Effect of IL-1ß, TNF-α and IGF-1 on trans-endothelial passage of synthetic vectors through an in vitro vascular endothelial barrier of striated muscle.

When administrated in the blood circulation, plasmid DNA (pDNA) complexed with synthetic vectors must pass through a vascular endothelium to transfect underlying tissues. Under inflammatory condition, cytokines can modify the endothelium integrity. Here, the trans-endothelial passage (TEP) of DNA complexes including polyplexes, lipoplexes and lipopolyplexes was investigated in the presence of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) or insulin-like growth factor-1 (IGF-1). The experiments were performed by using an in vitro model comprising a monolayer of mouse cardiac endothelial cells (MCEC) seeded on a trans-well insert and the transfection of C2C12 myoblasts cultured on the lower chamber as read out of TEP. We report that polyplexes made with a histidinylated derivative of lPEI (His-lPEI) exhibit the highest capacity (10.5 µg cm-2 h versus 0.324 µg cm-2 h) to cross TNF-α-induced inflamed endothelium model, but this positive effect is counterbalanced by the presence of IL-1ß. His-lPEI polyplex TEP is also increased in the presence of IGF-1 (2.58 µg cm-2 h). TEP of lipid-based DNA complexes including lipoplexes and lipopolyplexes was lowest compared with polymer-based DNA complexes. Overall, the results indicate that under inflammation, His-lPEI polyplexes have a good profile to cross a vascular endothelium of striated muscle with low cytotoxicity and high transfection efficiency of C2C12 myoblasts. These data provide insights concerning the endothelial passage of vectors in inflammatory conditions and can serve as a basis towards in vivo studies.

A Robust H ∞ Controller for an UAV Flight Control System.

The objective of this paper is the implementation and validation of a robust H ∞ controller for an UAV to track all types of manoeuvres in the presence of noisy environment. A robust inner-outer loop strategy is implemented. To design the H ∞ robust controller in the inner loop, H ∞ control methodology is used. The two controllers that conform the outer loop are designed using the H ∞ Loop Shaping technique. The reference vector used in the control architecture formed by vertical velocity, true airspeed, and heading angle, suggests a nontraditional way to pilot the aircraft. The simulation results show that the proposed control scheme works well despite the presence of noise and uncertainties, so the control system satisfies the requirements.

Depressed transient outward current in single hypertrophied cardiomyocytes isolated from the right ventricle of ferret heart.

OBJECTIVE: The aim of this study was to investigate transient outward potassium current (Ito) changes as a basis for the prolongation of the action potential associated with cardiac hypertrophy. METHODS: Right ventricular hypertrophy was induced by chronic pulmonary artery constriction in adult male ferrets. After 4-6 weeks, hearts were excised and single myocytes were isolated from the right ventricles of banded and sham-operated (control) animals by enzymatic dissociation. Ito was recorded by means of the whole cell patch clamp technique. RESULTS: Heart weight:body weight ratio and cell membrane capacitance, as indications of hypertrophy, were increased by 17% (P < 0.05) and 32% (P < 0.01) respectively in the banded group. Analysis of Ito showed that in hypertrophied myocytes compared to normal controls: (1) the density of current was significantly reduced; (2) both the time to peak and the time constant of inactivation were increased; (3) the voltage-dependent steady-state availability was not changed, with similar potentials for half activation (30.4 +/- 6.8 mV in control and 33.9 +/- 8.5 mV in hypertrophied cells) and half inactivation (-12.3 +/- 3.3 mV in control and -11.4 +/- 2.7 mV in hypertrophied cells); (4) the time constant for recovery from inactivation was significantly increased regardless of the holding potentials (-50 mV, -70 mV or -90 mV). CONCLUSIONS: Alterations of the transient outward potassium current with respect to its density, kinetics and recovery from inactivation can explain the prolongation of the action potential in myocytes isolated from pressure-overload hypertrophied heart and may thus be an important step in such cardiac adaptation.

Intracellular calcium transients from newborn rat cardiomyocytes in primary culture.

