Interleukin-6, C-reactive protein, fibrinogen, and risk of recurrence after ischaemic stroke: Systematic review and meta-analysis.

BACKGROUND: Recent randomised trials showed benefit for anti-inflammatory therapies in coronary disease but excluded stroke. The prognostic value of blood inflammatory markers after stroke is uncertain and guidelines do not recommend their routine measurement for risk stratification. METHODS: We performed a systematic review and meta-analysis of studies investigating the association of C-reactive protein (CRP), interleukin-6 (IL-6) and fibrinogen and risk of recurrent stroke or major vascular events (MVEs). We searched EMBASE and Ovid Medline until 10/1/19. Random-effects meta-analysis was performed for studies reporting comparable effect measures. RESULTS: Of 2,515 reports identified, 39 met eligibility criteria (IL-6, n = 10; CRP, n = 33; fibrinogen, n = 16). An association with recurrent stroke was reported in 12/26 studies (CRP), 2/11 (fibrinogen) and 3/6 (IL-6). On random-effects meta-analysis of comparable studies, CRP was associated with an increased risk of recurrent stroke [pooled hazard ratio (HR) per 1 standard-deviation (SD) increase in loge-CRP (1.14, 95% CI 1.06-1.22, p < 0.01)] and MVEs (pooled HR 1.21, CI 1.10-1.34, p < 0.01). Fibrinogen was also associated with recurrent stroke (HR 1.26, CI 1.07-1.47, p < 0.01) and MVEs (HR 1.31, 95% CI 1.15-1.49, p < 0.01). Trends were identified for IL-6 for recurrent stroke (HR per 1-SD increase 1.17, CI 0.97-1.41, p = 0.10) and MVEs (HR 1.22, CI 0.96-1.55, p = 0.10). CONCLUSION: Despite evidence suggesting an association between inflammatory markers and post-stroke vascular recurrence, substantial methodological heterogeneity was apparent between studies. Individual-patient pooled analysis and standardisation of methods are needed to determine the prognostic role of blood inflammatory markers and to improve patient selection for randomised trials of inflammatory therapies.

Thrombolysis for stroke in Ireland: increasing access and maintaining safety in a challenging environment.

BACKGROUND: In the setting of a national audit of acute stroke services, we examined the delivery of thrombolytic therapy for ischaemic stroke and whether current practice was achieving safe outcomes and consistent delivery for patients. METHOD: Data obtained from the recent national stroke audit was compared against previous Irish audit, the most recent SSNAP UK stroke audit and the Safe Implementation of Thrombolysis in Stroke-Monitoring Study (SITS-MOST) study. RESULTS: Thrombolysis was provided in 27 acute hospitals throughout Ireland during the period assessed with 82% (22/27) providing 24/7 access, the remaining sites using redirect policies. Decision to thrombolyse was made by stroke trained consultants in 63% (17/27) of units, with general physicians and emergency medicine consultants covering the other units. Thrombolysis rate for non-haemorrhagic stroke was 11% (n = 80/742, CI 95% ±2.23) versus a 1% rate in the 2008 audit. Sites receiving patients through a redirect policy had the highest thrombolysis rate, an average of 24%. Nearly 30% of cases were thrombolysed on the weekend. Eighty-three percent of cases were managed in a stroke unit at some time during admission versus 54% of the national total cases. Thirty-seven percent of patients were ≥80 years old. The mortality rate was 11.3% versus the national mortality rate for non-thrombolysed ischaemic strokes of 10% (p > 0.5), and this is comparable to the SITS-MOST 2007 study 3-month mortality rate of 11.3% (p > 0.5). CONCLUSION: Stroke thrombolysis is being effectively and safely provided in acute stroke services in Ireland despite regular involvement of non-specialist staff. There is still potential to improve thrombolysis rate.

Phospholipid oxidation and carotenoid supplementation in Alzheimer's disease patients.

