[A new treatment for vascular anomalies: Six cases treated with rapamycin]. / Une nouvelle approche thérapeutique des malformations vasculaires à faible débit : la rapamycine ­ à propos de 6 cas.

Vascular anomalies (VAs) result from the defective development of the embryonic vascular system and feature dysplastic malformed vessels, which are not always apparent at birth. They do not regress over the patient's lifetime; they usually have commensurate growth during childhood and may worsen over time if not treated. VAs may cause chronic painful swelling, bleeding, functional deficits or vital structure obstruction. These patients' quality of life is usually impaired because of the chronicity and recurrence of the disease. We report on six cases of complicated VAs, refractory to current treatments, treated with rapamycin, an mTor inhibitor recently used in VAs.

[Hemihypertrophy and scoliosis revealing a Chiari 1 malformation with syringomyelia]. / Hémi-hypertrophie et scoliose révélatrices d'une malformation de Chiari de type 1 avec syringomyélie.

We report the case of a 9-year-old boy with progressive thoracic scoliosis and crossed hemihypertrophy who was discovered with a Chiari 1 malformation and syringomyelia. These disorders are connected by complex physiopathological mechanisms; their association deserves attention. This observation reviews the importance of the clinical examination, particularly the neurological exam, in childhood scoliosis. The features suggesting a neurogenic background of spine deformation should be sought. Scoliosis with hemihypertrophy can be the sign of an underlying neurological abnormality.

Toward the management of inflammation: recent developments of mPGES-1 inhibitors.

For decades, the struggle against inflammation and related disorders has constituted an important field in medical practice, with strategies mainly aimed at inhibiting compounds produced through the arachidonic acid pathway. Thus, specific COX-2 inhibitors or "coxibs", were recently designed, that play an increasing but controversial role in reducing inflammatory phenomenon. Lately, several patents have been generated which target the specific inhibition of the microsomal Prostanglandin E synthase-1 (mPGES-1). This enzyme, which was cloned and characterized at the end of the nineties, catalyzes under inflammatory stimuli the last step of PGE2 synthesis. A corpus of data is now available illustrating the pivotal role played by this enzyme in numerous symptoms linked to inflammation such as fever, anorexia or pain. The present review highlights the current state of knowledge of the involvement of mPGES-1 in sickness behaviour and in other inflammation-related disorders and summarizes the recent patents related to mPGES-1 and its specific inhibition.

mPGES-1 knock-out mice are resistant to cancer-induced anorexia despite the absence of central mPGES-1 up-regulation in wild-type anorexic mice.

Anorexia-cachexia syndrome is a very common symptom observed in individuals affected by chronic inflammatory diseases. The present study was designed to address the possible involvement of the inducible microsomal prostaglandin E synthase-1 (mPGES-1) in the hypopaghia observed during these pathological states. To this end, we used a model of cancer-induced anorexia and we report here that despite the absence of up-regulation of the mPGES-1 enzyme within the brain during anorexia-cachexia syndrome, mPGES-1 knock-out mice exhibit resistance to tumor-induced anorexia and maintain their body mass.

c-Fos immunoreactivity induced by intraperitoneal LPS administration is reduced in the brain of mice lacking the microsomal prostaglandin E synthase-1 (mPGES-1).

The aim of the present study was to investigate the impact of the deletion of the microsomal prostaglandin E synthase-1 (mPGES-1) gene on lipopolysaccharide (LPS)-induced neuronal activation in central nervous structures. The mPGES-1 catalyses the conversion of COX-derived PGH(2) to PGE(2) and has been described as a regulated enzyme whose expression is stimulated by proinflammatory agents. Using the immediate-early gene c-fos as a marker of neuronal activation, we determined whether deletion of the mPGES-1 gene altered the neuronal activation induced by LPS in structures classically recognized as immunosensitive regions. No significant difference in the c-Fos immunostaining was observed in the brain of saline-treated mPGES-1+/+, mPGES-1+/- and mPGES-1-/- mice. However, we observed that LPS-induced neuronal activation was reduced in most of the centres known as immunosensitive nuclei in mPGES-1-/- mice compared with heterozygous and wild-type mice. The decrease in the number of c-Fos positive nuclei occurred particularly in the caudal ventrolateral medulla, the medial, intermediate and central parts of the nucleus tractus solitarius, area postrema, parabrachial nucleus, locus coeruleus, paraventricular nucleus of the hypothalamus, ventromedial preoptic area, central amygdala, bed nucleus of the stria terminalis and to a lesser extent in the ventrolateral part of the nucleus tractus solitarius and rostral ventrolateral medulla. These results suggest that the mPGES-1 enzyme is strongly needed to provide sufficient PGE(2) production required to stimulate immunosensitive brain regions and they are discussed with regard to the recent works reporting impaired sickness behavior in mPGES-1-/- mice.

