A hyperacute immune map of ischaemic stroke patients reveals alterations to circulating innate and adaptive cells.

Systemic immune changes following ischaemic stroke are associated with increased susceptibility to infection and poor patient outcome due to their role in exacerbating the ischaemic injury and long-term disability. Alterations to the abundance or function of almost all components of the immune system post-stroke have been identified, including lymphocytes, monocytes and granulocytes. However, subsequent infections have often confounded the identification of stroke-specific effects. Global understanding of very early changes to systemic immunity is critical to identify immune targets to improve clinical outcome. To this end, we performed a small, prospective, observational study in stroke patients with immunophenotyping at a hyperacute time point (< 3 h) to explore early changes to circulating immune cells. We report, for the first time, decreased frequencies of type 1 conventional dendritic cells (cDC1), haematopoietic stem and progenitor cells (HSPCs), unswitched memory B cells and terminally differentiated effector memory T cells re-expressing CD45RA (TEMRA). We also observed concomitant alterations to human leucocyte antigen D-related (HLA-DR), CD64 and CD14 expression in distinct myeloid subsets and a rapid activation of CD4+ T cells based on CD69 expression. The CD69+ CD4+ T cell phenotype inversely correlated with stroke severity and was associated with naive and central memory T (TCM) cells. Our findings highlight early changes in both the innate and adaptive immune compartments for further investigation as they could have implications the development of post-stroke infection and poorer patient outcomes.

Inflammation and brain injury: acute cerebral ischaemia, peripheral and central inflammation.

Inflammation is a classical host defence response to infection and injury that has many beneficial effects. However, inappropriate (in time, place and magnitude) inflammation is increasingly implicated in diverse disease states, now including cancer, diabetes, obesity, atherosclerosis, heart disease and, most relevant here, CNS disease. A growing literature shows strong correlations between inflammatory status and the risk of cerebral ischaemia (CI, most commonly stroke), as well as with outcome from an ischaemic event. Intervention studies to demonstrate a causal link between inflammation and CI (or its consequences) are limited but are beginning to emerge, while experimental studies of CI have provided direct evidence that key inflammatory mediators (cytokines, chemokines and inflammatory cells) contribute directly to ischaemic brain injury. However, it remains to be determined what the relative importance of systemic (largely peripheral) versus CNS inflammation is in CI. Animal models in which CI is driven by a CNS intervention may not accurately reflect the clinical condition; stroke being typically induced by atherosclerosis or cardiac dysfunction, and hence current experimental paradigms may underestimate the contribution of peripheral inflammation. Experimental studies have already identified a number of potential anti-inflammatory therapeutic interventions that may limit ischaemic brain damage, some of which have been tested in early clinical trials with potentially promising results. However, a greater understanding of the contribution of inflammation to CI is still required, and this review highlights some of the key mechanism that may offer future therapeutic targets.

Systemic infection, inflammation and acute ischemic stroke.

Extensive evidence implicates inflammation in multiple phases of stroke etiology and pathology. In particular, there is growing awareness that inflammatory events outside the brain have an important impact on stroke susceptibility and outcome. Numerous conditions, including infection and chronic non-infectious diseases, that are established risk factors for stroke are associated with an elevated systemic inflammatory profile. Recent clinical and pre-clinical studies support the concept that the systemic inflammatory status prior to and at the time of stroke is a key determinant of acute outcome and long-term prognosis. Here, we provide an overview of the impact of systemic inflammation on stroke susceptibility and outcome. We discuss potential mechanisms underlying the impact on ischemic brain injury and highlight the implications for stroke prevention, therapy and modeling.

Systemic inflammation affects reperfusion following transient cerebral ischaemia.