Alzheimer's disease (AD) is a progressive, neurodegenerative disease, characterised by decline of memory, cognitive function and changes in behaviour. Generic markers of lipid peroxidation are increased in AD and reactive oxygen species have been suggested to be involved in the aetiology of cognitive decline. Carotenoids are depleted in AD serum, therefore we have compared serum lipid oxidation between AD and age-matched control subjects before and after carotenoid supplementation. The novel oxidised phospholipid biomarker 1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC) was analysed using electrospray ionisation tandem mass spectrometry (MS) with multiple reaction monitoring (MRM), 8-isoprostane (IsoP) was measured by ELISA and ferric reducing antioxidant potential (FRAP) was measured by a colorimetric assay. AD patients (n=21) and healthy age-matched control subjects (n=16) were supplemented with either Macushield™ (10mg meso-zeaxanthin, 10mg lutein, 2mg zeaxanthin) or placebo (sunflower oil) for six months. The MRM-MS method determined serum POVPC sensitively (from 10µl serum) and reproducibly (CV=7.9%). At baseline, AD subjects had higher serum POVPC compared to age-matched controls, (p=0.017) and cognitive function was correlated inversely with POVPC (r=-0.37; p=0.04). After six months of carotenoid intervention, serum POVPC was not different in AD patients compared to healthy controls. However, POVPC was significantly higher in control subjects after six months of carotenoid intervention compared to their baseline (p=0.03). Serum IsoP concentration was unrelated to disease or supplementation. Serum FRAP was significantly lower in AD than healthy controls but was unchanged by carotenoid intervention (p=0.003). In conclusion, serum POVPC is higher in AD patients compared to control subjects, is not reduced by carotenoid supplementation and correlates with cognitive function.

Vascular risk assessment in older adults without a history of cardiovascular disease.

Modern cardiovascular risk prediction tools, which have their genesis in the Framingham Heart Study, have allowed more accurate risk stratification and targeting of treatments worldwide over the last seven decades. Better cardiovascular risk factor control during this time has led to a reduction in cardiovascular mortality and, at least in part, to improved life expectancy. As a result, western societies as a whole have seen a steady increase in the proportion of older persons in their populations. Unfortunately, several studies have shown that the same tools which have contributed to this increase cannot be reliably extrapolated for use in older generations. Recent work has allowed recalibration of existing models for use in older populations but these modified tools still require external validation before they can be confidently applied in clinical practice. Another complication is emerging evidence that aggressive risk factor modification in older adults, particularly more frail individuals, may actually be harmful. This review looks at currently available cardiovascular risk prediction models and the specific challenges faced with their use in older adults, followed by analysis of recent attempts at recalibration for this cohort. We discuss the issue of frailty, looking at our evolving understanding of its constituent features and various tools for its assessment. We also review work to date on the impact of frailty on cardiovascular risk modification and outline its potentially central role in determining the most sensible approach in older patients. We summarise the most promising novel markers of cardiovascular risk which may be of use in improving risk prediction in older adults in the future. These include markers of vascular compliance (such as aortic pulse wave velocity and pulse wave analysis), of endothelial function (such as flow mediated dilation, carotid intima-media thickness and coronary artery calcium scores), and also biochemical and circulating cellular markers.

Retrospective audit of a continuous infusion protocol for haemophilia A at a single haemophilia treatment centre.

Continuous infusion (CI) of factor concentrates has been suggested to decrease the risk of bleeding and reduce cost in the treatment of bleeding disorders. Concerns have also been raised regarding stability and sterility of products administered by CI, the risk of local thrombophlebitis and an association with the development of an inhibitor in mild haemophilia. A retrospective chart review was conducted to investigate a CI protocol regarding product use, maintenance of FVIII levels and the frequency of complications including inhibitor development. Twelve patients with haemophilia A received recombinant factor VIII by CI a total of 18 times between April 1998 and September 2003. Ages ranged from 4 months to 75 years and indications for treatment included severe bleeds and surgical prophylaxis. The protocol which was audited required a bolus of 50 U kg(-1) of FVIII followed by CI at an initial rate of 4 U kg(-1) hr(-1). All infusions were administered by i.v. infusion after diluting the reconstituted concentrate in saline. There were no documented cases of significant bleeding, adverse reactions, thrombophlebitis or infection. Two mild haemophilia A patients developed a low titre inhibitor after receiving CI. It is not clear in either case that CI was the main contributing factor. Our CI protocol will now be modified to use less product, aiming for more cost-effectiveness.

