Double entrapment of growth factors by nanoparticles loaded into polyelectrolyte multilayer films.

Delivery of growth factors and control of vascularization are prominent problems in regenerative medicine. Vascular endothelial growth factor (VEGF) has been used both in vitro and in vivo to promote angiogenesis but due to its short half-life its controlled delivery is a sought after method. In this study we present a new concept of degradable drug loaded nanoparticles entrapped into exponentially growing multilayer films. Through hydrolysis of the nanoparticles, the drug can be delivered over long periods in a controlled manner. Poly(ε-caprolactone) nanoparticles were loaded with VEGF and in turn the release of VEGF from a surface is controlled by a thick layer-by-layer polyelectrolyte film. Direct loading of VEGF inside the film was not efficient for long-term applications. When VEGF loaded nanoparticles were introduced into the film, the particles were equally distributed inside and were stable after several washes. Moreover, the presence of the film sustained the release of VEGF for 7 days. Addition of the nanoparticles to the film promoted endothelial cell proliferation, mainly due to the presence of VEGF. Mechanical properties of the film (Young's moduli) were also improved by the presence of nanoparticles. However, in the presence of the film loaded with nanoparticles and without any direct contact with this film, endothelial cell growth was also enhanced on polystyrene and on Transwell insert surfaces which demonstrates the effectiveness of the nanoparticles not only to improve the mechanical properties of the film but also to deliver active VEGF. An increase in nitric oxide levels as an indicator of endothelial cell activity was monitored and was correlated with the release of VEGF from the nanoparticle/film platform. Finally, such a system can be used as an auxiliary delivery body within implants to finely control the release of bioactive agent containing nanoparticles.

The comparison of the effects of T-piece and CPAP on hemodynamic parameters, arterial blood gases and success of weaning.

Weaning from mechanically ventilation is a period of transition from total ventilatory support to spontaneous breathing. The aim of this study was to compare the effects of T-Piece and continuous positive airway pressure (CPAP) on hemodynamic parameters, arterial blood gases and success of weaning. In a prospective, randomized, controlled trial, 40 consecutive patients requiring mechanically ventilation in our 8-bed adult general intensive care unit (ICU) for >48 hrs were considered eligible for this study. Patients were randomly divided into two groups (n: 20). Group T-piece received, 4 L/min, Group CPAP received, PEEP < or =5 cm H2O, FiO2 < or = 0.4. At the beginning of the weaning, duration of extubation and after 48 hours of extubation the arterial blood samples were taken for blood gases analysis, also the mean arterial pressure and heart rate were recorded. 40 patients in the ICU were included in the study. There were no significant differences within and between T-piece and CPAP groups according to hemodynamic parameters and arterial blood gases at the weaning period. The number of patients who could be unsuccessful weaned in the T-piece group was higher than the number of patients in the group CPAP (p < 0.001, p < 0.01). Whether, the technique used to wean patients, in this setting, resulted in a clinically relevant improvement in the outcomes addressed above requires further carefully designed, randomized, controlled trials (Tab. 4, Ref. 25).

A memantin HCL intoxication responsive to plasmapheresis therapy.

Memantin HCL (Ebixa) is a drug which antagonizes the effects of N-methyl-D-aspartat receptors and which is used for the treatment of acute Alzheimer patients. Plasmapheresis is a method of cleaning nonspecific extracorporeal blood and it is applied in many immunologic and toxicologic diseases. Female patient at the age of 35 was admitted to the emergency department with complaints of tendency to sleep and sensory loss. About 12 hours before her history she had taken 200 tablets of 10 mg memantin HCL (Ebixa) (2000 mg) and she was transferred to an intensive care department with the diagnosis of drug toxicity (400 mg toxic dose). Her memantin HCL (Ebixa) level in blood was 12,000 ng/mL. It was reported in her physical examination that she was unconscious, her general condition was bad, there were no cooperation and orientation, ahe hadmydriasis and reflexes of light, cornea and eyelash were bilaterally positive and she had horizontal nystagmus. Glascow Coma Scale of the patient was 6, body temperature was 37.5 degrees C and she had tachycardia (130/min) and hypertension (160/90 mmHg). Intravenous Diazepam was effective aginst recurring convulsions. Sinusoidal tachycardia was detected with electrocardiography (EKG) and respiratory alkalosis in arterial blood gases. Six cysles of plasmapheresis were aplied and in the sixth cycle the memantin HCL (Ebixa) level turned to normal. As a result of the sixth plasmapheresis the findings were normal and that is why she was discharged from the hospital. Plasmapheresis should be taken into consideration in case of drug overdose or high doses of plasmatic proteinous drug toxicities (Ref. 11).

