS: Plasma concentrations of IL-1ß, IL-1Ra, IL-10, TNF-α and TNF-R1 were similar to those in CSF. Plasma TNF-R2 and IL-1R2 concentrations were higher than in CSF. Concentrations of IL-8 and IL-6 in CSF were approximately10 to 1,000-fold higher than in plasma. There was a weak correlation between CSF and plasma IL-8 concentrations (r = 0.26), but no correlation for IL-6. Differences between the central and peripheral pattern of IL-6 were associated with episodes of ventriculostomy-related infection (VRI). A VRI was associated with CSF IL-6 >10,000 pg/mL (P = 0.0002), although peripheral infection was not significantly associated with plasma IL-6. CONCLUSIONS: These data suggest that plasma cytokine concentrations cannot be used to identify relative changes in the CSF, but that measurement of CSF IL-6 could provide a useful marker of VRI.

C-reactive protein in intracerebral hemorrhage: time course, tissue localization, and prognosis.

OBJECTIVES: We examined the C-reactive protein (CRP) response after spontaneous intracerebral hemorrhage (sICH) and its relationship to outcome. We additionally characterized early brain localization of CRP. METHODS: In this prospective, multicenter, international, collaborative, longitudinal study with cross-sectional immunohistochemical analysis of brain tissue, 223 patients (M/F: 132/91) were recruited during the 2010 calendar year. CRP was evaluated at admission (median 93 minutes from symptom onset), 24 hours, 48 hours, and 72 hours after sICH. Brains of 5 subjects with sICH were compared to brains of 2 aged controls without evidence of brain pathology and 7 patients with ischemic stroke. Plasma CRP was measured over 72 hours following sICH and its relationship to 30-day mortality and functional outcome at 30 days (Glasgow Outcome Scale) was determined. CRP immunostaining patterns were analyzed in samples of sICH autopsy brains. RESULTS: Plasma CRP increased over the 48 hours from admission and was significantly (p < 0.001) related to hematoma volume at later time points. The predictive utility of CRP for morbidity and mortality were maintained when adjusted for other risk factors and improved at 48 hours and 72 hours when compared with admission values. Although an early CRP localization was present in both ischemic and hemorrhagic lesions, an intense and diffuse neuropil staining was only present in sICH patients and particularly evident proximal to the hemorrhagic areas. CONCLUSIONS: Plasma CRP production increases markedly over the 48 hours to 72 hours period following sICH and is related to outcome. CRP is also present in large amounts around the hemorrhagic lesion and within neurons and glia of patients who died within 12 hours of sICH.

Interleukin-1 receptor antagonist reverses stroke-associated peripheral immune suppression.

INTRODUCTION: Infections are common following stroke and adversely affect outcome. Cellular immune suppression associated with acute stroke may increase susceptibility to infection. Cytokines are important contributors to both stroke pathology and the response to infection. Since interleukin (IL)-1 blockade is a candidate treatment for cerebral ischemia, we examined whether administration of interleukin-1 receptor antagonist (IL-1Ra) to patients with acute stroke affected innate cellular immune responses in a phase II placebo-controlled trial. METHODS: Venous blood samples were taken prior to treatment initiation, at 24h and 5 to 7d. Blood was also drawn from stroke-free controls. Lipopolysaccharide (LPS) stimulation of whole-blood cultures assessed the potential of leukocytes to produce cytokines. RESULTS: Induction of tumor necrosis factor (TNF)-α, IL-1ß, IL-6, IL-8 and IL-10 by LPS was significantly reduced in patients at admission, compared to controls. At 24h, cytokine induction remained suppressed in the placebo group. In contrast, for patients treated with IL-1Ra, induction of TNF-α, IL-6 and IL-10 was similar to controls and IL-1ß induction was significantly greater than in the placebo group. At 5 to 7d, TNF-α and IL-1ß induction remained suppressed only in the placebo group (p<0.05). Plasma cortisol concentrations, elevated at admission in patients compared to controls, were substantially reduced at 24h in the patients receiving IL-1Ra (p<0.05) and inversely correlated (p<0.001) with either TNF-α (r=-0.71) or IL-1ß induction (r=-0.67) at admission. CONCLUSION: Treatment with IL-1Ra reverses peripheral innate immune suppression in the acute phase of stroke, which is associated with attenuated cortisol production. The mechanisms underlying these observations, including the potential impact of IL-1Ra on stroke severity and the clinical significance of immune suppression, require further evaluation in larger studies.

Reconstituting National Institute for Biological Standards and Control (NIBSC) chemokines.

We observed significant discrepancies between immunoassay results when using different internally prepared reference preparations for interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) from the National Institute for Biological Standards and Control (NIBSC). To evaluate the reasons for this we prepared the chemokines using diluents that incorporated protein at different steps. This showed that even brief addition of water to these preparations, in the absence of additional protein, resulted in loss of immunoreactivity in assays. The data obtained emphasise the importance of adding protein at an early stage of preparation to avoid loss of material and potential loss of activity.