Involvement of central microsomal prostaglandin E synthase-1 in IL-1beta-induced anorexia.

In response to infection or inflammation, individuals develop a set of symptoms referred to as sickness behavior, which includes a decrease in food intake. The characterization of the molecular mechanisms underlying this hypophagia remains critical, because chronic anorexia may represent a significant health risk. Prostaglandins (PGs) constitute an important inflammatory mediator family whose levels increase in the brain during inflammatory states, and their involvement in inflammatory-induced anorexia has been proposed. The microsomal PGE synthase (mPGES)-1 enzyme is involved in the last step of PGE2 biosynthesis, and its expression is stimulated by proinflammatory agents. The present study attempted to determine whether an upregulation of mPGES-1 gene expression may account for the immune-induced anorexic behavior. We focused our study on mPGES-1 expression in the hypothalamus and dorsal vagal complex, two structures strongly activated during peripheral inflammation and involved in the regulation of food intake. We showed that mPGES-1 gene expression was robustly upregulated in these structures after intraperitoneal and intracerebroventricular injections of anorexigenic doses of IL-1beta. This increase was correlated with the onset of anorexia. The concomitant reduction in food intake and central mPGES-1 gene upregulation led us to test the feeding behavior of mice lacking mPGES-1 during inflammation. Interestingly, IL-1beta failed to decrease food intake in mPGES-1(-/-) mice, although these animals developed anorexia in response to a PGE2 injection. Taken together, our results demonstrate that mPGES-1, which is strongly upregulated during inflammation in central structures involved in feeding control, is essential for immune anorexic behavior and thus may constitute a potential therapeutic target.

[Evaluation of a new method for detection of obstructive disease in children asthma: the negative expiratory pressure (NEP)]. / Intérêt d'une nouvelle méthode de détection du trouble obstructif dans l'asthme de l'enfant: la pression négative expiratoire (NEP).

INTRODUCTION: To take in charge of an asthmatic child it is necessary to evaluate the lung function. METHODS: In this study, the Negative Expiratory Pressure (NEP) has been used for the first time in children with asthma. After lung spirometry by plethysmography, we have used the NEP to assess the prevalence of expiratory flow limitation (FL) during resting breath in 27 asthmatic children (mean age: 11 +/- 2,5 years) 3-4 days after a crisis in both sitting and supine positions. RESULTS: All the children presented an obstructive defect (FEV 1: 63 +/- 13% med) and a dynamic hyperinflation (FRC: 128 +/- 25% med). According to the NEP, 11 children presented an expiratory flow limitation (FL). Asthma was more severe in the FL than in non-FL children (GINA 2002 classification). Among the 11 FL children, 5 were FL in both sitting and supine position and 6 only in supine. Nine of the 27 children were FL with the conventional method. NEP seems a more accurate method to assess the clinical gravity of asthma than FEV 1. The reduction of FRC in the supine position probably explains the greater incidence of FL in supine position. CONCLUSION: Because of its easy execution, NEP seems to be well adapted for children. Links between FL detected by NEP and clinical signs of asthma has to be assessed by furthers studies including more patients.

Potassium efflux triggered by P2Y purinoceptor activation in cultured pituicytes.