H2O2-dependent activation of GCLC-ARE4 reporter occurs by mitogen-activated protein kinase pathways without oxidation of cellular glutathione or thioredoxin-1.

The gp91phox homologue Nox1 produces H2O2, which induces cell growth, transformation, and tumorigenicity. However, it has not been clear whether H2O2 effects are mediated indirectly via a generally oxidizing cellular environment or whether H2O2 more directly targets specific signaling pathways. Here, we investigated signaling by H2O2 induced by Nox1 overexpression using a luciferase reporter regulated by the antioxidant response element ARE4. Surprisingly, Nox1-derived H2O2 activated the reporter gene 15-fold with no effect on the redox state of the major thiol antioxidant substances, glutathione and thioredoxin. H2O2 signaling to ARE4 was mediated by activation of both the c-Jun N-terminal kinase and ERK1/2 pathways modulated by Ras. Thus, "redox signaling" resulting in kinase signaling pathways is distinct from "oxidative stress," and is mediated by discrete, localized redox circuitry.

Erk activation is required for Nrf2 nuclear localization during pyrrolidine dithiocarbamate induction of glutamate cysteine ligase modulatory gene expression in HepG2 cells.

Pyrrolidine dithiocarbamate (PDTC) induction of the human glutamate cysteine ligase modulatory (GCLM) gene is dependent on activation of the mitogen-activated protein kinases (MAPKs) extracellular regulated kinase (Erk) and p38, and is not affected by protein kinase C (PKC) or PI3K inhibitors. Nrf2 binding to the electrophile response element (EpRE) located within the GCLM promoter is decreased after MAPK inhibition, suggesting that Nrf2 could be a downstream target of activated MAPK. To evaluate this hypothesis, a series of Nrf2 proteins harboring mutations in conserved consensus MAPK phosphorylation sites were developed and used in multiple functional assays. All mutated Nrf2 proteins tested interacted with the cytoplasmic repressor Keap1 in a manner indistinguishable from wild-type Nrf2. Furthermore, the mutant and wild-type Nrf2 proteins were similarly capable of transactivating an EpRE-containing GCLM/luciferase reporter transgene. Collectively these functional assays suggest that Nrf2 is not likely to be a direct downstream target of activated MAPK in vivo. However, treatment of HepG2 cells with MAPK inhibitors PD98059 and/or SB202190 prior to exposure to PDTC, reduced Nrf2 translocation to the nucleus, suggesting that MAPK-directed phosphorylation is a requirement for nuclear localization during PDTC induction of GCLM gene expression.

Severe cardiac failure associated with presumed jellyfish sting. Irukandji syndrome?

We present a retrospective review of twelve cases of Irukandji syndrome associated with pulmonary oedema. This is a life-threatening envenoming due to a presumed jellyfish sting throughout Northern Australia, although only one case occurred outside North Queensland. Patients presented with significant and ongoing pain, tachycardia and hypertension. Half the patients became hypotensive requiring inotropic support. Cardiac echocardiography revealed significant cardiac dysfunction. Six patients required ventilatory support. There was no death reported due to pulmonary oedema, but one patient died of intracerebral haemorrhage. We believe patients may develop a toxin associated cardiomyopathy, and jellyfish other than Carukia barnesi may be responsible. As there is confusion with nomenclature, Carukia barnesi should be known as the Barnes jellyfish, and the diagnosis of cardiotoxic marine envenoming is suggested for any patient who has been stung by a jellyfish, develops no or minimal skin markings, and develops cardiogenic pulmonary oedema associated with Irukandji syndrome.

The Keap1 BTB/POZ dimerization function is required to sequester Nrf2 in cytoplasm.