Does inflammation predispose to recurrent vascular events after recent transient ischaemic attack and minor stroke? The North West of England transient ischaemic attack and minor stroke (NORTHSTAR) study.

BACKGROUND AND HYPOTHESIS: Inflammation is implicated in the pathogenesis and outcome of ischaemic injury. Poststroke inflammation is associated with outcome but it remains unclear whether such inflammation precedes or results from ischaemic injury. We hypothesised that inflammatory markers are associated with an increased risk of recurrent vascular events soon after transient ischaemic attack and minor stroke. METHODS: This was a multicentre, prospective, nested case-control study. Plasma concentrations of C-reactive protein, interleukin-6, interleukin-1-receptor antagonist and fibrinogen, leucocyte counts, erythrocyte sedimentation rate and inflammatory gene allele frequencies were analysed in 711 patients with recent transient ischaemic attack or minor stroke. Cases were defined by the incidence of one or more recurrent vascular events during the three-month follow-up. Association of inflammatory markers with case-status was determined using conditional logistic regression. RESULTS: Plasma concentrations of C-reactive protein, interleukin-1-receptor antagonist and interleukin-6 were not associated with case-status. In secondary analyses, only erythrocyte sedimentation rate was significantly associated with case-status (odds ratio 1·39, 95% confidence interval 1·03-1·85; P=0·03), but this effect did not persist after adjustment for smoking and past history of transient ischaemic attack or stroke. Single nucleotide polymorphisms in four inflammatory genes (interleukin-6, fibrinogen, P-selectin and vascular cell adhesion molecule-1) were nominally associated with case-status. CONCLUSIONS: Circulating inflammatory markers were not associated with recurrent vascular events. Nominally significant associations between genetic markers and case-status will require replication. These data provide little evidence for an inflammatory state predisposing to stroke and other vascular events in a susceptible population.

Brain inflammation is induced by co-morbidities and risk factors for stroke.

Chronic systemic inflammatory conditions, such as atherosclerosis, diabetes and obesity are associated with increased risk of stroke, which suggests that systemic inflammation may contribute to the development of stroke in humans. The hypothesis that systemic inflammation may induce brain pathology can be tested in animals, and this was the key objective of the present study. First, we assessed inflammatory changes in the brain in rodent models of chronic, systemic inflammation. PET imaging revealed increased microglia activation in the brain of JCR-LA (corpulent) rats, which develop atherosclerosis and obesity, compared to the control lean strain. Immunostaining against Iba1 confirmed reactive microgliosis in these animals. An atherogenic diet in apolipoprotein E knock-out (ApoE(-/-)) mice induced microglial activation in the brain parenchyma within 8 weeks and increased expression of vascular adhesion molecules. Focal lipid deposition and neuroinflammation in periventricular and cortical areas and profound recruitment of activated myeloid phagocytes, T cells and granulocytes into the choroid plexus were also observed. In a small, preliminary study, patients at risk of stroke (multiple risk factors for stroke, with chronically elevated C-reactive protein, but negative MRI for brain pathology) exhibited increased inflammation in the brain, as indicated by PET imaging. These findings show that brain inflammation occurs in animals, and tentatively in humans, harbouring risk factors for stroke associated with elevated systemic inflammation. Thus a "primed" inflammatory environment in the brain may exist in individuals at risk of stroke and this can be adequately recapitulated in appropriate co-morbid animal models.

Extreme conditions during multibubble cavitation: Sonoluminescence as a spectroscopic probe.

We review recent work on the use of sonoluminescence (SL) to probe spectroscopically the conditions created during cavitation, both in clouds of collapsing bubbles (multibubble sonoluminescence, (MBSL)) and in single bubble events. The effective MBSL temperature can be controlled by the vapor pressure of the liquid or the thermal conductivity of the dissolved gas over a range from ∼1600 to ∼9000K. The effective pressure during MBSL is ∼300bar, based on atomic line shifts. Given nanosecond emission times, this means that cooling rates are >10(12)K/s. In sulfuric and phosphoric acid, the low volatility and high solubility of any sonolysis products make bubble collapse more efficient and evidence for an optically opaque plasma core is found.

Off-resonance laser frequency stabilization using the Faraday effect.

We present a simple technique for stabilization of a laser frequency off resonance using the Faraday effect in a heated vapor cell with an applied magnetic field. In particular, we demonstrate stabilization of a 780 nm laser detuned up to 14 GHz from the (85)Rb D(2) 5(2)S(1/2) F = 2 to 5(2)P(3/2) F' = 3 transition. Control of the temperature of the vapor cell and the magnitude of the applied magnetic field allows locking ~6-14 GHz red and blue detuned from the atomic line. We obtain an rms fluctuation of 7 MHz over 1 h without stabilization of the cell temperature or magnetic field.