Reperfusion after stroke is critical for improved patient survival and recovery and can be achieved clinically through pharmacological (recombinant tissue plasminogen activator) or physical (endovascular intervention) means. Yet these approaches remain confined to a small percentage of stroke patients, often with incomplete reperfusion, and therefore there is an urgent need to learn more about the mechanisms underlying the no-reflow phenomenon that prevents restoration of adequate microvascular perfusion. Recent evidence suggests systemic inflammation as an important contributor to no-reflow and to further investigate this here we inject interleukin 1 (IL-1) i.p. 30 min prior to an ischaemic challenge using a remote filament to occlude the middle cerebral artery (MCA) in mice. Before, during and after the injection of IL-1 and occlusion we use two-dimensional optical imaging spectroscopy to record the spatial and temporal dynamics of oxyhaemoglobin concentration in the cortical areas supplied by the MCA. Our results reveal that systemic inflammation significantly reduces oxyhaemoglobin reperfusion as early as 3h after filament removal compared to vehicle injected animals. CD41 immunohistochemistry shows a significant increase of hyper-coagulated platelets within the microvessels in the stroked cortex of the IL-1 group compared to vehicle. We also observed an increase of pathophysiological biomarkers of ischaemic damage including elevated microglial activation co-localized with interleukin 1α (IL-1α), increased blood brain barrier breakdown as shown by IgG infiltration and increased pyknotic morphological changes of cresyl violet stained neurons. These data confirm systemic inflammation as an underlying cause of no-reflow in the post-ischaemic brain and that appropriate anti-inflammatory approaches could be beneficial in treating ischaemic stroke.

Case report: myoepithelial carcinoma of the breast: a case report with imaging and pathological findings.

We present a case of myoepithelial carcinoma of the breast together with illustrations of the imaging and pathological appearances as well as discussion on the management of this condition.

Cortical death caused by striatal administration of AMPA and interleukin-1 is mediated by activation of cortical NMDA receptors.

Striatal coadministration of interleukin-1beta (IL-1beta) with alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (S-AMPA) in rats results in widespread cortical cell death not caused by either treatment alone. This cortical damage was unaffected by cortical infusion of the AMPA-receptor antagonist NBQX. Cortical infusion of an NMDA-receptor antagonist D-AP5 significantly inhibited (57%; P < 0.05) cortical death, but had no effect on the local striatal death. Thus, cortical neuronal death induced by striatal S-AMPA and human recombinant interleukin-1beta (hrIL-1beta) is mediated by activation of NMDA receptors in the cortex. The authors propose that IL-1beta actions on AMPA-receptor mediated cell death may involve the activation of polysynaptic pathways from the striatum to the cortex.

The interleukin-1 system: an attractive and viable therapeutic target in neurodegenerative disease.

Inflammatory processes in the brain have been implicated in both acute and chronic neurodegenerative disease. One of the most studied inflammatory mediators in this respect is the cytokine interleukin-1 (IL-1), which has diverse actions in the central nervous system and mediates a wide variety of effects, including the host defense responses to local and systemic disease and injury. Both pre-clinical and clinical data suggest a role for IL-1 as a key mediator of cell death in acute neurodegenerative conditions, such as stroke and head injury. IL-1 has also been implicated in a number of chronic diseases, including Parkinson's and Alzheimer's disease, as well as in epilepsy. Constitutive expression of IL-1 is very low in normal brain, but is up-regulated rapidly in response to local or peripheral insults. The mechanisms regulating the expression IL-1 are not well defined, but appear to involve multiple effects on neuronal, glial and endothelial cell function. Therefore, the IL-1 system represents an attractive and intensely competitive therapeutic target.

Selective increases in cytokine expression in the rat brain in response to striatal injection of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate and interleukin-1.