Resting and transient levels of intracellular free calcium concentrations were recorded in indo-1 loaded neonatal rat ventricular cardiomyocytes in primary culture by means of an interactive laser cytometer. The calcium transients were induced by high potassium and caffeine applications. The resting level of intracellular calcium remained constant (about 140 nM) throughout the culture (up to 7 days). The calcium transients induced by 100 mM K+ changed during culture from a low, cobalt sensitive response at 2 days, to a strong biphasic response at 7 days. At 2 days the response was fully blocked by cobalt. At 7 days the transient phase was abolished by cobalt and ryanodine, whereas the second sustained phase was only partially blocked. The calcium transient induced by caffeine was present as early as the first days, and increased with the age of the culture. This transient was blocked by ryanodine. The calcium influx through sarcolemmal calcium channels could be responsible for intracellular calcium transients in 2 day-old cells, whereas in 7 day-old cells, they seem to be only the trigger for sarcoplasmic reticulum calcium release via a mechanism such as 'calcium-induced calcium-release'. Other mechanisms, such as the sodium-calcium exchange mechanism activated by sarcolemmal depolarisation, seem to be implicated too and therefore could explain the sustained level of intracellular calcium during 100 mM K+ stimulation. The developmental changes through differentiation and maturation of myocytes in culture could account for the age dependent evolution of the responses obtained. From these results it is possible to conclude that calcium movements implicated in the excitation-contraction coupling mechanism in the development of rat neonatal cardiomyocytes are similar in primary culture and in the postnatal period in vivo.

The action of calcium channel blockers on recombinant L-type calcium channel alpha1-subunits.

1. CHO cells expressing the alpha(1C-a) subunit (cardiac isoform) and the alpha(1C-b) subunit (vascular isoform) of the voltage-dependent L-type Ca2+ channel were used to investigate whether tissue selectivity of Ca2+ channel blockers could be related to different affinities for alpha1C isoforms. 2. Inward current evoked by the transfected alpha1 subunit was recorded by the patch-clamp technique in the whole-cell configuration. 3. Neutral dihydropyridines (nifedipine, nisoldipine, (+)-PN200-110) were more potent inhibitors of alpha(1C-)b-subunit than of alpha(1C-a)-subunit. This difference was more marked at a holding potential of -100 mV than at -50 mV. SDZ 207-180 (an ionized dihydropyridine) exhibited the same potency on the two isoforms. 4. Pinaverium (ionized non-dihydropyridine derivative) was 2 and 4 fold more potent on alpha(1C-a) than on alpha(1C-b) subunit at Vh of -100 mV and -50 mV, respectively. Effects of verapamil were identical on the two isoforms at both voltages. 5. [3H]-(+)-PN 200-110 binding experiments showed that neutral dihydropyridines had a higher affinity for the alpha(1C-b) than for the alpha(1C-a) subunit. SDZ 207-180 had the same affinity for the two isoforms and pinaverium had a higher affinity for the alpha(1C-a) subunit than for the alpha(1C-b) subunit. 6. These results indicate marked differences among Ca2+ channel blockers in their selectivity for the alpha(1C-a) and alpha(1C-b) subunits of the Ca2+ channel.

Clinician attributions associated with the diagnosis of schizophrenia in African American and non-African American patients.

The authors examined the schizophrenia diagnosis in 292 psychiatric inpatients in a largely African American community. Clinicians completed a free-response questionnaire that described their diagnostic decisions. Psychotic symptoms such as hallucinations, which were attributed to African American and non-African American patients at different rates, did not necessarily correspond to differences in diagnostic rates. Rather, symptoms not differentially attributed between groups often corresponded with higher rates of schizophrenia for African American patients. Attributions of negative symptoms showed the largest differences between African American and non-African American patients in rates of schizophrenia diagnosis; thought disorder equalized rates of the diagnosis between the 2 groups of patients. Logistic regression analyses suggested that different aggregate decision models were applied to patients of differing race.

Scorpion envenoming in two regions of Colombia: clinical, epidemiological and therapeutic aspects.

