The peripheral immune response in hyperglycemic stroke.

OBJECTIVES: Hyperglycemia is common in acute ischemic stroke patients and is associated with poor clinical outcome. However, aggressive reduction of post-stroke hyperglycemia did not improve clinical outcome, suggesting that other mechanisms are playing a detrimental role in hyperglycemic stroke. We hypothesize that the acute post-stroke immune response is altered in the hyperglycemic state leading to higher mortality and morbidity. The objective of this study was to characterize temporal changes in circulating immune cells after stroke and their association with clinical outcomes in hyperglycemic compared to euglycemic patients. PATIENTS AND METHODS: This retrospective study included 97 (58 % euglycemic, 42 % hyperglycemic) patients presenting within 12 h of symptom onset of stroke. Blood neutrophil, monocyte and lymphocyte concentrations were measured sequentially for 96 h post stroke. Primary clinical outcome was the difference in the NIH stroke scale at admission compared to discharge. Secondary outcome measures included discharge disposition and the modified Rankin Scale (mRS) at 90 days. RESULTS: Circulating neutrophils were significantly higher in hyperglycemic than in euglycemic patients within the first 48 h post stroke, while lymphocyte counts trended to be lower. Hyperglycemic patients had higher mortality rates, less favorable discharge disposition and worse neurological function at 90 days. In both groups, the neutrophil to lymphocytes ratio ((NLR) remained strongly associated with neurological function at discharge within the first 24 h (p < 0.001), and remained significant in hyperglycemic patients up to 48 h (p < 0.001). Multivariate regression analysis showed no confounding by other factors and a significant correlation with differences in NIHSS score (CI; - 9.287 to -1.46, p = 0.0077**) and NLR (CL; 0.6058-6.901, p = 0.0203*) in hyperglycemic patients. CONCLUSIONS: Our data suggests that circulating immune cells play an important role in mediating poor clinical outcome in hyperglycemic patients following stroke. The NLR is a strong predictor of neurological outcomes in hyperglycemic patients. Thus, the modulation of immune cells may be a viable therapeutic approach to improve outcomes for this high risk group.

Does the response to aspirin and clopidogrel vary over 6 months in patients with ischemic heart disease?

BACKGROUND: Dual-antiplatelet therapy (DAPT) with aspirin and a P2Y12 inhibitor, mostly clopidogrel, is the default therapy in both acute coronary syndrome (ACS) and after intracoronary stents. It is well established that responses to antiplatelet therapy (APT), particularly clopidogrel, are subject to considerable interindividual variability. OBJECTIVES: We investigated whether responses to APT in individuals vary significantly over time. METHODS: Simultaneous assay with VerifyNow(™) and short thrombelastography (s-TEG) was performed before and at four time points over 6 months after hospital discharge in 40 patients receiving DAPT. Serum thromboxane B2 levels were also measured. RESULTS: While aspirin response units (ARU) by VerifyNow(™) and serum thromboxane B2 levels remained stable over time, arachidonic acid (AA)-mediated platelet aggregation with s-TEG (i.e. area under the curve at 15 min in AA channel, AUC15AA ) increased at 1 week compared with predischarge (P < 0.008). In addition, platelet reactivity units (PRU) by VerifyNow(™) (P = 0.046) and adenosine diphosphate (ADP)-mediated platelet aggregation with s-TEG (i.e. AUC15ADP ) also increased at 1 week compared with predischarge (P = 0.026). There were no significant changes in either platelet reactivity or rates of high on-treatment platelet reactivity while receiving clopidogrel beyond 1 week. CONCLUSIONS: This study demonstrates important variability in responses to APT within individuals between predischarge and 1 week but not thereafter. The use of a single early (predischarge) platelet function assay as an indicator of future response may therefore be flawed. The design of future strategies to assess individual responses for tailored therapy needs to take this into account.

Takotsubo (apical ballooning) syndrome in the recovery period following dobutamine stress echocardiography: a first report.

