Prospective validation of the ABCD2 score for patients in the emergency department with transient ischemic attack

Background:

The ABCD2 score (Age, Blood pressure, Clinical features, Duration of symptoms and Diabetes) is used to identify patients having a transient ischemic attack who are at high risk for imminent stroke. However, despite its widespread implementation, the ABCD2 score has not yet been prospectively validated. We assessed the accuracy of the ABCD2 score for predicting stroke at 7 (primary outcome) and 90 days.

Methods:

This prospective cohort study enrolled adults from eight Canadian emergency departments who had received a diagnosis of transient ischemic attack. Physicians completed data forms with the ABCD2 score before disposition. The outcome criterion, stroke, was established by a treating neurologist or by an Adjudication Committee. We calculated the sensitivity and specificity for predicting stroke 7 and 90 days after visiting the emergency department using the original “high-risk” cutpoint of an ABCD2 score of more than 5, and the American Heart Association recommendation of a score of more than 2.

Results:

We enrolled 2056 patients (mean age 68.0 yr, 1046 (50.9%) women) who had a rate of stroke of 1.8% at 7 days and 3.2% at 90 days. An ABCD2 score of more than 5 had a sensitivity of 31.6% (95% confidence interval [CI] 19.1–47.5) for stroke at 7 days and 29.2% (95% CI 19.6–41.2) for stroke at 90 days. An ABCD2 score of more than 2 resulted in sensitivity of 94.7% (95% CI 82.7–98.5) for stroke at 7 days with a specificity of 12.5% (95% CI 11.2–14.1). The accuracy of the ABCD2 score as calculated by either the enrolling physician (area under the curve 0.56; 95% CI 0.47–0.65) or the coordinating centre (area under the curve 0.65; 95% CI 0.57–0.73) was poor.

Interpretation:

This multicentre prospective study involving patients in emergency departments with transient ischemic attack found the ABCD2 score to be inaccurate, at any cut-point, as a predictor of imminent stroke. Furthermore, the ABCD2 score of more than 2 that is recommended by the American Heart Association is nonspecific.There are approximately 100 visits to the emergency department per 100 000 population for transient ischemic attack each year.¹ Although often considered benign, transient ischemic attack carries a risk of imminent stroke. Studies have shown that the risk of stroke is 0.2%–10% within 7 days of the first transient ischemic attack, and this risk increases to 1.2%–12% at 90 days.^2–9 Stroke continues to be the leading cause of disability among adults and the third-leading cause of death in North America.^10,11 Identifying people with transient ischemic attack who are at high risk of stroke is an opportunity to prevent stroke.^3,4 However, urgent investigation of all transient ischemic attacks would require substantial resources. Three studies have attempted to develop clinical decision rules (i.e., scores) for assessing whether a patient with transient ischemic attack is at high risk of stroke.^9,12,13 Combined, these studies led to the development of the ABCD2 (Age, Blood pressure, Clinical features, Duration of symptoms and Diabetes) score. However, despite its widespread implementation, the ABCD2 score has not yet been prospectively validated.^12,14–18 This essential step in the development of rules for making clinical predictions has recently been requested.^14,19–21The objective of this study was to externally validate the ABCD2 score as a tool for identifying patients seen in the emergency department with transient ischemic attack who are at high risk of stroke within 7 (primary outcome) and 90 days (one of the secondary outcomes).     

Cloning and optimization of induction conditions for mature PsaA (pneumococcal surface adhesin A) expression in Escherichia coli and recombinant protein stability during long-term storage

The gene corresponding to mature PsaA from Streptococcus pneumoniae serotype 14 was cloned into a plasmid with kanamycin resistance and without a purification tag in Escherichia coli to express high levels of the recombinant protein for large-scale production as a potential vaccine candidate or as a carrier for polysaccharide conjugation at Bio-Manguinhos/Fiocruz. The evaluation of induction conditions (IPTG concentration, temperature and time) in E. coli was accomplished by experimental design techniques to enhance the expression level of mature recombinant PsaA (rPsaA). The optimization of induction process conditions led us to perform the recombinant protein induction at 25°C for 16 h, with 0.1mM IPTG in Terrific Broth medium. At these conditions, the level of mature rPsaA expression obtained in E. coli BL21 (DE3) Star by pET28a induction with IPTG was in the range of 0.8 g/L of culture medium, with a 10-fold lower concentration of inducer than usually employed, which contributes to a less expensive process. Mature rPsaA expressed in E. coli BL21 (DE3) Star accounted for approximately 30-35% of the total protein. rPsaA purification by ion exchange allowed the production of high-purity recombinant protein without fusion tags. The results presented in this work confirm that the purified recombinant protein maintains its stability and integrity for long periods of time in various storage conditions (temperatures of 4 or -70°C using different cryoprotectors) and for at least 3 years at 4 or -70°C in PBS. The conformation of the stored protein was confirmed using circular dichroism. Mature rPsaA antigenicity was proven by anti-rPsaA mouse serum recognition through western blot analysis, and no protein degradation was detected after long periods of storage.     