We have previously investigated the effects of extracellular ATP on the concentration of free cytosolic calcium ([Ca2+]i) from rat cultured neurohypophysial astrocytes (pituicytes). We demonstrated that ATP acts via a P2Y receptor to increase [Ca2+]i. In the present study, we examine the effect of ATP on K+ efflux using 86Rb+ as an isotopic tracer, in order to characterize the possible presence of a Ca2+-activated K+ conductance and to establish the implications of pituicytes in the regulation of stimulus-secretion coupling. ATP evoked an increase in 86Rb+ efflux from cultured pituicytes. This effect was Ca2+ dependent, as indicated by the unresponsiveness of cells loaded with BAPTA/AM (20 microM). Furthermore, the effect of ATP was mimicked by 2-methylthio-adenosine-5'-triphosphate (2MeSATP), a P2 purinoceptor agonist, and abolished by Reactive Blue 2 (RB-2), a selective P2Y antagonist, implying a role for the P2Y purinoreceptor. A pharmacological study revealed that Ba2+ and tetraethylammonium (TEA), two inhibitors of K+ channels, both strongly reduced the ATP-stimulated 86Rb+ efflux. In addition, the effect of ATP was modulated by different peptidic toxins. Apamin (100 nM), an inhibitor of the small-conductance Ca2+-activated K+ channels, partly blocked ATP-induced 86Rb+ efflux. Leiurus quinquestriatus hebraeus (LQH) scorpion venom (20 microg/ml) and Buthus tamulus (BT) scorpion venom (20-200 microg/ml) inhibited ATP-induced 86Rb+ efflux. The specificity of the effects of the crude venoms was checked using charybdotoxin (100 nM) and iberiotoxin (1 pM), which are the active toxins extracted from the LQH and BT venoms, respectively. These data indicate the involvement of several types of Ca2+-activated K+ channels in the ATP-dependent K+ efflux, and lead to the proposal that, in the neurohypophysis, extracellular ATP released by nerve terminals may act directly on the pituicytes and induce a K+ efflux via a P2Y purinoreceptor.

[Utilization of an equimolar mixture of oxygen-nitrous oxide in a general pediatric ward]. / Utilisation du mélange équimolaire oxygène-protoxyde d'azote dans un service de pédiatrie générale.

UNLABELLED: We report our experience of the utilization of the 50% oxygen-nitrous oxide mixture (nitrous oxide 50%) in our general pediatric ward after one year of use. PATIENTS AND METHODS: Between 1st April 1997 and 31st March 1998, children who had to undergo a painful procedure were proposed to inhale 50% nitrous oxide before the procedure. We evaluate pain, restlessness and adverse effects. RESULTS: The procedures (127 of them) were carried out in 90 children (61 boys). They were aged from 5 months to 15 years (mean: 5.7 years; median: 4.1 years). Indications were: lumbar puncture (n = 45), burning dressing (n = 29), venous cannulation (n = 12), minor surgery (n = 27), and miscellaneous (n = 14). Inhalation time was between 2 to 70 min (mean: 14.4 min; median: 11 min). Pain was absent or low in 106 cases (83.4%). Restlessness was absent or low in 100 cases (78.8%). Averse events were observed 12 times, but they were always minor and quickly reversible. CONCLUSION: Nitrous oxide (50%) can be used successfully in a general pediatric ward. Other studies are necessary to define the best conditions.

ATP acting on P2Y receptors triggers calcium mobilization in primary cultures of rat neurohypophysial astrocytes (pituicytes).