A number of cytokines contribute to acute experimental neurodegeneration. The cytokine response can have detrimental or beneficial effects depending on the temporal profile and balance between pro- and anti-inflammatory molecules. Our recent data suggest that the pro-inflammatory cytokine interleukin-1beta (IL-1beta) acts at specific sites (e.g., the striatum) in the rat brain to cause distant cortical injury, when co-administered with the potent excitotoxin alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (S-AMPA). The objective of the present study was to investigate changes in the expression of several cytokines simultaneously in the rat striatum and cortex after intrastriatal administration of vehicle, S-AMPA or human recombinant (hr) IL-1beta alone or S-AMPA co-injected with hrIL-1beta using reverse transcription-polymerase chain reaction (RT-PCR; Taqman fluorogenic probes) and enzyme-linked immunosorbent assay (ELISA). Injection of S-AMPA alone increased IL-6 mRNA expression in the ipsilateral striatum after 8 h, whilst striatal injection of IL-1beta alone increased local IL-1beta and IL-1ra mRNAs. The levels of mRNA encoding IL-1alpha, IL-1beta, IL-1ra, IL-6, IL-10 and TNFalpha were markedly elevated in the ipsilateral cortex 8 h after co-injection of S-AMPA and hrIL-1beta. Cortical mRNA levels for IL-4, IL-18, TGFbeta and IFNgamma were not significantly different between treatment groups after 2 h or 8 h. A similar pattern of change in the levels of IL-1alpha and IL-6 protein was observed 8 h after treatment. These data demonstrate selective increases in the expression of cytokines in areas of remote cell death in response to administration of hrIL-1beta and S-AMPA. Such cytokines may be involved in the ensuing damage, and further clarification of their actions could aid future therapeutic strategies for several acute neurodegenerative disorders.

The role of pro- and antiinflammatory cytokines in neurodegeneration.

Experimental and clinical damage to the brain leads to rapid upregulation of an array of cytokines predominantly by glia. These cytokines may exert neurotoxic or neuroprotective actions. This paper will focus on the pro-inflammatory cytokine interleukin-1 (IL-1), which participates in diverse forms of brain damage including ischemia, brain trauma, and excitotoxic injury. Administration of low doses of IL-1 markedly exacerbates these forms of brain damage, whereas blocking IL-1 release or actions reduces neuronal death. IL-1 receptor antagonist (IL-1ra) is also upregulated by brain damage (mainly by neurons) and acts as an endogenous inhibitor of neurodegeneration, presumably by blocking IL-1 actions on its receptor. We have studied the actions of both IL-1 and IL-1ra in experimental models of ischemic and neurotoxic injury in rats, and have found site-specific effects within the striatum. On the basis of this and further work, we propose that IL-1 can exacerbate cell death in these conditions by modifying polysynaptic anterograde pathways leading from the striatum to the cortex. The precise nature of these pathways remains undetermined, as do the underlying mechanisms by which IL-1 can exert its effects, but appear to involve induction of IL-1 in specific brain regions and activation of cortical glutamatergic pathways.

Interleukin-1 in the brain: mechanisms of action in acute neurodegeneration.

Interleukin-1 (IL-1) exerts a number of diverse actions in the brain, and it is currently well accepted that it contributes to experimentally induced neurodegeneration. Much of this is based on studies using the IL-1 receptor antagonist, which inhibits cell death caused by ischemia, brain injury, or excitotoxins. Our aim is to determine how and where in the brain IL-1 acts to produce these effects. Most of the neurodegenerative effects of IL-1 are thought to be through IL-1 beta. However, we have data implicating IL-1 alpha in excitotoxic cell death. Furthermore mice lacking both IL-1 alpha and IL-1 beta show dramatically reduced ischemic cell death, whereas deletion of IL-1 alpha or IL-1 beta alone fails to modify damage. It has also been demonstrated that IL-1 exacerbates ischemic injury in mice in the absence of the type I IL-1 receptor, suggesting the existence of novel IL-1 receptors in the brain. IL-1 also dramatically exacerbates neuronal loss in response to intrastriatal administration of the excitotoxin AMPA in the rat brain, an effect accompanied by marked increases in cytokine expression in the frontoparietal cortex, which precedes subsequent cell death in this region. Intrastriatal AMPA also results in limbic seizures that are exacerbated by IL-1, and we hypothesize, therefore, that IL-1 exacerbates cell death through increased seizure activity. Therefore, IL-1 appears to induce acute neurodegeneration through a number of mechanisms.