To determine clinical and epidemiological features of scorpion stings in two departments of Colombia, a descriptive study was performed in the hospitals of 10 towns from Antioquia (2 256 071 inhabitants) and five from Tolima (630 424 inhabitants). One hundred and twenty-nine cases were admitted during one year, 51 in Antioquia, 78 in Tolima and 41 were children less than 15 years old. Most stings (70.5%) occurred inside the house; 27.9% were on the hands and 26.4% on the feet. The scorpion species involved were Tityus pachyurus (51), Centruroides gracilis (31), T. fuehrmanni (29), T. asthenes (7) and Chactas spp. (1). In 10 cases the scorpion involved was not identified. Systemic envenoming signs (e.g. vomiting, tachypnea) were significantly more frequent in children than in adults (P < 0.05). Four children had hypertension, but none developed pulmonary oedema. One 3-year-old girl, stung by T. asthenes, had acute oedematous pancreatitis. Ninety-eight patients had mild envenoming. Moderate (27 patients) and severe (four patients) envenoming was significantly more frequent in children than in adults (P = 0.003; relative risk = 2.97). A pepsin-digested anti-Centruroides spp. antivenom was administered to 19 of 31 patients presenting systemic envenoming signs. No adverse reactions to antivenom were observed.

[Idiopathic mediastinal fibrosis associated with hypercoagulability: a case report]. / Fibrosis mediastínica idiopática asociada a hipercoagulabilidad. A propósito de un caso.

Mediastinal fibrosis is a rare and unknown disease characterized by the presence of a fibrotic mass in the anterior mediastinum that can invade close structures as gullet, trachea, recurrent nerve, superior cava vein etc. Only 1-2% of patients with mediastinal fibrosis associate superior cava vein obstruction. When it appears, it is produced by extrinsic compression for fibrotic magma in almost all the cases. A case of hypercoagulability disorder associated with mediastinal fibrosis and superior cava vein obstruction by thrombosis (intrinsec mechanism), and the satisfactory evolution after the treatment with oral anticoagulation, corticosteroids and tamoxifen is presented.

Effects of thapsigargin and cyclopiazonic acid on intracellular calcium activity in newborn rat cardiomyocytes during their development in primary culture.

The effects of specific inhibitors of sarcoplasmic reticulum (SR) calcium ATPase, thapsigargin (TG), and cyclopiazonic acid (CPA) were investigated on the resting and transient levels of intracellular free calcium concentrations recorded in Indo-1-loaded ventricular myocytes of newborn rat heart in primary culture. The calcium transients were induced by caffeine (10 mM) or high potassium (100 mM) solutions. In 2 day- as in 7-day-old cultured cells, the calcium transients induced by 10 mM caffeine were blocked dose dependently by TG and CPA. The dose-response curves suggest that TG was more efficient than CPA and that both drugs were more efficient in 7-day- than in 2-day-old cells. The calcium transients induced by 100 mM K+ were also strongly inhibited by these agents. The lack of effect on sarcolemmal calcium currents, as shown by whole-cell patch-clamp experiments, suggests that these drugs affect only SR function. In cells exhibiting spontaneous activity, the associated calcium transients were not affected by TG or CPA at the beginning of the culture (2-day-old cells) but were fully blocked at the end (7-day-old cells). These results confirm that TG and CPA specifically inhibit the cardiac SR Ca2+ pump without affecting the sarcolemmal calcium current. Their blocking effect of the calcium transients as a function of the developmental stage of neonatal cardiac cells in culture suggests an increasing role of the SR in the regulation of intracellular calcium. This argues for developmental changes of the SR through the differentiation and maturation of newborn cardiomyocytes at the early stage of the postnatal life, leading to a predominant role of the SR in excitation-contraction coupling mechanisms in adult cells.

Changes of the potassium currents in rat aortic smooth muscle cells during postnatal development.

This study was designed to investigate the role and regulation of arterial K+ channels during postnatal development. Rat thoracic aortic segments were suspended for isometric tension and resting membrane potential (RMP) recording. Contraction in response to 4-aminopyridine (4-AP) was similar in 4-, 8- and 12-week-old rats but was higher in 1-day-old rats. Contraction in response to tetraethylammonium (TEA) increased after 4 weeks. TEA increased the contractions evoked by noradrenaline in the aorta from 8- and 12-week-old rats but not from 1-day- and 4-week-old rats. RMP did not change during development. Patch-clamp studies of freshly isolated smooth muscle cells from the same aortas bathed in Ca2+-free medium showed a voltage-dependent K+ current (IK) sensitive to 4-AP. This current remained stable at all ages whereas the density of the total IK, recorded in the presence of Ca2+, showed a twofold increase between 4 and 8 weeks. This current was highly sensitive to TEA and charybdotoxin. The binding site density of 125I-labelled charybdotoxin was threefold higher in the membranes of aortas from 12-week-old compared to 4-week-old rats. These results indicate that changes in K+ channel distribution occur in the rat aorta during postnatal development. These are related to an increase in the expression of charybdotoxin-sensitive Ca2+-activated K+ channels.