The effect of adenosine triphosphate (ATP) on the intracellular Ca2+ concentration ([Ca2+]i) of cultured neurohypophysial astrocytes (pituicytes) was studied by fluorescence videomicroscopy. ATP evoked a [Ca2+]i increase, which was dose dependent in the 2.5-50 microM range (EC50=4.3 microM). The ATP-evoked [Ca2+]i rise was not modified during the first minute following the removal of external Ca2+. Application of 500 nM thapsigargin inhibited the ATP-dependent [Ca2+]i increase. Caffeine (10 mM) and ryanodine (1 microM) did not affect the ATP-induced [Ca2+]i rise. The pituicytes responded to various P2 purinoceptor agonists with the following order of potency: ATP=ATP[gamma-S]=2-MeSATP>/=ADP, where ATP[gamma-S] is adenosine 5'-O-(3-thiotriphosphate) and 2-MeSATP is 2-methylthio-adenosine-5'-triphosphate. Adenosine, AMP, alpha, beta-methylene adenosine-5'-triphosphate (alpha,beta-MeATP), beta, gamma methylene adenosine-5'-triphosphate (beta,gamma-MeATP) and uridine 5'-triphosphate (UTP) were ineffective. The P2 purinoceptor antagonists blocked the ATP-evoked [Ca2+]i increase with the following selectivity: RB-2>suramin>PPADS, where RB-2 is Reactive Blue 2 and PPADS is pyridoxal-phosphate-6-azophenyl-2', 4'-disulphonic acid. The ATP-evoked [Ca2+]i increase was substantially blocked by pertussis toxin treatment, suggesting that it might be mediated by a pertussis-toxin-sensitive G protein. The phospholipase C (PLC) inhibitor U-73122 (0.5 microM) abolished the ATP-evoked [Ca2+]i rise, whereas its inactive stereoisomer U-73343 (0.5 microM) remained ineffective. Our results indicate that, in rat cultured pituicytes, ATP stimulation induces an increase in [Ca2+]i due to PLC-mediated release from intracellular stores through activation of a pertussis-toxin-sensitive, G-protein-linked P2Y receptor.

Stimulus-secretion coupling in neurohypophysial nerve endings: a role for intravesicular sodium?

It is generally accepted that Ca is essentially involved in regulated secretion, but the role of this cation, as well as others such as Na, is not well understood. An illustrative example occurs in neurohypophysial secretion, where an experimentally induced increase in the cytosolic concentration of Na+ can induce continuous neuropeptide release. In contrast, an increase in cytosolic Ca2+ will have only a transient stimulatory effect. The secretion-promoting targets for Ca2+ are not known; they may be cytosolic, as is usually assumed, but they may also be intravesicular, especially in view of evidence that Ca-rich secretory vesicles are preferentially secreted. In the present work, we have investigated the movements of these cations into and out of secretory vesicles during stimulus-secretion coupling. Isolated rat neurohypophysial nerve endings were stimulated by potassium (55 mM) depolarization, and at 6 min (peak secretion) and 20 min after the onset of stimulation, the elemental content of individual secretory vesicles was measured by quantitative x-ray microanalysis. A depolarization-induced transient increase in intravesicular Na+ concentration was found to coincide with the onset of secretion. Moreover, only a predicted small fraction of peripheral vesicles-presumably the docked ones-were Na+-loaded. The low sulfur concentration of Na+-rich vesicles most likely resulted from vesicle swelling. The results suggest that high intravesicular Na+ concentrations in docked vesicles, occurring by Na+/Ca2+ exchange or by transient fusion pore opening, is a proximal event in exocytosis.

Calcium-induced calcium increase in secretory vesicles of permeabilized rat neurohypophysial nerve terminals.

Digitonin-permeabilized isolated neurohypophysial nerve terminals are known to release their secretory vesicle content under calcium challenge. On this preparation, we monitored intra-organelle Ca2+ concentration using digital fluorescence microscopy of Fura-2. The superfusion of artificial intracellular solution containing 10 to 50 microM Ca2+ induced an intra-organelle [Ca2+] increase. Two major organelles are candidates for this increase: secretory vesicles and mitochondria. In an attempt to detect calcium changes in the vesicles, ruthenium red was used to impair mitochondrial calcium uptake. Part of the ruthenium red-insensitive intra-organelle [Ca2+] increase was abolished by raising sodium in the solution. Removing sodium boosted the intra-organelle [Ca2+] increase. These results taken together suggest the participation of Na/Ca exchange, known to exist in the membrane of these secretory vesicles. In addition to Na/Ca exchange, there would be at least another mechanism of vesicular calcium intake, as suggested by the partial inhibition of intra-organelle [Ca2+] increase obtained under acidic compartments: neutralization with NH4Cl. This mechanism remains to be defined. The main conclusion presented here, that an intravesicular [Ca2+] increase takes place at the rate of secretion, was predicted by the hypothesis that intravesicular Ca2+ changes would be involved in stimulus-secretion coupling.