We report a case of Takotsubo syndrome occurring in the recovery phase after a dobutamine stress echocardiogram. Takotsubo syndrome is a widely acknowledged cause of reversible left ventricular systolic dysfunction. It has garnered much attention from the cardiological community since its presentation frequently mimics that of ST-segment elevation myocardial infarction. The exact aetiology remains incompletely defined, although stress is recognized frequently as a precipitating factor. In recent years it has emerged that stress testing, as part of a patient's investigative assessment, can also induce Takotsubo's syndrome. All prior reports of dobutamine-induced Takotsubo's syndrome have described apical ballooning at peak stress. We describe the case of an 85-year-old lady who developed apical ballooning in the recovery period after a dobutamine stress echocardiogram, despite having normal left ventricular wall motion at rest and at peak stress. We believe this to be the first such case reported in the literature. Dobutamine stress testing can precipitate Takotsubo's syndrome not just at peak stress but also during the recovery period. All those performing dobutamine stress tests should be aware of this rare but potentially important complication.

Improved membrane protein solubilization and clean-up for optimum two-dimensional electrophoresis utilizing GLUT-1 as a classic integral membrane protein.

Two-dimensional (2-D) electrophoresis remains a primary resolving tool for proteomic analyses. The final number of proteins resolved by 2-D electrophoresis depends on their respective solubility, size, charge, and isoelectric point. While water-soluble cytosolic proteins have often been well represented in 2-D maps, the same is not true with membrane proteins. Highly hydrophobic in nature, membrane proteins are poorly resolved in 2-D gels due to problems associated primarily with sample preparation. This is of especial concern in neuroscience studies where many proteins of interest are membrane bound. In the current work, we present a substantially improved sample preparation protocol for membrane proteins utilizing the GLUT-1 glucose transporter from brain microvessels as an example of a typical membrane protein. GLUT-1 (SLC2A1; solute carrier family 2 (facilitated glucose transporter), member 1) is a 55kD glycoprotein that contains 12 membrane-spanning alpha helices that impart the protein its characteristic hydrophobicity. GLUT-1 based on its amino acid sequence has a theoretical isoelectric point (pI) of 8.94. Using a combination of the non-ionic detergents, n-dodecyl-beta-maltoside (DDM) and amido sulphobetaine-14 (ASB-14) for sample solubilization, and a modification of the Bio-Rad 2-D clean-up protocol involving trichloroacetic acid (TCA)/acetone, we obtained near complete solubilization of GLUT-1 and greater than 90% recovery of this membrane protein in 1-D and 2-D Western blots. The total number of proteins resolved also increased dramatically in Deep Purple total protein stains using our improved protocol.

Ferritin: a novel mechanism for delivery of iron to the brain and other organs.

Traditionally, transferrin has been considered the primary mechanism for cellular iron delivery, despite suggestive evidence for additional iron delivery mechanisms. In this study we examined ferritin, considered an iron storage protein, as a possible delivery protein. Ferritin consists of H- and L-subunits, and we demonstrated iron uptake by ferritin into multiple organs and that the uptake of iron is greater when the iron is delivered via H-ferritin compared with L-ferritin. The delivery of iron via H-ferritin but not L-ferritin was significantly decreased in mice with compromised iron storage compared with control, indicating that a feedback mechanism exists for H-ferritin iron delivery. To further evaluate the mechanism of ferritin iron delivery into the brain, we used a cell culture model of the blood-brain barrier to demonstrate that ferritin is transported across endothelial cells. There are receptors that prefer H-ferritin on the endothelial cells in culture and on rat brain microvasculature. These studies identify H-ferritin as an iron transport protein and suggest the presence of an H-ferritin receptor for mediating iron delivery. The relative amount of iron that could be delivered via H-ferritin could make this protein a predominant player in cellular iron delivery.

Improving the positive predictive value of exercise testing in women.

