Correlations between gaseous and liquid phase chemistries induced by cold atmospheric plasmas in a physiological buffer.

The understanding of plasma-liquid interactions is of major importance, not only in physical chemistry, chemical engineering and polymer science, but in biomedicine as well as to better control the biological processes induced on/in biological samples by Cold Atmospheric Plasmas (CAPs). Moreover, plasma-air interactions have to be particularly considered since these CAPs propagate in the ambient air. Herein, we developed a helium-based CAP setup equipped with a shielding-gas device, which allows the control of plasma-air interactions. Thanks to this device, we obtained specific diffuse CAPs, with the ability to propagate along several centimetres in the ambient air at atmospheric pressure. Optical Emission Spectroscopy (OES) measurements were performed on these CAPs during their interaction with a liquid medium (phosphate-buffered saline PBS 10 mM, pH 7.4) giving valuable information about the induced chemistry as a function of the shielding gas composition (variable O2/(O2 + N2) ratio). Several excited species were detected including N2+(First Negative System, FNS), N2(Second Positive System, SPS) and HO˙ radical. The ratios between nitrogen/oxygen excited species strongly depend on the O2/(O2 + N2) ratio. The liquid chemistry developed after CAP treatment was investigated by combining electrochemical and UV-visible absorption spectroscopy methods. We detected and quantified stable oxygen and nitrogen species (H2O2, NO2-, NO3-) along with Reactive Nitrogen Species (RNS) such as the peroxynitrite anion ONOO-. It appears that the RNS/ROS (Reactive Oxygen Species) ratio in the treated liquid depends also on the shielding gas composition. Eventually, the composition of the surrounding environment of CAPs seems to be crucial for the induced plasma chemistry and consequently, for the liquid chemistry. All these results demonstrate clearly that for physical, chemical and biomedical applications, which are usually achieved in ambient air environments, it is necessary to realize an effective control of plasma-air interactions.

Young Sprague Dawley rats infected by Plasmodium berghei: A relevant experimental model to study cerebral malaria.

Cerebral malaria (CM) is the most severe manifestation of human malaria yet is still poorly understood. Mouse models have been developed to address the subject. However, their relevance to mimic human pathogenesis is largely debated. Here we study an alternative cerebral malaria model with an experimental Plasmodium berghei Keyberg 173 (K173) infection in Sprague Dawley rats. As in Human, not all infected subjects showed cerebral malaria, with 45% of the rats exhibiting Experimental Cerebral Malaria (ECM) symptoms while the majority (55%) of the remaining rats developed severe anemia and hyperparasitemia (NoECM). These results allow, within the same population, a comparison of the noxious effects of the infection between ECM and severe malaria without ECM. Among the ECM rats, 77.8% died between day 5 and day 12 post-infection, while the remaining rats were spontaneously cured of neurological signs within 24-48 hours. The clinical ECM signs observed were paresis quickly evolving to limb paralysis, global paralysis associated with respiratory distress, and coma. The red blood cell (RBC) count remained normal but a drastic decrease of platelet count and an increase of white blood cell numbers were noted. ECM rats also showed a decrease of glucose and total CO2 levels and an increase of creatinine levels compared to control rats or rats with no ECM. Assessment of the blood-brain barrier revealed loss of integrity, and interestingly histopathological analysis highlighted cyto-adherence and sequestration of infected RBCs in brain vessels from ECM rats only. Overall, this ECM rat model showed numerous clinical and histopathological features similar to Human CM and appears to be a promising model to achieve further understanding the CM pathophysiology in Humans and to evaluate the activity of specific antimalarial drugs in avoiding/limiting cerebral damages from malaria.

Oxidative modification and electrochemical inactivation of Escherichia coli upon cold atmospheric pressure plasma exposure.

