Impact of arterial procedures on coagulation and fibrinolysis : a pilot study

Abstract Objective: The main goal of our study was to assess the impact of vascular procedures on the activity of hemostatic and fibrinolytic pathways. Methods: We enrolled 38 patients with ≥ 45 years old undergoing surgery for abdominal aortic aneurysm or peripheral artery disease under general or regional anesthesia and who were hospitalized at least one night after the procedure. Patients undergoing carotid artery surgery and those who had acute bypass graft thrombosis, cancer, renal failure defined as estimated glomerular filtration rate < 30 ml/min/1.73m2, venous thromboembolism three months prior to surgery, or acute infection were excluded from the study. We measured levels of markers of hemostasis (factor VIII, von Willebrand factor:ristocetin cofactor [vWF:CoR], antithrombin), fibrinolysis (D-dimer, tissue plasminogen activator [tPA], plasmin-antiplasmin complexes), and soluble cluster of differentiation 40 ligand (sCD40L) before and 6-12h after vascular procedure. Results: Significant differences between preoperative and postoperative levels of factor VIII (158.0 vs. 103.3, P<0.001), antithrombin (92.1 vs. 74.8, P<0.001), D-dimer (938.0 vs. 2406.0, P=0.005), tPA (10.1 vs. 12.8, P=0.002), and sCD40L (9092.9 vs. 1249.6, P<0.001) were observed. There were no significant differences between pre- and postoperative levels of vWF:CoR (140.6 vs. 162.8, P=0.17) and plasmin-antiplasmin complexes (749.6 vs. 863.7, P=0.21). Conclusion: Vascular surgery leads to significant alterations in hemostatic and fibrinolytic systems. However, the direction of these changes in both pathways remains unclear and seems to be different depending on the type of surgery. A study utilizing dynamic methods of coagulation and fibrinolysis assessment performed on a larger population is warranted

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234. Treatment of melanoma patients with genetically modified tumor vaccines (GMTV) does not decrease the spontaneous apoptosis of CD4+ and CD8+ T cells

In this study, the spontaneous apoptosis of CD4+ and CD8+ T-cells in patients with melanoma were examined. Fifty-seven patients enrolled in the study were participating in an experimental immunotherapy study and were administered vaccines consisting of irradiated, gene modified allogenic melanoma cells: MICH I H6/ GMCSF + MICH II H6/GMCSF. The control group consisted of 20 healthy volunteers. Venous blood was obtained on the day of the administration of the first vaccine, and then approximately once a month. Blood was obtained in EDTA tubes, after which the Iymphocytes were isolated by Ficoll-Hypaque gradient centrifugation. The Iymphocytes were then incubated for 24hrs in medium containing fetal calf serum at 37°C in 5% CO2 to allow for spontaneous apoptosis to take place. The Iymphocytes were then stained with PE-Iabeled CD4 or CD8 and next, stained for apoptosis using the FITC-labeled Annexin V binding method. A two color flow cytometry was used to measure the proportion of CD4+ and CD8+ Iymphocytes that underwent apoptosis. SD) of CD4+ T cells were Annexin±6.3% (mean ±In patients with melanoma 21 6.3% in healthy controls (P±V+ compared with 15 < 0.0001). For CD8+ T cells, 4.5%±8.1% of T cells were Annexin V+ and in healthy controls 19±in patients 31 of T cells were Annexin V+(P < 0.0001). There were no significant changes with time in the proportion of CD4+ and CD8+ Iymphocytes undergoing spontaneous apoptosis after the start of the gene modified tumor vaccine therapy

Epigenetics of Epileptogenesis-Evoked Upregulation of Matrix Metalloproteinase-9 in Hippocampus

