Predictors of systemic inflammatory response syndrome in ischemic stroke undergoing systemic thrombolysis with intravenous tissue plasminogen activator

Background: Systemic inflammatory response syndrome (SIRS) is an inflammatory process associated with poor outcomes in acute ischemic stroke (AIS) patients. However, no study to date has investigated predictors of SIRS in AIS patients treated with intravenous (IV) tissue plasminogen activator (tPA). Methods: Consecutive patients were retrospectively reviewed for evidence of SIRS during their acute hospitalization. SIRS was defined as the presence of 2 or more of the following: (1) body temperature less than 36°C or greater than 38°C, (2) heart rate greater than 90, (3) respiratory rate greater than 20, or (4) white blood cell count less than 4000/mm or greater than 12,000/mm or more than 10% bands for more than 24 hours. Those diagnosed with an infection were excluded. A scoring system was created to predict SIRS based on patient characteristics available at the time of admission. Logistic regression was used to evaluate potential predictors of SIRS using a sensitivity cutoff of ≥65% or area under the curve of .6 or more. Results: Of 212 patients, 44 had evidence of SIRS (21%). Patients with SIRS were more likely to be black (61% versus 54%; P = .011), have lower median total cholesterol at baseline (143 versus 167 mg/dL; P = .0207), and have history of previous stroke (51% versus 35%; P = .0810). Ranging from 0 to 6, the SIRS prediction score consists of African American (2 points), history of hypertension (1 point), history of previous stroke (1 point), and admission total cholesterol less than 200 (2 points). Patients with an SIRS score of 4 or more were 3 times as likely to develop SIRS when compared with patients with a score of ≤3 (odds ratio = 2.815, 95% confidence interval 1.43-5.56, P = .0029). Conclusions: In our sample of IV tPA-treated AIS patients, clinical and laboratory characteristics available on presentation were able to identify patients likely to develop SIRS during their acute hospitalization. Validation is required in other populations. If validated, this score could assist providers in predicting who will develop SIRS after treatment with IV tPA

Mangiferin has an additive effect on the apoptotic properties of hesperidin in Cyclopia sp. tea extracts.

A variety of biological pro-health activities have been reported for mangiferin and hesperidin, two major phenolic compounds of Honeybush (Cyclopia sp.) tea extracts. Given their increasing popularity, there is a need for understanding the mechanisms underlying the biological effects of these compounds. In this study, we used real-time cytotoxicity cellular analysis of the Cyclopia sp. extracts on HeLa cells and found that the higher hesperidin content in non-fermented "green" extracts correlated with their higher cytotoxicity compared to the fermented extracts. We also found that mangiferin had a modulatory effect on the apoptotic effects of hesperidin. Quantitative PCR analysis of hesperidin-induced changes in apoptotic gene expression profile indicated that two death receptor pathway members, TRADD and TRAMP, were up regulated. The results of this study suggest that hesperidin mediates apoptosis in HeLa cells through extrinsic pathway for programmed cell death

The unfolded protein response (UPR)-activated transcription factor X-box-binding protein 1 (XBP1) induces microRNA-346 expression that targets the human antigen peptide transporter 1 (TAP1) mRNA and governs immune regulatory genes

To identify endoplasmic reticulum (ER) stress-induced microRNAs (miRNA) that govern ER protein influx during the adaptive phase of unfolded protein response, we performed miRNA microarray profiling and analysis in human airway epithelial cells following ER stress induction using proteasome inhibition or tunicamycin treatment. We identified miR-346 as the most significantly induced miRNA by both classic stressors. miR-346 is encoded within an intron of the glutamate receptor ionotropic delta-1 gene (GRID1), but its ER stress-associated expression is independent of GRID1. We demonstrated that the spliced X-box-binding protein-1 (sXBP1) is necessary and sufficient for ER stress-associated miR-346 induction, revealing a novel role for this unfolded protein response-activated transcription factor. In mRNA profiling arrays, we identified 21 mRNAs that were reduced by both ER stress and miR-346. The target genes of miR-346 regulate immune responses and include the major histocompatibility complex (MHC) class I gene products, interferon-induced genes, and the ER antigen peptide transporter 1 (TAP1). Although most of the repressed mRNAs appear to be indirect targets because they lack specific seeding sites for miR-346, we demonstrate that the human TAP1 mRNA is a direct target of miR-346. The human TAP1 mRNA 3'-UTR contains a 6-mer canonical seeding site for miR-346. Importantly, the ER stress-associated reduction in human TAP1 mRNA and protein levels could be reversed with an miR-346 antagomir. Because TAP function is necessary for proper MHC class I-associated antigen presentation, our results provide a novel mechanistic explanation for reduced MHC class I-associated antigen presentation that was observed during ER stress

Two PKC consensus sites on human acid-sensing ion channel 1b differentially regulate its function