Cellular dimensions and cell dynamics, or the difficulty over capturing time and space in the era of electron microscopy

The introduction of electron microscopy profoundly altered biomedical research, providing a tool for a more detailed but at the same time a spatially and temporally more restricted visual analysis. Examining the case study of Golgi apparatus research in the 1950s and 1960s, it will be shown how microscopists handled these challenges, and how these confrontations modified the general concept of cellular organization. This will also shed light on the artifact debate and on the question of scientific realism in the field of microscopy.     

The proteolipid protein promoter drives expression outside of the oligodendrocyte lineage during embryonic and early postnatal development

The proteolipid protein (Plp) gene promoter is responsible for driving expression of one of the major components of myelin--PLP and its splice variant DM-20. Both products are classically thought to express predominantly in oligodendrocytes. However, accumulating evidence suggests Plp expression is more widespread than previously thought. In an attempt to create a mouse model for inducing oligodendrocyte-specific gene deletions, we have generated transgenic mice expressing a Cre recombinase cDNA under control of the mouse Plp promoter. We demonstrate Plp promoter driven Cre expression is restricted predominantly to mature oligodendrocytes of the central nervous system (CNS) at postnatal day 28. However, crosses into the Rosa26(LacZ) and mT/mG reporter mouse lines reveal robust and widespread Cre activity in neuronal tissues at E15.5 and E10.5 that is not strictly oligodendrocyte lineage specific. By P28, all CNS tissues examined displayed high levels of reporter gene expression well outside of defined white matter zones. Importantly, our study reinforces the emerging idea that Plp promoter activity is not restricted to the myelinating cell lineage, but rather, has widespread activity both during embryonic and early postnatal development in the CNS. Specificity of the promoter to the oligodendrocyte cell lineage, as shown through the use of a tamoxifen inducible Plp-CreER(t) line, occurs only at later postnatal stages. Understanding the temporal shift in Plp driven expression is of consequence when designing experimental models to study oligodendrocyte biology.     

Treatment of poor-risk myelodysplastic syndromes and acute myeloid leukemia with a combination of 5-azacytidine and valproic acid

5-azacytidine (AZA) has become standard treatment for patients with higher-risk myelodysplastic syndrome (MDS). Response rate is about 50% and response duration is limited. Histone deactylase (HDAC) inhibitors are attractive partners for epigenetic combination therapy. We treated 24 patients with AZA (100?mg/m(2), 5?days) plus valproate (VPA; continuous dosing, trough serum level 80-110?μg/ml). According to WHO classification, 5 patients had MDS, 2 had MDS/MPD, and 17 had acute myeloid leukemia (AML). Seven patients (29%) had previously received intensive chemotherapy, and five had previous HDAC inhibitor treatment. The overall response rate was 37% in the entire cohort but significantly higher (57%) in previously untreated patients, especially those with MDS (64%). Seven (29%) patients achieved CR (29%) and two PR (8%), respectively. Hematological CR was accompanied by complete cytogenetic remission according to conventional cytogenetics in all evaluable cases. Some patients also showed complete remission according to FISH on bone marrow mononuclear cells and CD34(+) peripheral blood cells, as well as by follow-up of somatic mitochondrial DNA mutations. Four additional patients achieved at least marrow remissions. Factors influencing response were AML (vs. MDS), marrow blast count, pretreatment, transfusion dependency, concomitant medication with hydroxyurea, and valproic acid (VPA) serum level. This trial is the first to assess the combination of AZA plus VPA without additional ATRA. A comparatively good CR rate, relatively short time to response, and the influence of VPA serum levels on response suggest that VPA provided substantial additional benefit. However, the importance of HDAC inhibitors in epigenetic combination therapy can only be proven by randomized trials.     

YeeI, a novel protein involved in modulation of the activity of the glucose-phosphotransferase system in Escherichia coli K-12

The membrane-bound protein EIICB(Glc) encoded by the ptsG gene is the major glucose transporter in Escherichia coli. This protein is part of the phosphoenolpyruvate:glucose-phosphotransferase system, a very important transport and signal transduction system in bacteria. The regulation of ptsG expression is very complex. Among others, two major regulators, the repressor Mlc and the cyclic AMP-cyclic AMP receptor protein activator complex, have been identified. Here we report identification of a novel protein, YeeI, that is involved in the regulation of ptsG by interacting with Mlc. Mutants with reduced activity of the glucose-phosphotransferase system were isolated by transposon mutagenesis. One class of mutations was located in the open reading frame yeeI at 44.1 min on the E. coli K-12 chromosome. The yeeI mutants exhibited increased generation times during growth on glucose, reduced transport of methyl-alpha-d-glucopyranoside, a substrate of EIICB(Glc), reduced induction of a ptsG-lacZ operon fusion, and reduced catabolite repression in lactose/glucose diauxic growth experiments. These observations were the result of decreased ptsG expression and a decrease in the amount of EIICB(Glc). In contrast, overexpression of yeeI resulted in higher expression of ptsG, of a ptsG-lacZ operon fusion, and of the autoregulated dgsA gene. The effect of a yeeI mutation could be suppressed by introducing a dgsA deletion, implying that the two proteins belong to the same signal transduction pathway and that Mlc is epistatic to YeeI. By measuring the surface plasmon resonance, we found that YeeI (proposed gene designation, mtfA) directly interacts with Mlc with high affinity.     