Cold atmospheric pressure plasmas (CAPPs) are known to have bactericidal effects but the mechanism of their interaction with microorganisms remains poorly understood. In this study the bacteria Escherichia coli were used as a model and were exposed to CAPPs. Different gas compositions, helium with or without adjunctions of nitrogen or oxygen, were used. Our results indicated that CAPP induced bacterial death at decontamination levels depend on the duration, post-treatment storage and the gas mixture composition used for the treatment. The plasma containing O2 in the feeding gas was the most aggressive and showed faster bactericidal effects. Structural modifications of treated bacteria were observed, especially significant was membrane leakage and morphological changes. Oxidative stress caused by plasma treatment led to significant damage of E. coli. Biochemical analyses of bacterial macromolecules indicated massive intracellular protein oxidation. However, reactive oxygen and nitrogen species (RONS) are not the only actors involved in E. coli's death, electrical field and charged particles could play a significant role especially for He-O2 CAPP.

Cold Atmospheric Plasma Induces a Predominantly Necrotic Cell Death via the Microenvironment.

INTRODUCTION: Cold plasma is a partially ionized gas generated by an electric field at atmospheric pressure that was initially used in medicine for decontamination and sterilization of inert surfaces. There is currently growing interest in using cold plasma for more direct medical applications, mainly due to the possibility of tuning it to obtain selective biological effects in absence of toxicity for surrounding normal tissues,. While the therapeutic potential of cold plasma in chronic wound, blood coagulation, and cancer treatment is beginning to be documented, information on plasma/cell interaction is so far limited and controversial. METHODS AND RESULTS: Using normal primary human fibroblast cultures isolated from oral tissue, we sought to decipher the effects on cell behavior of a proprietary cold plasma device generating guided ionization waves carried by helium. In this model, cold plasma treatment induces a predominantly necrotic cell death. Interestingly, death is not triggered by a direct interaction of the cold plasma with cells, but rather via a transient modification in the microenvironment. We show that modification of the microenvironment redox status suppresses treatment toxicity and protects cells from death. Moreover, necrosis is not accidental and seems to be an active response to an environmental cue, as its execution can be inhibited to rescue cells. CONCLUSION: These observations will need to be taken into account when studying in vitro plasma/cell interaction and may have implications for the design and future evaluation of the efficacy and safety of this new treatment strategy.

Improving anticoagulation control in hospitalized elderly patients on warfarin.

OBJECTIVES: To determine the effect of patient characteristics and of specific guidelines that were developed for managing warfarin therapy in older adults and included in an in-house computer program on anticoagulation quality. DESIGN: Thirteen-month observational study. SETTING: Acute care, extended care, and rehabilitation geriatric wards of a teaching hospital in Paris, France. PARTICIPANTS: Hospitalized patients (N=307, mean age 86.1 +/- 6.1) treated with warfarin with a therapeutic international normalized ratio range of 2.0 to 3.0. INTERVENTION: Patients were assigned according to care unit to the computer-generated dosing group (CGD) or the standard management group (SM; usual physician-based care). MEASUREMENTS: Relationships between anticoagulation quality criteria and covariates (age, sex, warfarin indication, treatment phase, follow-up duration, model of care). RESULTS: According to multivariate analysis, only model of care and follow-up duration independently influenced anticoagulation control; the proportion of time within therapeutic INR range 2.0 to 3.0 was significantly greater in the CGD group than in the SM group (59% vs 48%, P=.004). When a wider INR range was analyzed (1.8-3.2), the proportion of time within range was 73% versus 64% (P=.006). Use of the computer was associated with fewer days with INRs greater than 3, a smaller percentage of INRs of 4 or greater, a longer time to the first INR of 4.0 or greater, and a smaller mean number of INRs per month than SM (all P<.01). CONCLUSION: Initiation regimen and long-term rules that have specifically been developed and included in a computerized dosage program improve quality of anticoagulation in elderly inpatients, allowing them to benefit from a quality of care as high as that of younger ambulatory patients.