Human acid-sensing ion channel 1b (hASIC1b) is a H+-gated amiloride-sensitive cation channel. We have previously shown that glioma cells exhibit an amiloride-sensitive cation conductance. Amiloride and the ASIC1 blocker psalmotoxin-1 decrease the migration and proliferation of glioma cells. PKC also abolishes the amiloride-sensitive conductance of glioma cells and inhibits hASIC1b open probability in planar lipid bilayers. In addition, hASIC1b's COOH terminus has been shown to interact with protein interacting with C kinase (PICK)1, which targets PKC to the plasma membrane. Therefore, we tested the hypothesis that PKC regulation of hASIC1b at specific PKC consensus sites inhibits hASIC1b function. We mutated three consensus PKC phosphorylation sites (T26, S40, and S499) in hASIC1b to alanine, to prevent phosphorylation, and to glutamic acid or aspartic acid, to mimic phosphorylation. Our data suggest that S40 and S499 are critical sites mediating the modulation of hASIC1b by PKC. We expressed mutant hASIC1b constructs in Xenopus oocytes and measured acid-activated currents by two-electrode voltage clamp. T26A and T26E did not exhibit acid-activated currents. S40A was indistinguishable from wild type (WT), whereas S40E, S499A, and S499D currents were decreased. The PKC activators PMA and phorbol 12,13-dibutyrate inhibited WT hASIC1b and S499A, and PMA had no effect on S40A or on WT hASIC1b in oocytes pretreated with the PKC inhibitor chelerythrine. Chelerythrine inhibited WT hASIC1b and S40A but had no effect on S499A or S40A/S499A. PKC activators or the inhibitor did not affect the surface expression of WT hASIC1b. These data show that the two PKC consensus sites S40 and S499 differentially regulate hASIC1b and mediate the effects of PKC activation or PKC inhibition on hASIC1b. This will result in a deeper understanding of PKC regulation of this channel in glioma cells, information that may help in designing potentially beneficial therapies in their treatment

Two PKC consensus sites on human acid-sensing ion channel 1b differentially regulate its function

Human acid-sensing ion channel 1b (hASIC1b) is a H+-gated amiloride-sensitive cation channel. We have previously shown that glioma cells exhibit an amiloride-sensitive cation conductance. Amiloride and the ASIC1 blocker psalmotoxin-1 decrease the migration and proliferation of glioma cells. PKC also abolishes the amiloride-sensitive conductance of glioma cells and inhibits hASIC1b open probability in planar lipid bilayers. In addition, hASIC1b's COOH terminus has been shown to interact with protein interacting with C kinase (PICK)1, which targets PKC to the plasma membrane. Therefore, we tested the hypothesis that PKC regulation of hASIC1b at specific PKC consensus sites inhibits hASIC1b function. We mutated three consensus PKC phosphorylation sites (T26, S40, and S499) in hASIC1b to alanine, to prevent phosphorylation, and to glutamic acid or aspartic acid, to mimic phosphorylation. Our data suggest that S40 and S499 are critical sites mediating the modulation of hASIC1b by PKC. We expressed mutant hASIC1b constructs in Xenopus oocytes and measured acid-activated currents by two-electrode voltage clamp. T26A and T26E did not exhibit acid-activated currents. S40A was indistinguishable from wild type (WT), whereas S40E, S499A, and S499D currents were decreased. The PKC activators PMA and phorbol 12,13-dibutyrate inhibited WT hASIC1b and S499A, and PMA had no effect on S40A or on WT hASIC1b in oocytes pretreated with the PKC inhibitor chelerythrine. Chelerythrine inhibited WT hASIC1b and S40A but had no effect on S499A or S40A/S499A. PKC activators or the inhibitor did not affect the surface expression of WT hASIC1b. These data show that the two PKC consensus sites S40 and S499 differentially regulate hASIC1b and mediate the effects of PKC activation or PKC inhibition on hASIC1b. This will result in a deeper understanding of PKC regulation of this channel in glioma cells, information that may help in designing potentially beneficial therapies in their treatment

RTCA measurement of IC50 value of A) mangiferin, B) hesperidin and C) hesperidin in the presence of 5 μg/ml mangiferin in HeLa cells, after 12h of treatment.

<p>Error bars represent the standard derivations. IC50 values were calculated in RTCA software with use of non-linear regression (sigmoidal dose response model). Each extract concentration was tested in triplicate and experiments were repeated twice. Obtained IC50 values were validated with the MTT method in independent experiments (HeLa, 12h exposure). MTT recorded IC50 values were 4.210 +/–0.320 μg/ml for hesperidin, 20.087 +/–0.320 μg/ml for mangiferin, and 1.623 +/– 0.323 μg/ml for hesperidin in presence of mangiferin (5 μg/ml).</p

Cyclopia sp. extracts induce apoptotic changes in HeLa cells.

<p><b>A</b>) Hoechst 33342 staining was used to detect the formation of condensed pycnotic nucleus. The cells were incubated in a presence of extracts (at concentrations equal to IC50 values) for 12h; <b>B</b>) Characterization of DNA fragmentation induced by extracts (at concentrations equal to IC50 values) was examined by agarose gel electrophoresis. In untreated cells (ctrl), there was no DNA fragmentation. Under treatment with the different extracts, DNA fragmentation occurs (M - DNA standard, Fermentas SM0333).</p

Representation of validated PCR apoptotic gene expression array results.

<p>The heat map left column marked <b>h</b> and <b>h+m,</b> represents relative expression fold changes (relative to 18S, GAPDH, HRTP1 and GUSB) versus EtOH control) in HeLa treated with hesperidin <b>(h)</b> (2.5 μg/ml, <u>4.01 μM</u>) and hesperidin (2.5 μg/ml, <u>4.01 μM</u>) in a presence of mangiferin (5 μg/ml, <u>11.84 μM</u>) <b>(h+m)</b>, respectively. HeLa cells were incubated for 12h in presence of EtOH (EtOH concentration was adjusted to combined treatment conditions) or specified compounds. "*" denotes relative fold change ≤ –2 and ≥2;"▴" denotes relative fold change ≤ –1.5 and ≥1.5. For heat map generation RQ values were converted to fold change and PermutMatrix software was used <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092128#pone.0092128-Caraux1" target="_blank">[49]</a>.</p

Real-time cellular analysis of Cyclopia sp. extracts on cell growth curves.

<p>Impact of extract treatment (5 μg/ml final concentration for each extracts) on HeLa cells growth curves is presented in the top panel. Each curve represents an average of 3 replicates from 2 independent experiments. The bottom panel represents changes in cell proliferation during 12h treatment expressed as the growth curve slope. The error bars represent the standard derivation values.</p