Dissociation between the effects of interleukin-1 on excitotoxic brain damage and body temperature in the rat.

The cytokine interleukin-1beta (IL-1beta) mediates and exacerbates excitotoxic brain damage in the rat striatum. Co-injection of the selective glutamate receptor agonist alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (S-AMPA) with human recombinant IL-1beta (hrIL-1beta) in the striatum of rats results in both local (striatal) damage and extensive, distant neuronal death in the cortex. The objective of the present study was to investigate the mechanisms underlying IL-1beta actions on excitotoxic damage, and to determine whether this effect of IL-1beta, a potent pyrogen, is due to modification of body temperature. Striatal infusion of S-AMPA (7.5 nmol) in anaesthetised rats produced localised striatal damage. Intrastriatal co-infusion of hrIL-1beta (10 ng) with S-AMPA caused similar striatal damage, but also produced extensive cortical damage, together with a modest increase in body temperature (0.9 degrees C) compared to rats infused with S-AMPA alone. Infusion of S-AMPA into the striatum, together with intracerebroventricular (i.c.v.) injection of hrIL-1beta, produced a similar rise in temperature to striatal co-infusion of S-AMPA and hrIL-1beta, but resulted in only local (striatal) neuronal damage, that was similar to that caused by striatal infusion of S-AMPA alone. These data suggest that the effects of IL-1beta on AMPA-receptor mediated neuronal damage in the striatum can be dissociated from its pyrogenic effects on body temperature.

Influence of corticotrophin releasing factor on neuronal cell death in vitro and in vivo.

Several studies have demonstrated that antagonists of the corticotrophin releasing factor (CRF) receptor markedly inhibit experimentally induced excitotoxic, ischaemic and traumatic brain injury in the rat, and that CRF expression is elevated in response to experimentally induced stroke or traumatic brain injury. CRF is also induced by the pro-inflammatory cytokine interleukin 1 (IL-1), which participates in various forms of neurodegeneration. The aim of this study was to test the hypothesis that CRF is toxic directly in vivo or in vitro. In primary cultures of rat cortical neurons, exposure to CRF (10 pM-100 nM) for 24 h failed to cause cell death directly, or to modify the neurotoxic effects of N-methyl-D-aspartate (NMDA). Similarly, infusion of CRF (0.3-5 microg) into specific brain regions of the rat did not induce cell death and did not significantly alter the neuronal damage produced by infusion of excitatory amino acids. These data demonstrate that CRF is not directly neurotoxic, and suggest that either CRF mediates neuronal damage by indirect actions (e.g. on the vasculature) and/or that CRF is not the endogenous ligand which contributes to neurodegeneration through activation of CRF receptors.

Measurement of valproic acid in serum as the 4-bromophenacyl ester by high performance liquid chromatography.

A method for the analysis of valproic acid in plasma or serum has been developed. The specimen is deproteinized with acetonitrile containing nonanoic acid as internal standard and an aliquot of supernate is heated at 70 degrees C for 15 min with 4-bromophenacyl bromide at pH 7. An aliquot of the reaction mixture is subjected to reverse phase high performance liquid chromatography at ambient temperature and a detector wavelength of 254 nm. Drug and internal standard eluted within 6 min. The method correlated well with a gas chromatographic procedure. Recoveries ranged from 85 to 108% and the day-to-day coefficients of variation ranged from 3.8 to 10.7%.

Scintimammography: a pictorial review of the variable normal, post surgical and pathological appearances.

Scintimammography has a high specificity and sensitivity for the detection of primary and metastatic breast carcinoma and in the evaluation of the postoperative breast. This review highlights the spectrum of pathological features as well as the normal postoperative and postreconstruction changes and the variable normal appearances that may be encountered with this technique.