In vitro and in vivo evaluation of the effects of demineralized bone matrix or calcium sulfate addition to polycaprolactone-bioglass composites.

The objective of this study was to improve the efficacy of polycaprolactone/bioglass (PCL/BG) bone substitute using demineralized bone matrix (DBM) or calcium sulfate (CS) as a third component. Composite discs involving either DBM or CS were prepared by compression moulding. Bioactivity of discs was evaluated by energy dispersive X-ray spectroscopy (ESCA) and scanning electron microscopy (SEM) following simulated body fluid incubation. The closest Calcium/Phosphate ratio to that of hydroxyl carbonate apatite crystals was observed for PCL/ BG/DBM group (1.53) after 15 day incubation. Addition of fillers increased microhardness and compressive modulus of discs. However, after 4 and 6-week PBS incubations, PCL/BG/DBM discs showed significant decrease in modulus (from 266.23 to 54.04 and 33.45 MPa, respectively) in parallel with its highest water uptakes (36.3 and 34.7%). Discs preserved their integrity with only considerable weight loss (7.5-14.5%) in PCL/BG/DBM group. In vitro cytotoxicity tests showed that all discs were biocompatible.

[Seronin syndrome and cardiac arrest caused by high-dose moclobemide (case report)].

Serotonin syndrome is the syndrome resulting from brain tissue serotonin accumulation and accompanying by central nervous system dysfunction and circulatory collapse, which leads to a serious mortal danger to life. A female patient aged 31 years, diagnosed as having chronic psychosis in the history, was admitted to an intensive care unit in a critical state for having taking an increased moclobemide dose. The patient developed cardiac arrest and cardiopulmonary resuscitation (CPR) was initiated. A 15-minute CPR recovered sinus rhythm and pulse on the peripheral arteries of the limbs. When consciousness and respiration improved, the patient was weaned from resuscitation and extubated on the second day. On day 4, the patient was transferred from the intensive care unit to the department of psychiatry. The authors consider that patients with overdosage of antipsychotic agents at a risk for such serious complications, such as cardiac arrest, should be necessarily monitored in the intensive care unit.

Transient palsy of peripheral cranial nerves following open heart surgery.

A 32-year-old man developed hoarseness of voice, inability to swallow and restricted movement of the tongue after open heart surgery. Peripheral injury of the cranial nerves IX, X and XII was suspected, and it was thought that the duration of the surgery together with the endotracheal tube cuff and trans-oesophageal echocardiography probe pressure, as well as the head and neck position might have been the causes of this complication.

Laryngeal mask airway insertion with remifentanil.

INTRODUCTION: We conducted a study to find out the best conditions for LMA insertion with two different doses of remifentanil added to propofol and propofol administered alone. METHODS: Following hospital clinical research ethics committee approval, 60 ASA I-II patients were included in the randomized double-blind study. Following premedication, patients received i.v. 0.25 microg kg(-1) remifentanil (Group R1), 0.50 microg kg(-1) remifentanil (Group R2) or normal saline (Group P) in 60 sec. Then following 20 mg lidocaine, propofol 2 mg kg(-1) were administered in R1 and R2 groups and 2.5 mg kg(-1) in P group. Ease of insertion of LMA and airway quality at first attempt was assessed. Number of attempts of LMA insertion, apnea time, additional propofol requirement and hemodynamic changes were recorded. RESULTS: There were no significant differences in demographic data among the patients. Apnea time (mean +/- SEM) was significantly shorter in P group (34.09 +/- 5.5 sec) compared to R1 (82.5 +/- 12.7 sec) and R2 (87.2 +/- 6.6 sec) groups (p = 0.01 and p = 0.001). Ease of LMA insertion was assessed as grade 1 in 100% of patients in R2 group while 65% in R1 and 30% in P groups. Undesirable responses following LMA insertion were observed in 54% of patients in P group. CONCLUSION: Propofol given 2.5 mg kg(-1) alone is not a good agent for LMA insertion. Remifentanil used in both doses combined with propofol provides good and excellent conditions for insertion of LMA with minimal hemodynamic disturbances.