OBJECTIVE: To identify exercise test variables that can improve the positive predictive value of exercise testing in women. DESIGN: Cohort study. SETTING: Regional cardiothoracic centre. SUBJECTS: 1286 women and 1801 men referred by primary care physicians to a rapid access chest pain clinic, of whom 160 women and 406 men had ST depression of at least 1 mm during exercise testing. The results for 136 women and 124 men with positive exercise tests were analysed. MAIN OUTCOME MEASURES: The proportion of women with a positive exercise test who could be identified as being at low risk for prognostic coronary heart disease and the resulting improvement in the positive predictive value. RESULTS: Independently of age, an exercise time of more than six minutes, a maximum heart rate of more than 150 beats/min, and an ST recovery time of less than one minute were the variables that best identified women at low risk. One to three of these variables identified between 11.8% and 41.2% of women as being at low risk, with a risk for prognostic disease of between 0-11.5%. The positive predictive value for the remaining women was improved from 47.8% up to 61.5%, and the number of normal angiograms was potentially reducible by between 21.1-54.9%. By the same criteria, men had higher risks for prognostic disease. CONCLUSIONS: A strategy of discriminating true from false positive exercise tests is worthwhile in women but less successful in men.

Distribution of divalent metal transporter 1 and metal transport protein 1 in the normal and Belgrade rat.

Iron accumulation in the brain occurs in a number of neurodegenerative diseases. Two new iron transport proteins have been identified that may help elucidate the mechanism of abnormal iron accumulation. The Divalent Metal Transporter 1 (DMT1), is responsible for iron uptake from the gut and transport from endosomes. The Metal Transport Protein 1 (MTP1) promotes iron export. In this study we determined the cellular and regional expression of these two transporters in the brains of normal adult and Belgrade rats. Belgrade rats have a defect in DMT1 that is associated with lower levels of iron in the brain. In the normal rat, DMT1 expression is highest in neurons in the striatum, cerebellum, thalamus, ependymal cells lining the third ventricle, and vascular cells throughout the brain. The staining in the ependymal cells and endothelial cells suggests that DMT1 has an important role in iron transport into the brain. In Belgrade rats, there is generalized decrease in immunodetectable DMT1 compared to normal rats except in the ependymal cells. This decrease in immunoreactivity, however, was absent on immunoblots. The immunoblot analysis indicates that this protein did not upregulate to compensate for the chronic defect in iron transport. MTP1 staining is found in most brain regions. MTP1 expression in the brain is robust in pyramidal neurons of the cerebral cortex but is not detected in the vascular endothelial cells and ependymal cells. MTP1 staining in Belgrade rats was decreased compared to normal, but similar to DMT1 this decrease was not corroborated by immunoblotting. These results indicate that DMT1 and MTP1 are involved in brain iron transport and this involvement is regionally and cellularly specific.

Glucose transporter asymmetries in the bovine blood-brain barrier.

The transport of glucose across the mammalian blood-brain barrier is mediated by the GLUT1 glucose transporter, which is concentrated in the endothelial cells of the cerebral microvessels. Several studies supported an asymmetric distribution of GLUT1 protein between the luminal and abluminal membranes (1:4) with a significant proportion of intracellular transporters. In this study we investigated the activity and concentration of GLUT1 in isolated luminal and abluminal membrane fractions of bovine brain endothelial cells. Glucose transport activity and glucose transporter concentration, as determined by cytochalasin B binding, were 2-fold greater in the luminal than in the abluminal membranes. In contrast, Western blot analysis using a rabbit polyclonal antibody raised against the C-terminal 20 amino acids of GLUT1 indicated a 1:5 luminal:abluminal distribution. Western blot analysis with antibodies raised against either the intracellular loop of GLUT1 or the purified erythrocyte protein exhibited luminal:abluminal ratios of 1:1. A similar ratio was observed when the luminal and abluminal fractions were exposed to the 2-N-4[(3)H](1-azi-2,2,2,-trifluoroethyl)benzoxyl-1,3-bis-(d-mannos-4-yloxyl)-2-propylamine ([(3)H]ATB-BMPA) photoaffinity label. These observations suggest that either an additional glucose transporter isoform is present in the luminal membrane of the bovine blood-brain barrier or the C-terminal epitope of GLUT1 is "masked" in the luminal membrane but not in the abluminal membranes.

Experimental stroke in the female diabetic, db/db, mouse.