Transactivation of phase II detoxification enzymes and antioxidant proteins is mediated by the Cap'N'Collar transcription factor, Nrf2, which is sequestered in the cytoplasm by the actin-binding protein Keap1. Mutation of a conserved serine (S104A) within the Keap1 BTB/POZ domain disrupts Keap1 dimerization and eliminates the ability of Keap1 to sequester Nrf2 in the cytoplasm and repress Nrf2 transactivation. Disruption of endogenous Keap1 dimerization using BTB/POZ dominant negative proteins also inhibits the ability of Keap1 to retain Nrf2 in the cytoplasm. Exposure to an electrophilic agent that induces Nrf2 release and nuclear translocation disrupts formation of a Keap1 complex in vivo. Collectively, these data support the conclusion that Keap1 dimerization is required for Nrf2 sequestration and transcriptional repression. Furthermore, exposure to inducing agents disrupts the Keap1 dimerization function and results in Nrf2 release.

Identification of a variant antioxidant response element in the promoter of the human glutamate-cysteine ligase modifier subunit gene. Revision of the ARE consensus sequence.

Constitutive and inducible expression of the gene encoding the modulator subunit of human glutamate-cysteine ligase (GCLM) is regulated by either of two regions of the promoter; an antioxidant response element (ARE) at -302:-291 and a 44-bp fragment (-346:-303) upstream of the ARE. This second region includes a consensus AP-1 site previously considered responsible for the enhancer activity of the upstream fragment. Deletion of a 165-bp fragment (-348:-183) including the ARE and upstream 44-bp fragment totally ablated t-butyl hydroquinone (tBHQ) inducibility of a GCLM promoter/luciferase transgene. Mutation analyses confirmed that both the ARE and the -346:-303 fragment could support induction following tBHQ exposure but demonstrated that induction in the latter case did not involve the AP-1 site at -341:-335. A region sharing significant homology with the consensus ARE sequence except for a single nucleotide mismatch at -330 (5'-TTACnnnGCA-3' versus 5'-TGACnnnGCA-3') was identified at the 5'-end of the 44-bp fragment immediately adjacent to the AP-1 site. A G in this position has been considered an invariant requirement of functional ARE sequences. Mutation of T(-330) to A (a substitution known to ablate ARE function) or C eliminated basal and inducible expression. Substitution of a G at -330 enhanced basal expression relative to the wild-type sequence, but induction following tBHQ exposure was comparable, indicating that either sequence (5'-TTACnnnGCA-3' versus 5'-TGACnnnGCA-3') may function as an ARE, although the former sequence is less effective at directing basal expression. This possibility was confirmed by similar mutational analyses of the core sequence of hNQO1, a prototypic ARE. Electrophoretic mobility shift competition assays revealed that the 5'-TTACnnnGCA-3' sequence could compete with the hNQO1 ARE for protein binding but was less effective than a similar probe containing the 5'-TGACnnnGCA-3' motif. Probes including the T(-330)A or T(-330)C mutations were ineffective. These results reveal that the GCLM promoter includes two functional AREs, one having a variant sequence. The results indicate that the consensus ARE sequence should be revised to 5'-RTKAYnnnGCR-3'.

Differential induction of mafF, mafG and mafK expression by electrophile-response-element activators.

The three small Maf proteins, MafF, MafG and MafK, have been implicated in a number of physiological processes, including development, differentiation, haematopoiesis and stress response. Here we report the constitutive expression of mafF, mafG and mafK in six human cell lines derived from various tissues (HepG2, IMR-32, K-562, HEK-293, RD and A549). The expression patterns of mafF, mafG and mafK varied widely among cell lines. Because small Maf proteins have been implicated in electrophile response element (EpRE)-mediated stress response, the ability of three EpRE activators [pyrrolidinedithiocarbamate (PDTC), phenylethyl isothiocyanate (PEITC) and t-butylhydroquinone (tBHQ)] to induce small Maf expression was examined in detail in HepG2 cells. Both PDTC and PEITC induced mafF, mafG and mafK expression, whereas tBHQ failed to markedly induce any of the three small Mafs. Where a response was observed, mafF was induced to the greatest extent compared with mafG and mafK, and this response was transcriptionally mediated. PDTC also induced small Maf expression in the other cell lines examined, with patterns of induction varying among cell lines. The differences in expression among the cell lines examined, coupled with the induction patterns observed, indicate that the three small maf genes are stress-responsive, but may be regulated via differing mechanisms. Furthermore, the fact that tBHQ, PDTC and PEITC induce EpRE activity, but that tBHQ fails to markedly induce any of the small Mafs, suggests that up-regulation of small Mafs is not an absolute requirement for EpRE-mediated gene expression.