The effect of propofol and thiopentone on impairment by reactive oxygen species of endothelium-dependent relaxation in rat aortic rings.

The effect in isolated rat aorta of propofol, thiopentone, midazolam, etomidate and fentanyl on the impairment of endothelium-dependent relaxation by reactive oxygen species is reported. Aortic rings were exposed to reactive oxygen species by the electrolysis of the bathing physiological buffer, and endothelium-dependent relaxation in response to acetylcholine was inhibited. This inhibition was countered by incubation before electrolysis with propofol (2 x 10(-5) and 6 x 10(-5) M) or thiopentone (10(-5)-10(-4) M), but not with midazolam (3 x 10(-4) M), etomidate (3 x 10(-4) M) or fentanyl (3 x 10(-5) M). We suggest that the protective effect of propofol and thiopentone against the loss of endothelium-dependent relaxation caused by reactive oxygen species may be because of their antioxidant and free radical scavenging properties, which could be clinically relevant during surgical procedures in which ischaemia is unavoidable.

The pulmonary vascular response to propofol in the isolated perfused rat lung.

This study investigated the effect of propofol on the pulmonary vascular bed of the rat. Propofol (5 x 10(-6) to 5 x 10(-4) M) did not alter the basal perfusion pressure in isolated rat lungs perfused at a constant flow (0.03 mL g body wt-1 min-1) with Krebs-Henseleit solution. When perfusion pressure was elevated by raising the K+ concentration to 30 mM (depolarizing Krebs-Henseleit solution), propofol decreased it in a concentration-dependent manner. Indomethacin (3 x 10(-6) M) and NG-nitro-L-arginine methyl ester (10(-4) M) did not affect the response to propofol, which excluded the role of cyclo-oxygenase metabolites and nitric oxide, respectively. The ATP-sensitive K+ (K+ATP) channel blocker glibenclamide (3 x 10(-6) and 10(-5) M) inhibited the vasodilator effect of propofol. When lungs were perfused with Ca(2+)-free depolarizing Krebs-Henseleit solution, 0.1-2.5 mM Ca+2 produced a concentration-dependent pressor response. Propofol (5 x 10(-5) M) attenuated the vasopressor response to Ca2+ significantly. In conclusion, the activation of K+ATP channels is probably the major mechanism of the vasodilator effect of propofol, at clinically relevant concentrations, in the rat lung. The Ca2+ antagonistic property of propofol is evident only at higher concentrations.

Role of G proteins in the vasodilator response to endothelin isopeptides in vivo.

The purpose of the present study was to determine the influence of pertussis toxin (PTX) on the pulmonary and systemic vasodilator responses to endothelin (ET) isopeptides in the intact cat under conditions of constant pulmonary blood flow and left atrial pressure. When pulmonary vasomotor tone was actively increased by an intralobar arterial infusion of U-46619, intralobar arterial bolus injections of ET-1, ET-2, and ET-3 decreased lobar arterial pressure and systemic vascular resistance in a dose-related manner. The vasodilator responses to ET-1 and ET-2 in the cat lung were abolished by PTX pretreatment, whereas PTX pretreatment did not alter the pulmonary vasodilator response to ET-3 and cromakalim, a specific ATP-sensitive potassium (KATP) channel activator, and the systemic vasodilator responses to all ET isopeptides studied. Glipizide, an inhibitor of KATP channels, inhibited the pulmonary vasodilator responses to ET-1, ET-2, and ET-3, whereas the systemic vasodilator responses to these isopeptides were not changed. The present data are the first to provide a functional correlate in vivo suggesting the existence of different signal transduction mechanisms for two pulmonary vascular ET receptor subtypes, ETA-like that is PTX sensitive and has greater sensitivity to ET-1 and ET-2 (than to ET-3) and ETc-like that is PTX insensitive and has sensitivity to ET-3 (than to ET-1 and ET-2). However, both ET-receptor subtypes promote vasodilation in the adult pulmonary vascular bed by activating KATP channels.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of nifedipine, verapamil and diltiazem on pulmonary vascular resistance and vasoconstrictors in cats.