Diabetic hyperglycemia increases brain damage after cerebral ischemia in animals and humans, although the underlying mechanisms remain unclear. Gender-linked differences in ischemic tolerance have been described but have not been studied in the context of diabetes. In the current study, we used a model of unilateral common carotid artery ligation, combined with systemic hypoxia, to study the effects of diabetes and gender on hypoxic-ischemic (HI) brain damage in the genetic model of Type II diabetes, the db/db, mouse. Male and female, control and db/db, mice were subjected to right common carotid artery ligation followed by varying periods of hypoxia (8% oxygen/92% nitrogen) to assess mortality, infarct volume, and tissue damage by light microscopic techniques. End-ischemic regional cerebral blood flow (CBF) was determined using [14C] iodoantipyrine autoradiography. Glycolytic and high energy phosphate compounds were measured in blood and brain by enzymatic and fluorometric techniques. Gender and diabetes had significant effects on mortality from HI and extent of brain damage in the survivors. Female mice were more resistant than their male counterparts, such that the severity (mortality and infarction size) in the male diabetics > female diabetics - male controls > female controls. Endischemic CBF and depletion of cerebral high energy reserves were comparable among all groups. Surprisingly, female diabetic mice were more hyperglycemic and demonstrated a greater prolonged lactacidosis than the males; however, they were more resistant to damage. The results suggest a unique pathophysiology of hypoxia-ischemia in the female diabetic brain.

Sex differences in investigation results and treatment in subjects referred for investigation of chest pain.

OBJECTIVE: To evaluate differences in investigation results and treatment between men and women referred for diagnostic treadmill exercise testing and coronary arteriography. DESIGN: Cohort study. SETTING: Tertiary cardiology centre. SUBJECTS: 1522 subjects referred by primary care physicians to an open access chest pain clinic for initial investigation of chest pain, of whom 485 were subsequently referred for coronary arteriography; and a similar cohort of 107 subjects referred directly by secondary care physicians for diagnostic coronary arteriography. MAIN OUTCOME MEASURES: Rates of positive exercise tests and rates for referral for arteriography and revascularisation according to sex. RESULTS: Overall, women were less likely to be referred for arteriography and revascularisation than men. However, men were more likely to have positive exercise tests, and for various exercise test diagnostic end points men were also more likely to have significant coronary artery disease. After taking this into account, there was no sex difference in referral rates for arteriography or revascularisation. CONCLUSIONS: There was no evidence of a sex bias resulting in inappropriate underinvestigation or undertreatment of women. However, the positive predictive value of treadmill exercise testing is low for women and further research is needed into how best to investigate women with chest pain.

An unusual atrial mass.

Giant coronary artery aneurysm is a rare manifestation of coronary artery disease. This patient presented at echocardiography and was successfully managed by surgical resection.

Prenatal expression of the GLUT4 glucose transporter in the mouse.

The GLUT4 glucose transporter is primarily expressed in skeletal muscle, heart and adipose tissue, where its expression is postnatal, coincident with the acquisition of insulin-regulated glucose transport. In muscle, contraction also regulates GLUT4 activity in the postnatal animal. Here we demonstrate that GLUT4 is expressed in the developing mouse embryo with specific tissue and spatiotemporal patterns. From embryonic day 9 (E9; E1 = day of copulation plug) to postnatal day 70 (P70), mice were analyzed for GLUT4 mRNA and protein expression by in situ hybridization, immunohistochemistry and immunoblot. Specificity was confirmed with sense riboprobe hybridization and peptide competition, respectively. At E9, GLUT4 was detected in the cranial neural folds in the outer (mantle) layer of the neuroepithelium. At E10, expression was present throughout the developing heart and was prominent in the endocardial cushions through E12. At E10-12, GLUT4 was also prominent in craniofacial mesenchyme. GLUT4 expression in cartilage and bone was evident at E12 and was maintained throughout early postnatal life. GLUT4 was apparent throughout embryonic development in the ventricular epithelium, choroid plexus and in the developing cerebellum. At birth, cardiac expression was reduced and GLUT4 was most evident in cartilage, bone and specific brain regions. In the latter, GLUT4 expression was most evident in the cerebellum, specifically in the external granular layer through P7 and in the internal granular layer thereafter. Maximal GLUT4 protein levels in the cerebellum were measured between P14 and P21 and were reduced in the adult brain. These findings suggest that GLUT4-mediated glucose transport may play important roles during development of the brain and nonneuronal tissues in the mouse embryo.