Phosphoprotein enriched in astrocytes-15 kDa expression inhibits astrocyte migration by a protein kinase C delta-dependent mechanism

Phosphoprotein enriched in astrocytes-15 kDa (PEA-15), a phosphoprotein enriched in astrocytes, inhibits both apoptosis and proliferation in normal and cancerous cells. Here, analysis of PEA-15 expression in glioblastoma organotypic cultures revealed low levels of PEA-15 in tumor cells migrating away from the explants, regardless of the expression levels in the originating explants. Because glioblastomas are highly invasive primary brain tumors that can originate from astrocytes, we explored the involvement of PEA-15 in the control of astrocyte migration. PEA-15-/- astrocytes presented an enhanced motility in vitro compared with their wild-type counterparts. Accordingly, NIH-3T3 cells transfected by green fluorescent protein-PEA-15 displayed a reduced migration. Reexpression of PEA-15 restored PEA-15-/- astrocyte motility to wild-type levels. Pharmacological manipulations excluded a participation of extracellular signal-regulated kinase/mitogen-activated protein kinase, phosphatidylinositol 3-kinase/Akt, and calcium/calmodulin-dependent protein kinase II in this effect of PEA-15. In contrast, treatment by bisindolylmaleimide, G?6976, and rottlerin, and chronic application of phorbol 12-myristate 13-acetate and/or bryostatin-1 indicated that PKC delta mediated PEA-15 inhibition of astrocyte migration. PEA-15-/- astrocytes constitutively expressed a 40-kDa form of PKC delta that was down-regulated upon PEA-15 reexpression. Together, these data reveal a new function for PEA-15 in the inhibitory control of astrocyte motility through a PKC delta-dependent pathway involving the constitutive expression of a catalytic fragment of PKC delta.     

CB1 cannabinoid receptor antagonism: a new strategy for the treatment of liver fibrosis

Hepatic fibrosis, the common response associated with chronic liver diseases, ultimately leads to cirrhosis, a major public health problem worldwide. We recently showed that activation of hepatic cannabinoid CB2 receptors limits progression of experimental liver fibrosis. We also found that during the course of chronic hepatitis C, daily cannabis use is an independent predictor of fibrosis progression. Overall, these results suggest that endocannabinoids may drive both CB2-mediated antifibrogenic effects and CB2-independent profibrogenic effects. Here we investigated whether activation of cannabinoid CB1 receptors (encoded by Cnr1) promotes progression of fibrosis. CB1 receptors were highly induced in human cirrhotic samples and in liver fibrogenic cells. Treatment with the CB1 receptor antagonist SR141716A decreased the wound-healing response to acute liver injury and inhibited progression of fibrosis in three models of chronic liver injury. We saw similar changes in Cnr1-/- mice as compared to wild-type mice. Genetic or pharmacological inactivation of CB1 receptors decreased fibrogenesis by lowering hepatic transforming growth factor (TGF)-beta1 and reducing accumulation of fibrogenic cells in the liver after apoptosis and growth inhibition of hepatic myofibroblasts. In conclusion, our study shows that CB1 receptor antagonists hold promise for the treatment of liver fibrosis.     

Functional Exhaustion of CD4+ T Lymphocytes during Primary Cytomegalovirus Infection

Human CMV establishes lifelong persistence after primary infection. Chronic CMV infection is associated with intermittent viral reactivation inducing high frequencies of CD4(+) T lymphocytes with potent antiviral and helper properties. Primary CMV infection is characterized by an intense viral replication lasting for several months. The impact of this prolonged exposure to high Ag loads on the functionality of CD4(+) T cells remains incompletely understood. In pregnant women with primary CMV infection, we observed that CMV-specific CD4(+) T lymphocytes had a decreased capacity to proliferate and to produce IL-2. A very large proportion of CMV-specific CD4(+) T cells had downregulated the expression of CD28, a costimulatory molecule centrally involved in the production of IL-2. Unexpectedly, both CD28(-) and CD28(+)CD4(+) T cells produced low levels of IL-2. This defective production of IL-2 was part of a larger downregulation of cytokine production. Indeed, CMV-specific CD4(+) T cells produced lower amounts of IFN-γ and TNF-α and showed lower functional avidity during primary as compared with chronic infection. Increased programmed death-1 expression was observed in CD28(+) CMV-specific CD4(+) T cells, and programmed death-1 inhibition increased proliferative responses. These results indicate that primary CMV infection is associated with the exhaustion of CMV-specific CD4(+) T cells displaying low functional avidity for viral Ags.