T-type calcium current is expressed in dedifferentiated adult rat ventricular cells in primary culture.

Ventricular myocytes isolated from the heart of adult rats were able do dedifferentiate within 7 days in primary culture using a medium containing 10% fetal calf serum. Calcium current was recorded in these cells by means of the whole cell patch clamp technique and compared to the calcium current obtained in freshly isolated myocytes and in non dedifferentiated cultured myocytes. In dedifferentiated cardiomyocytes, both T-type (ICa-T) and L-type (ICa-L) calcium current components were recorded while only L-type was observed in freshly isolated and in non dedifferentiated cultured myocytes. ICa-T was separated from ICa-L through its voltage dependence and its pharmacology. These results demonstrate that ICa-T which has been shown to be present at the neonatal stage but absent in adult ventricular cells can be reexpressed when adult cells were dedifferentiated in culture. Its possible involvement in development of cardiac cells and in electrophysiological properties leading to spontaneous activity is discussed.

Action of a NO donor on the excitation-contraction pathway activated by noradrenaline in rat superior mesenteric artery.

The aim of the present study was to investigate the actions of NO donors in ratsuperior mesenteric artery stimulated with noradrenaline by studying their effects on isometric tension, membrane potential (Vm), cytosolic calcium concentration ([Ca2+]cyt) and accumulation of inositol phosphates. In unstimulated arteries, SNAP (S-nitroso-N-acetylpenicillamine, 10 microM) hyperpolarised Vm by 3.0 +/- 0.5 mV (n = 9). In KCl-stimulated arteries, SNAP relaxed contraction without changing Vm and [Ca2+]cyt. In noradrenaline-stimulated arteries, SNAP relaxed tension, repolarised Vm and decreased [Ca2+]cyt with the same potency. Responses to SNAP were unaffected by the following K+ channel blockers: glibenclamide, 4-aminopyridine, apamin and charybdotoxin, and by increasing the KCl concentration to 25 mM. In SNAP-pretreated arteries, the production of inositol phosphates and the contraction stimulated by noradrenaline were inhibited similarly. The guanylate cyclase inhibitor ODQ abolished the increase in cyclic GMP content evoked by SNAP and inhibited the effects of SNAP on contraction, Vm and accumulation of inositol phosphates in noradrenaline-stimulated artery. These results indicate that, in rat superior mesenteric arteries activated by noradrenaline, inhibition of production of inositol phosphates is responsible for the effects of the NO donor SNAP on membrane potential, [Ca2+]cyt and contraction through a cyclic GMP-dependent mechanism.

Effects of Bay K 8644 on L-type calcium current from newborn rat cardiomyocytes in primary culture.

We examined the effects of the dihydropyridine agonist Bay K 8644 on L-type calcium channels in newborn rat ventricular cardiomyocytes during their development in primary culture. Experiments were performed at day 2 and 7 of the culture which constituted the early postnatal and maximally developed stages, respectively, of isolated cells in our experimental conditions. In the presence of racemic Bay K 8644 (10(-6) M), L-type calcium current (ICa-L) density recorded by perforated patch-clamp technique was increased by 127 +/- 4% (n = 8) in 2-day-old cells. The increase was only 103 +/- 5% (n = 10) in 7-day-old cells, resulting in a current density similar to that observed in freshly-dissociated adult cells (90 +/- 7%; n = 10). At every stage of the culture Bay K 8644 increased ICa-L with a 10-mV shift of the peak current towards hyperpolarized levels but without change in activation threshold and reversal potential. This shift can be explained by the corresponding change in steady-state activation and inactivation relationships towards negative potentials. The potentiating effect of Bay K 8644 was further studied as a function of phosphorylation levels of calcium channels. When calcium channels were phosphorylated by dibutyryl cyclic AMP (2 x 10(-4) M), increase of ICa-L density by Bay K 8644 was comparable at every stage of cell culture. However, direct activation of beta-receptors by isoproterenol did not increase ICa-L density in 2-day-old cultured cells as it did in 7-day-old cells, while direct activation of adenylate cyclase by forskolin similarly increased ICa-L, at both stages of culture. From these results, it can be suggested that the higher increase of ICa-L density by Bay K 8644 in 2-day- than in 7-day-old cultured cells could be interpreted as the result of a difference in the phosphorylation level of calcium channels for each stage of development. The possible increase in the basal phosphorylation level of calcium channels during culture of neonatal cardiac cells is discussed with respect to changes in functional properties of calcium channels during postnatal maturation of these cardiac cells.