In the present study, the effects of three classes of L-type calcium channel-blocking agents, nifedipine, verapamil and diltiazem, on the lobar arterial pressure and the vasoconstrictor responses in the pulmonary vascular bed were compared to those of cromakalim, a KATP channel activator, in the anaesthetized cat under controlled pulmonary blood flow and constant left atrial pressure. These drugs were infused intralobarly in doses selected which did not raise left atrial pressure, change cardiac output or alter systemic arterial pressure. Intralobar bolus injections of calcium channel-blocking agents and of the K+ channel activator decreased the lobar arterial pressure in a dose-related manner when pulmonary vasomotor tone was actively elevated by intralobar arterial infusion of U46619. The pulmonary vasodilator response to these agents was accompanied by a dose-related decrease of systemic arterial pressure. In decreasing lobar arterial pressure at elevated pulmonary vasomotor tone, the order of potency was nifedipine > verapamil > diltiazem, whereas in reducing systemic arterial pressure, the order of potency was nifedipine > diltiazem > verapamil. The calcium channel-blocking agents were less active than the reference drug, cromakalim, in both vascular beds. Intralobar arterial infusions of nifedipine, verapamil and diltiazem, at the rates of 0.03 mumol/min, 0.2 mumol/min and 0.1 mumol/min, respectively, caused no changes in cardiac output and in systemic and pulmonary arterial pressure. Infusion of all three calcium-channel-blocking agents blocked the pulmonary vasoconstrictor responses to BAY K 8644 (calcium entry promoter) and U46619 (thromboxane A2 mimic). Nifedipine infusion also reduced the pulmonary vasoconstrictor responses to methoxamine and BHT933 (alpha 1- and alpha 2-adrenoceptor agonists, respectively), whereas verapamil infusion reduced the responses only to methoxamine. Infusion of diltiazem caused no significant decrease of responses to either alpha-adrenoceptor agonist. The results of the present study suggest that the dihydropyridine, nifedipine, is more potent than the non-dihydropyridines, verapamil and diltiazem, in reducing the pulmonary vascular resistance and more effective in inhibiting the vasoconstrictor responses to the alpha-adrenoceptor agonists, to U46619 and to BAY K 8644 in the feline pulmonary circulation at the infusion rates which cause no or little hemodynamic changes.

Resolution and kinetic characterization of glutathione S-transferases from human jejunal mucosa.

Cytosolic glutathione S-transferases were purified from human jejunal mucosa by affinity chromatography on S-hexylglutathione-Sepharose 4B. Chromatofocusing in the pH range 7-4 yielded peaks with apparent pI's of 7.2 (peak 1), 5.2 (peak 2), and 4.4 (peak 3). Each enzymatic fraction was shown to have a homodimeric structure, with subunit mass of 24.9 +/- 0.5 (P1), 27.9 +/- 0.9 (P2), and 23.4 +/- 0.8 (P3) kDa, as determined by SDS-PAGE. The substrate specificity of each peak was tested using discriminating substrates for basic, near-neutral, and acidic GSTs. With cumene hydroperoxide, the diagnostic substrate for the alpha (basic) class of GSTs, P1 showed 8- to 36-fold higher activity than P2 and P3. Ethacrynic acid, the selective substrate for the acidic enzyme (pi), gave highest activity with P3. The inhibitory potentials of sulfobromophthalein, cibacron blue, tributyltin acetate, triphenyltin chloride, and bromphenol blue were also tested. A qualitative resemblance between P1 and alpha, and P3 and pi GSTs was noted. The substrate specificity and inhibiton parameters of P2 corresponded most closely to those of mu-GST. The relative abundances of P1, P2, and P3 (based on CDNB-conjugating activity) were 35, 5, and 60%, respectively.