ATP-evoked increases in [Ca2+]i and peptide release from rat isolated neurohypophysial terminals via a P2X2 purinoceptor.

1. The effect of externally applied ATP on cytosolic free Ca2+ concentration ([Ca2+]i) was tested in single isolated rat neurohypophysial nerve terminals by fura-2 imaging. The release of vasopressin (AVP) and oxytocin (OT) upon ATP stimulation was also studied from a population of terminals using specific radioimmunoassays. 2. ATP evoked a sustained [Ca2+]i increase, which was dose dependent in the 1-100 microM range (EC50 = 4.8 microM). This effect was observed in only approximately 40 % of the terminals. 3. Interestingly, ATP, in the same range (EC50 = 8.6 microM), evoked AVP, but no significant OT, release from these terminals. 4. Both the [Ca2+]i increase and AVP release induced by ATP were highly and reversibly inhibited by suramin, suggesting the involvement of a P2 purinergic receptor in the ATP-induced responses. Pyridoxal-5-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), another P2 purinergic receptor antagonist, strongly reduced the ATP-induced [Ca2+]i response. 5. To further characterize the receptor, different agonists were tested, with the following efficacy: ATP = 2-methylthio-ATP > ATP-gamma-S > alpha, beta-methylene-ATP > ADP. The compounds adenosine, AMP, beta, gamma-methylene-ATP and UTP were ineffective. 6. The ATP-dependent [Ca2+]i increase was dependent on extracellular Ca2+ concentration ([Ca2+]o). Fluorescence-quenching experiments with Mn2+ showed that externally applied ATP triggered a Mn2+ influx. The ATP-induced [Ca2+]i increase and AVP release were independent of and additive to a K+-induced response, in addition to being insensitive to Cd2+. The ATP-induced [Ca2+]i increase was strongly reduced in the presence of Gd3+. These results suggest that the observed [Ca2+]i increases were elicited by Ca2+ entry through a P2X channel receptor rather than via a voltage-dependent Ca2+ channel. 7. We propose that ATP, co-released with neuropeptides, could act as a paracrine-autocrine messenger, stimulating, via Ca2+ entry through a P2X2 receptor, the secretion of AVP, in particular, from neurohypophysial nerve terminals.

Calcium in secretory vesicles of neurohypophysial nerve endings: quantitative comparison by X-ray microanalysis of cryosectioned and freeze-substituted specimens.

The calcium content of individual secretory vesicles in rat neurohypophysial nerve endings was measured by quantitative electron probe X-ray microanalysis. Directly frozen control and potassium-depolarized isolated endings were analysed using two presumably equivalent preparative techniques: (1) freeze-substitution in presence of oxalic acid followed by sectioning of resin-embedded pellets; or (2) direct cryosectioning of the frozen pellets followed by freeze-drying in the column of the microscope. In the pellets of stimulated endings, both approaches revealed an increase in the calcium content of neurosecretory vesicles. This increase was statistically more significant in the specimens prepared by cryosectioning, probably because in this case the contribution of 'dead' nerve endings could be eliminated on the basis of excessive cytoplasmic sodium and chloride. The results demonstrate that an increase in cytosolic calcium can lead to an increase in intravesicular calcium, and that when this occurs, it occurs within a subpopulation of vesicles in a given nerve ending. In addition, measured intravesicular calcium was dispersed over a wide range of concentrations, as predicted by the hypothesis of intravesicular calcium priming. When the vesicular calcium content was averaged per nerve ending, a relatively wide distribution of concentrations was again observed, indicating that some nerve endings respond more strongly to the stimulation than others.

Mono- and bispecific single-chain antibody fragments for cancer therapy.