<div><p>Enhanced levels of Matrix Metalloproteinase-9 (MMP-9) have been implicated in the pathogenesis of epilepsy in humans and rodents. Lack of Mmp-9 impoverishes, whereas excess of Mmp-9 facilitates epileptogenesis. Epigenetic mechanisms driving the epileptogenesis-related upregulation of MMP-9 expression are virtually unknown. The aim of this study was to reveal these mechanisms. We analyzed hippocampi extracted from adult and pediatric patients with temporal lobe epilepsy as well as from partially and fully pentylenetetrazole kindled rats. We used a unique approach to the analysis of the kindling model results (inclusion in the analysis of rats being during kindling, and not only a group of fully kindled animals), which allowed us to separate the molecular effects exerted by the epileptogenesis from those related to epilepsy and epileptic activity. Consequently, it allowed for a disclosure of molecular mechanisms underlying causes, and not consequences, of epilepsy. Our data show that the epileptogenesis-evoked upregulation of Mmp-9 expression is regulated by removal from Mmp-9 gene proximal promoter of the two, interweaved potent silencing mechanisms–DNA methylation and Polycomb Repressive Complex 2 (PRC2)-related repression. Demethylation depends on a gradual dissociation of the DNA methyltransferases, Dnmt3a and Dnmt3b, and on progressive association of the DNA demethylation promoting protein Gadd45β to Mmp-9 proximal gene promoter <i>in vivo</i>. The PRC2-related mechanism relies on dissociation of the repressive transcription factor YY1 and the dissipation of the PRC2-evoked trimethylation on Lys27 of the histone H3 from the proximal <i>Mmp-9</i> promoter chromatin <i>in vivo</i>. Moreover, we show that the DNA hydroxymethylation, a new epigenetic DNA modification, which is localized predominantly in the gene promoters and is particularly abundant in the brain, is not involved in a regulation of MMP-9 expression during the epileptogenesis in the rat hippocampus as well as in the hippocampi of pediatric and adult epileptic patients. Additionally, we have also found that despite of its transient nature, the histone modification H3S10ph is strongly and gradually accumulated during epileptogenesis in the cell nuclei and in the proximal Mmp-9 gene promoter in the hippocampus, which suggests that H3S10ph can be involved in DNA demethylation in mammals, and not only in <i>Neurospora</i>. The study identifies <i>MMP-9</i> as the first protein coding gene which expression is regulated by DNA methylation in human epilepsy. We present a detailed epigenetic model of the epileptogenesis-evoked upregulation of <i>MMP-9</i> expression in the hippocampus. To our knowledge, it is the most complex and most detailed mechanism of epigenetic regulation of gene expression ever revealed for a particular gene in epileptogenesis. Our results also suggest for the first time that dysregulation of DNA methylation found in epilepsy is a cause rather than a consequence of this condition.</p></div

H3S10ph, transcriptionally activating histone modification, is strongly induced during epileptogenesis in the hippocampal chromatin.

<p><b>(A) <i>The phosphorylation of the histone H3 on serine 10 (H3S10ph) gradually and strongly increases in chromatin of the Mmp-9 proximal promoter during epileptogenesis in the rat hippocampus in vivo</i>.</b> DNA was isolated from the hippocampal samples obtained by chromatin immunoprecipitation with anti-H3S10ph antibody from the unstimulated (control), as well as from the partially kindled and fully kindled rats. <i>Mmp-9</i> proximal promoter content was evaluated by qPCR. Control ChIP reaction was performed using isotype antibody. Values are means ± SEM (*, <i>p</i> < 0.05; **, <i>p</i> < 0.01; <i>n</i> = 4). (<b>B) <i>During epileptogenesis in the rat hippocampus</i>, <i>the histone H3 is strongly phosphorylated on serine 10</i>.</b> Equal amounts (20 μg) of nuclear cell lysates obtained from the unstimulated (control) as well as the partially kindled and fully kindled rat hippocampi were analyzed by Western blot with anti-H3S10ph antibody. As a loading control, histone H3 was used. Representative Western blot analyses are shown.</p

Mmp-9 mRNA expression and activity are increased progressively during epileptogenesis in the rat hippocampus.