Activation of the {beta}-catenin/T-cell-specific transcription factor/lymphoid enhancer factor-1 pathway by plasminogen activators in ECV304 carcinoma cells.

Besides its involvement in clot lysis, the plasminogen activator (PA) system elicits various cellular responses involved in cell migration, adhesion, and proliferation and plays a key role in the progression of cancers. beta-Catenin interacts with E-cadherins and functions as transcriptional coactivator of the Wnt-signaling pathway, which is implicated in tumor formation when aberrantly activated. We report that tissue-type plasminogen activator (tPA) elicited tyrosine phosphorylation and cytosolic accumulation of an active (non-serine-threonin phosphorylated, nonubiquitinated) form of beta-catenin in ECV304 carcinoma cells. tPA-dependent beta-catenin activation is mediated through epidermal growth factor receptor (EGFR) transactivation (via Src), suggested by the inhibitory effects of AG1478 and PP2 (specific inhibitors of EGFR and Src, respectively) and by the lack of beta-catenin activation in EGFR-negative B82 fibroblasts. EGFR phosphorylation and beta-catenin activation were inhibited by plasminogen activator inhibitor 1 and pertussis toxin, two inhibitors of the urokinase-type plasminogen activator (uPA)/uPA receptor system. beta-Catenin activation was correlated with the phosphorylation of glycogen synthase kinase-3beta through a phosphatidylinositol 3-kinase/Akt-dependent mechanism. Gel shift experiments revealed the activation of beta-catenin/T-cell-specific transcription factor (Tcf)/lymphoid enhancer factor-1 (Lef) transcriptional complex, evidenced by an increased binding of nuclear extracts to oligonucleotides containing the cyclin D1 Lef/Tcf site. beta-Catenin silencing through small interfering RNA and antisense oligonucleotides inhibited both the tPA-mediated cyclin D1 expression and cell proliferation. A similar activation of the beta-catenin pathway was triggered by amino-terminal fragment, the NH(2)-terminal catalytically inactive fragment of tPA, thus suggesting that this effect was independent of the proteolytic activity of plasminogen activators. In conclusion, the beta-catenin/Lef/Tcf pathway is activated by tPA and is involved in cell cycle progression and proliferation.

The sphingomyelin/ceramide pathway is involved in ERK1/2 phosphorylation, cell proliferation, and uPAR overexpression induced by tissue-type plasminogen activator.

Plasminogen activators (tPA and uPA) are serine proteases that convert the circulating zymogen plasminogen to active plasmin and mediate fibrin degradation. These multifunctional proteins trigger various biological events such as extracellular matrix degradation, cell adhesion, migration, and proliferation, through not yet fully characterized mechanisms. We report that, in smooth muscle cells and ECV-304 carcinoma cells, tPA and ATF (the N-terminal catalytically inactive fragment of tPA) elicited DNA synthesis that requires activation of the sphingomyelin/ceramide/sphingosine-1-phosphate (Spm/Cer/S1P), signaling pathway and was blocked by D-erythro-2-(N-myristoylamino)-1-phenyl-propanol (D-MAPP) and N-N'-dimethyl sphingosine (DMS), two classical inhibitors of sphingosine-1-phosphate biosynthesis. Binding of tPA to its receptor uPAR triggered the coordinated activation of two key enzymes of the Spm/Cer/S1P pathway, the neutral sphingomyelinase and the sphingosine kinase-1 that was mediated by a common pertussis toxin (PTX)-sensitive mechanism. The tPA-induced sphingosine kinase-1 activation was mediated by Src, since it was inhibited by herbimycin A and in SrcK- cells (overexpressing a dominant negative kinase defective form of Src) and by ERK1/2 (early phase peaking at 15 min). Sphingosine kinase-1 activation was followed by a second phase of ERK1/2 phosphorylation (peaking at 120 min) and subsequent DNA synthesis, which were inhibited by D-MAPP and DMS, by anti-EGD-1 antibodies and in SrcK- cells (in which the mitogenic signaling was rescued by sphingosine-1-phosphate). Altogether, these data underline a pivotal role for the Spm/Cer/S1P pathway in the tPA-induced mitogenic signaling.