Especially when dealing with solid cancers, single-chain antibody fragments (scFvs) have a lot of advantages. Due to their small size (27 kDa), these proteins clear more rapidly from the blood, and penetrate faster and deeper into tissues, than whole antibodies. Furthermore, the lack of constant regions ensures that they are not retained in tissues such as the liver and kidney. This reduces possible toxic side-effects. Single-chain construction is normally done by polymerase chain reaction (PCR). To decrease the overall cost of oligonucleotide primer synthesis, time-consuming primer design, multiple PCR reactions and individual PCR optimization, we designed a universal single-step overlap extension PCR protocol using hybridoma cDNA as a template. To overcome the lack of effector function, bispecific scFvs, consisting of an scFv produced against a tumour-associated antigen fused to a T cell marker-specific scFv, are being created, starting from already assembled scFv, by means of two additional PCR reactions. In this paper we describe both PCR methods that were successfully used to create scFvs against the human transferrin receptor, the human interleukin-2 receptor, the human CD3 molecule, a breast tumour-associated antigen and an anti-transferrin-anti-CD3 bispecific scFv.

Immunotherapy for cancer: construction, expression and functional characterization of chimeric antibodies.

Monoclonal antibodies (Mabs) are a potential key component for the treatment of cancer, because of their specificity and multiple effector functions. Hybridoma technology and progress in genetic engineering made it possible to customize antibody molecules, rendering them more suitable for selective application. A widely used technique is the construction of mouse-human hybrid molecules by recombinant DNA techniques. These so-called chimeric antibodies contain the murine variable (V) regions fused to the human constant (C) regions. In this report, a general approach is described for the production of chimeric antibodies. The gene segments encoding the murine variable heavy and light chain are isolated by the polymerase chain reaction and cloned into expression vectors containing the human gamma 1 heavy chain gene and the human K light chain gene, respectively. Subsequently, these constructs are transfected into a non-Ig-producing murine hybridoma, eg SP2/0 cells. The in vitro study of the functional characteristics and biological properties of the thus obtained chimeric antibodies are discussed.

Cytochemical localization of ecto-ATPases in rat neurohypophysis.

We studied the distribution of Ca(2+)- or Mg(2+)-dependent ATPase activity in rat neurohypophysis using the lead cytochemical method of Ando et al. In electron microscopy, precipitates were found lining the outer surface of the plasma membrane surrounding nerve endings and pituicytes. These precipitates were believed to represent the activity of ecto-ATPases (as opposed to Ca pump ATPases) for the following reasons: there was equal activation by Ca2+ in the absence of Mg2+ or Mg2+ in the absence of Ca2+; the effects of the two ions were not additive; there was activation by ATP or GTP; and there was resistance to glutaraldehyde fixation, to high (10 mM) Ca2+ concentrations, and to various inhibitors such as NEM, vanadate, oligomycin, quercetin, p-chloromercuribenzoate, ouabain, and levamisole. Cytosolic activity observed in certain nerve endings in the same conditions of incubation but more sensitive to NEM is also described and discussed.

Calcium loading of secretory granules in stimulated neurohypophysial nerve endings.

The total calcium content of secretory granules, Cag, was evaluated in isolated neurohypophysial nerve endings. The Cag in the resting state, as measured by X-ray microanalysis, is relatively high with an average of 7.4 +/- 0.6 mmol/kg wet weight. Following a depolarizing potassium challenge, a subpopulation of granules with even higher Cag could be detected, dispersed over a wider range of concentrations (up to 70 mmol/kg wet weight). After subsequent rinsing in physiological saline, Cag decreased to control values. This could have resulted from Ca2+ extrusion, or from preferential secretion of calcium-enriched granules. Our data can be interpreted in favor of the second explanation since no decrease in Cag was observed when secretion was blocked by a hyperosmotic saline. The effect of hyperosmotic conditions on isolated nerve endings was further studied by monitoring free cytoplasmic Ca2+ with the calcium-sensitive dye Fura-2 and by conventional electron microscopy. It was demonstrated that hyperosmotic treatment alone did not increase basal cytosolic Ca2+ concentrations but did significantly reduce the potassium-induced cytosolic rise in Ca2+. Electron microscopy of nerve endings in hyperosmotic conditions showed numerous exocytotic figures at various stages. The observed changes in Cag are in accord with a published hypothesis which proposes that intragranular calcium is a significant variable in regulated secretion.