<p>30 mg/kg of PTZ was administrated intraperitoneally at least 10 times to partially and fully kindled study group. Single PTZ dose study group received only one PTZ administration at 30 mg/kg dose. Rats were sacrificed 24 h after the final dose. (<b>A) <i>Mmp-9 mRNA accumulates progressively during epileptogenesis in the hippocampus</i></b>. For each analysis equal amounts of RNA samples isolated from naive (control) and PTZ-treated (single PTZ dose, partially kindled, fully kindled) rat hippocampi were used. Data is presented as fold change in mRNA expression. Values are means ± SEM (*, <i>p</i> < 0.05; **, <i>p</i> < 0.01; <i>n</i> = 4). (<b>B) <i>Mmp-9</i>, <i>but not Mmp-2</i>, <i>gelatynolytic activity augments strongly in the hippocampi of PTZ-kindled rats</i></b>. All of the study groups showed unchanged hippocampal MMP-2 activity, whereas MMP-9 activity was substantially modified. For gelatine zymography analysis equal amounts of protein samples isolated from naive (control) and PTZ-treated (single PTZ dose, partially and fully kindled) rat hippocampi were used. Graph data is presented as fold change in gelatynolytic activity. Values are means ± SEM (*, <i>p</i> < 0.05; <i>n</i> = 4). Representative cropped gel image of Mmp-9 and Mmp-2 gelatynolytic activity is showed.</p

Model of the epileptogenesis-evoked upregulation of Mmp-9 expression in the hippocampus.

<p><b>(A)</b> In the control rat hippocampus, Mmp-9 proximal promoter is bound by YY1, which nucleates the DNA methyltransferases, Dnmt3a and Dnmt3b, as well as PRC2, leading to the strong promoter DNA methylation and simultaneous trimethylation on lysine 27 of histone H3 in the surrounding chromatin. (<b>B)</b> In partially kindled rat hippocampus, YY1 dissociates out of the gene promoter, leading to a partial removal from the chromatin region other silencing proteins Dnmt3a, Dnmt3b, and PRC2. Concomitantly, DNA-demethylation-related protein Gadd45β and the activating histone mark H3S10ph start to accumulate in the chromatin. Consequently, it induces partial demethylation of the Mmp-9 gene promoter and leads to a reduction in its chromatin of the repressive chromatin mark H3K27me3. These complex molecular events lead to a moderate stimulation of the Mmp-9 gene expression. <b>(C)</b> In the fully kindled rat hippocampus, Mmp-9 expression is highly upregulated as a consequence of a much more profound proximal promoter demethylation (due to a complete dissociation from its chromatin of Dnmt3a and Dnmt3b as well as a strongly increased accumulation of Gadd45β), disappearance of the PRC2-related repression with a complete removal of H3K27me3, and a significant increase in the abundance of H3S10ph in the chromatin region.</p

Mmp-9 upregulation during epilepsy development is strictly dependent on epileptogenesis-evoked demethylation of its gene promoter.

<p>To block epilepsy development, we used dizocilpine (the NMDA receptor antagonist displaying anticonvulsant activity). 0.1 mg/kg of dizocilpine or saline was intraperitoneally injected to rats 30 min before each PTZ dose administration (30 mg/kg). (<b>A) <i>Dizocilpine treatment effectively suppresses the development of PTZ-evoked epilepsy in rats</i></b>. Animals were observed up to 2 h after each PTZ injection and seizures were scored according to a modified Racine’s scale. Values are means ± SEM (*, <i>p</i><0.05; <i>n</i> = 11). (<b>B) <i>PTZ kindling-evoked upregulation of the Mmp-9 mRNA expression is fully inhibited by dizocilpine administration in the rat hippocampus</i>.</b> Dizocilpine administration suppresses the PTZ kindling–evoked augmentation in the hippocampal Mmp-9 mRNA expression, whereas repeated PTZ treatment without dizocilpine injections leads to significant upregulation of Mmp-9 mRNA level. For RT-qPCR analysis equal amounts of RNA isolated from naive (control), PTZ-treated (saline + PTZ), and dizocilpine-treated (dizocilpine + PTZ) rat hippocampi were used. Data is presented as fold change in mRNA expression. Values are means ± SEM (*, <i>p</i><0.05; ***, <i>p</i><0.001; <i>n</i> = 8). <b>(C) <i>Dizocilpine treatment completely inhibits the PTZ kindling-dependent Mmp-9 proximal promoter demethylation in the rat hippocampus</i></b>. <i>Mmp-9</i> proximal promoter methylation level was evaluated using qPCR in DNA samples obtained by immunoprecipitation of methylated DNA from naive (control), PTZ-treated (saline+PTZ), and dizocilpine-treated (dizocilpine+PTZ) rat hippocampi. Data is presented as a percent of input. Values are means ± SEM (*, <i>p</i><0.05; <i>n</i> = 5).</p