Comparison of the diagnostic performance of three soluble fibrin monomer tests and a D-dimer assay in patients with clinically suspected deep vein thrombosis of the lower limbs.

We assessed three soluble fibrin monomer (SFM) assays in 231 in and out-patients with clinically suspected deep-vein thrombosis. Thrombosis was confirmed or excluded by complete lower-limb ultrasound. SFM assay were less accurate than VIDAS D-dimer and in patients with small thrombosis or under anticoagulation. Specificity was lower for a similar sensitivity.

Pharmacokinetics and pharmacodynamics of intramuscular dermatan sulfate revisited : a single- and repeated-dose study in healthy volunteers.

OBJECTIVE: To assess the pharmacokinetics and effects on blood coagulation of dermatan sulfate (DS), a glycosaminoglycan with antithrombotic properties, following intravenous and single and repeated intramuscular administrations. The mean molecular weight of DS is currently 22kD, i.e. 5kD lower than batches used in the early development of the compound. SUBJECTS AND METHODS: Each of 14 male healthy volunteers received DS 300mg as an 8-hour intravenous infusion and as single and repeated (once daily for 9 days) intramuscular injections. Nine of the same subjects were also given DS 100mg and 200mg as single intramuscular doses. Plasma DS concentrations were measured with a specific chromogenic assay. Activated partial thromboplastin time (aPTT) and thrombin clotting time (TCT) responses were also determined. RESULTS: The mean +/- SD volume of distribution and terminal half-life of DS were 6.0 +/- 1.4L and 0.9 +/- 0.2h after intravenous infusion. Maximum plasma concentration (C(max)) and area under the plasma concentration-time curve after single intramuscular injections were dose-proportional. After repeated intramuscular administration, steady state was reached by day 3. On day 9, plasma DS fluctuated between 4.3 +/- 1.5 (C(max)) and 0.9 +/- 0.4 (minimum plasma concentration at steady state) mg/L, time to C(max) was 3.4 +/- 0.8h, terminal half-life was 12.2 +/- 4.1h and the accumulation factor was 2.0 +/- 0.5. Geometric mean bioavailability of intramuscular DS was 54% and 79% after single and repeated 300mg administration, respectively. aPTT and TCT responses were both linearly related to plasma DS concentrations, with TCT showing greater responsivity. CONCLUSION: As compared with earlier DS studies, the present data showed a greater extent of DS absorption after single intramuscular administration and a faster absorption rate after repeated dosing, and provided evidence of dose-response predictability. These findings may explain the improved antithrombotic efficacy of DS observed in a recent clinical trial.

The pharmacodynamics of tinzaparin in healthy volunteers.

We report the pharmacodynamic properties of tinzaparin (175 U/kg antifactor Xa) given as a single daily administration for 5 consecutive days to 14 healthy volunteers as a known safe, effective treatment of deep vein thrombosis and pulmonary embolism. The Cmax for antifactor Xa (0.87 +/- 0.15 U/ml) was associated with a 2.4 +/- 0.5-fold prolongation of the activated partial thromboplastin time (APTT) and a high antithrombin activity (0.38 +/- 0.1 U/ml). The Cmax value of antifactor Xa was 1.5- and twofold lower than those generated by similar doses of nadroparin and enoxaparin respectively. The clearance of antifactor Xa activity (1.29 +/- 0.2 l/h) was 1.5- and twofold greater than those reported for nadroparin and enoxaparin respectively. These results indicated that the antithrombotic and prohaemorrhagic effects of a low molecular weight heparin were independent from the absolute levels of antifactor Xa activities and from the prolongation of the APTT.