Biphasic effects of 15-deoxy-delta(12,14)-prostaglandin J(2) on glutathione induction and apoptosis in human endothelial cells.

The lipid products derived from the cyclooxygenase pathway can have diverse and often contrasting effects on vascular cell function. Cyclopentenone prostaglandins (cyPGs), such as 15-deoxy-Delta(12,14)-prostaglandin-J(2) (15d-PGJ(2)), a peroxisome proliferator-activated receptor-gamma (PPARgamma) agonist, have been reported to cause endothelial cell apoptosis, yet in other cell types, cyPGs induce cytoprotective mediators, such as heat shock proteins, heme oxygenase-1, and glutathione (GSH). Herein, we show in human endothelial cells that low micromolar concentrations of 15d-PGJ(2) enhance GSH-dependent cytoprotection through the upregulation of glutamate-cysteine ligase, the rate-limiting enzyme of GSH synthesis, as well as GSH reductase. The effect of 15d-PGJ(2) on GSH synthesis is attributable to the cyPG structure but is independent of PPAR, inasmuch as the other cyPGs, but not PPARgamma or PPARalpha agonists, are able to increase GSH. The increase in cellular GSH is accompanied by abrogation of the proapoptotic effects of 4-hydroxynonenal, a product of lipid peroxidation present in atherosclerotic lesions. However, higher concentrations of 15d-PGJ(2) (10 micromol/L) cause endothelial cell apoptosis, which is further enhanced by depletion of cellular GSH by buthionine sulfoximine. We propose that the GSH-dependent cytoprotective pathways induced by 15d-PGJ(2) contribute to its antiatherogenic effects and that these pathways are distinct from those leading to apoptosis.

Cell-type specific differences in glutamate cysteine ligase transcriptional regulation demonstrate independent subunit control.

Glutamate cysteine ligase (GCL; also referred to as gamma-glutamylcysteine synthetase, GCS) catalyzes the rate-limiting step of glutathione synthesis. The GCL holoenzyme is composed of a catalytic (GCLC; also called GCS(h)) and a modifier (GCLM; also called GCS(l)) subunit, each encoded by a unique gene. Wild-type and mutant promoter/luciferase reporter transgenes containing the promoter region of each GCL subunit gene were transfected into A549 (lung carcinoma), HEK 293 (transformed embryonic kidney), HepG2 (hepatocellular carcinoma), and RD (skeletal muscle rhabdomyosarcoma) cells to examine potential cell-type related differences in transcriptional regulation. In A549, HepG2, and RD cells, maximal basal expression of the GCLC transgene required the full-length (-3802 bp) promoter. Maximal expression in HEK 293 cells was uniquely directed by cis-elements contained within the -2752 to -1286 bp fragment of the promoter. No differences in GLCM promoter function were detected among these 4 cell lines. GCL subunit induction in each cell line by pyrrolidine dithiocarbamate (PDTC), phenethyl isothiocyanate (PEITC), and beta-naphthoflavone (beta-NF) was examined by RNAse protection assays. Although both genes were similarly induced in HepG2 cells by beta-NF, PDTC, and PEITC, neither was induced by beta-NF in A549, HEK 293, and RD cells. PDTC and PEITC induced GCLM to a much greater extent than GCLC in HEK 293 cells and failed to induce GCLC in RD cells. Neither subunit was induced by any of the agents in A549 cells. These studies indicate that the GCL subunit genes are independently regulated and display cell-type specific differences in both basal and inducible expression.

Life-threatening cardiac failure in a healthy young female with Irukandji syndrome.

We present a case of a previously well 24-year-old female patient who developed severe and life-threatening Irukandji syndrome that required ventilation and inotropic support. This case provides further evidence that there are jellyfish other than the Irukandji jellyfish that can cause cardiac decompensation, and there is a suggestion that application of a pressure immobilization bandage may worsen the envenomation. We include our recommended treatment for the Irukandji syndrome.