Has the breast cancer 'two week wait' guarantee for assessment made any difference?

AIMS: To determine the referral practice of GPs to a Fast Access Breast Clinic before and after the implementation of the 'two week wait' and to demonstrate the impact of this guarantee on the detection rate of breast cancer and access to the Breast Clinic. METHODS: A complete audit cycle was performed in a District General hospital (Eastbourne District General Hospital). The main outcome measures were detected breast cancer, clinical accuracy of the GPs and the waiting time for a Fast Access breast clinic. RESULTS: Prior to the implementation of the Government's Directive, the detection rate of breast cancer was 22% and all cancers were seen within 1 week of referral letter. The routine waiting time for non-urgent assessment was 4 weeks. The clinical accuracy of GP referrals was 42%. On the basis of the findings in the first part of the audit, guidelines were sent to the GPs to aid in their referrals to FABC, prior to the 1 April deadline. After the implementation the re-audit showed that the cancer detection rate had dropped to 19% and only 85% of patients were seen in 2 weeks. The routine wait for non-urgent assessment had gone up to a minimum of 8 weeks. The clinical accuracy had slightly increased to 49%. CONCLUSION: With the transfer of power on deciding the urgency of referrals from the specialist to the GP, there has been a decline in the cancer detection rate and an increase in the waiting time for patients not deemed urgent by the GPs. The decision on the urgency of referral should be in the hands of the relevant specialist who individually can decide the best way to run their Fast Access Breast Clinics and thus achieve the government's guarantee of a maximum two week wait for patients suspected of having breast cancer.

Audit of referral practice to a fast-access breast clinic before the guaranteed 2-week wait.

Currently in the fast-access breast clinic at Eastbourne District General Hospital, the specialist determines the level of urgency of a referral. With the new '2-week wait' imposed by the Government since 1 April 1999, the determination of urgency has transferred to the general practitioner. Therefore, we decided to audit the current situation to see whether the guidelines were adequate for this change of emphasis. A total of 100 consecutive patients referred to the fast-access breast clinic were evaluated to assess the quality of referrals and the effectiveness of the clinic. Only 80% of the referrals adhered to the guidelines; 73% of the referrals were deemed by the specialist to be urgent and seen within the 2-week time period. All patients who were subsequently diagnosed as having breast cancer were seen within 2 weeks. The audit indicates that further specific changes could be made to the guidelines to improve the referral practice.

Prolene plug repair for femoral hernia.

We present the technique and our results in using a plug of Prolene mesh to repair femoral hernias. By filling the femoral canal with a Prolene plug we achieved a sound hernia repair. Our results allow us to recommend the technique to others.

An Endovascular Perforation Model of Subarachnoid Haemorrhage in Rat Produces Heterogeneous Infarcts that Increase with Blood Load.

Subarachnoid haemorrhage (SAH) is a devastating disease and a major burden on society. Despite this, pharmacological treatment options are limited. Appropriate animal modelling of SAH is essential for the development of neuroprotective drugs, but experimental SAH often fails to produce widespread neuronal loss, as frequently seen in humans. We report that a recently described modification of the endovascular perforation model in rat produced widespread heterogeneous infarcts 72 h after SAH. Cerebral blood flow (CBF) was monitored, with or without intracranial pressure (ICP) measurement, for 1 h after induction of SAH. Blood load size was assessed, and brain injury was quantified at 72 h using histological staining, blood brain barrier breakdown assessment and immunofluorescent imaging of neuronal viability and microglial activation. Results showed that ICP measurement allowed for faster recovery of CBF, potentially reducing brain injury. Larger subarachnoid blood loads predicted more extensive neuronal damage which was easily quantified with the combination of histological and immunohistochemical techniques. Thus, for the investigation of neuroprotective strategies after SAH, the present protocol produces quantifiable, clinically relevant, heterogeneous patterns of infarct due to large blood loads, high ICP and low CBF.