Developmental changes in Ca2+ currents from newborn rat cardiomyocytes in primary culture.

Electrophysiological characteristics of neonatal rat ventricular cardiomyocytes in primary culture were studied using the whole-cell patch-clamp recording technique. Cell size, estimated by measurement of membrane capacitance, was significantly increased throughout the culture from 22.4 +/- 5.4 pF at day 2 to 55.0 +/- 16.1 pF at day 7, reflecting the hypertrophic process which characterises postnatal cell development. The Ca2+ current was investigated at day 2 and 7 of the culture which constituted the early postnatal and maximally developed stages, respectively, of isolated cells in our experimental conditions. At 2 days of culture, two types of Ca2+ current could be distinguished, as also observed in freshly dissociated newborn ventricular cells. From their potential dependence and pharmacological characteristics, they could be attributed to the T- (ICa-T) and L-type (ICa-L) Ca2+ current components. After 7 days of culture, only the latter ICa-L was present and its density was significantly increased when compared to the density in 2-day-old cells, but lower than that obtained in freshly dissociated adult cells. As the age of the culture progressed, the steady-state inactivation curve was shifted toward negative potentials, in the direction of the inactivation curve obtained for adult cells. Compared to the serum-free control conditions, the density of ICa-L was significantly increased in the presence of fetal calf serum throughout the culture. Consequently, the density of ICa-L obtained in 7-day-old cells was similar to the density of ICa-L obtained in freshly dissociated adult cardiac cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative efficacy and safety of calcium carbasalate plus metoclopramide versus ergotamine tartrate plus caffeine in the treatment of acute migraine attacks.

This randomized, double-blind, double-dummy, multicenter, parallel-group study aimed at comparing the efficacy and safety of calcium carbasalate (equivalent to 900 mg aspirin) plus metoclopramide 10 mg (CM) with ergotamine tartrate 1 mg plus caffeine 100 mg (EC) administered in the treatment of 2 acute migraine attacks. A total of 296 patients fulfilling the International Headache Society diagnostic criteria for migraine were enrolled. In total, one or two migraine attacks were treated in 268 and 235 patients, respectively. The primary endpoint for the first treated attack was headache relief, with intensity decreasing from moderate or severe to mild or absent 2 h after drug intake. Usual secondary efficacy endpoints were assessed. A superiority of CM over EC was observed for both treated attacks for the main endpoint: success in 54 versus 36%, p = 0.003 for the first attack and 60 versus 44%, p = 0.02 for the second attack. CM was also significantly superior to EC during the first attack for complete headache relief (20 vs. 8%, p = 0.006), nausea (42 vs. 63%, p = 0. 007) and willingness to take the drug again (90 vs. 80%, p = 0.043). The global efficacy evaluation, rated by the investigators, was significantly more favorable to CM for both attacks (p = 0.001 for the first attack and p = 0.02 for the second). The patients' evaluation was significant for the first attack (p = 0.002). The global incidence of adverse events was 45% higher with EC, though not significant (32 vs. 22%, p = 0.075). They were most often unspecific and mild to moderate in intensity. Gastrointestinal side effects were significantly less frequent with CM than EC (7 vs. 21%, p = 0.001). Thus, CM is more effective and has a better gastrointestinal safety than EC in the acute treatment of migraine attacks.

[Medicine, mathematics and statistics. Practical aspects]. / Medicina, matemáticas y estadística. Aspectos prácticos.

To identify the statistical methods more frequently used in the medical literature, we reviewed 4,218 papers equivalent to 26 years/periodical. 26% of the papers did not have any statistical method. In the remaining 74% only descriptive (24.5%) or inferential (75.5%) methods were used. Overall there were 3,882 inferential test done. Based on a probability analysis we propose a framework to rationalize the teaching of statistics to improve the decision making process in medicine.