From bench to bedside: in vitro and in vivo evaluation of a neonate-focused nebulized surfactant delivery strategy.

BACKGROUND: Non-invasive delivery of nebulized surfactant has been a neonatology long-pursued goal. Nevertheless, the clinical efficacy of nebulized surfactant remains inconclusive, in part, due to the great technical challenges of depositing nebulized drugs in the lungs of preterm infants. The aim of this study was to investigate the feasibility of delivering nebulized surfactant (poractant alfa) in vitro and in vivo with an adapted, neonate-tailored aerosol delivery strategy. METHODS: Particle size distribution of undiluted poractant alfa aerosols generated by a customized eFlow-Neos nebulizer system was determined by laser diffraction. The theoretical nebulized surfactant lung dose was estimated in vitro in a clinical setting replica including a neonatal continuous positive airway pressure (CPAP) circuit, a cast of the upper airways of a preterm neonate, and a breath simulator programmed with the tidal breathing pattern of an infant with mild respiratory distress syndrome (RDS). A dose-response study with nebulized surfactant covering the 100-600 mg/kg nominal dose-range was conducted in RDS-modelling, lung-lavaged spontaneously-breathing rabbits managed with nasal CPAP. The effects of nebulized poractant alfa on arterial gas exchange and lung mechanics were assessed. Exogenous alveolar disaturated-phosphatidylcholine (DSPC) in the lungs was measured as a proxy of surfactant deposition efficacy. RESULTS: Laser diffraction studies demonstrated suitable aerosol characteristics for inhalation (mass median diameter, MMD = 3 µm). The mean surfactant lung dose determined in vitro was 13.7% ± 4.0 of the 200 mg/kg nominal dose. Nebulized surfactant delivered to spontaneously-breathing rabbits during nasal CPAP significantly improved arterial oxygenation compared to animals receiving CPAP only. Particularly, the groups of animals treated with 200 mg/kg and 400 mg/kg of nebulized poractant alfa achieved an equivalent pulmonary response in terms of oxygenation and lung mechanics as the group of animals treated with instilled surfactant (200 mg/kg). CONCLUSIONS: The customized eFlow-Neos vibrating-membrane nebulizer system efficiently generated respirable aerosols of undiluted poractant alfa. Nebulized surfactant delivered at doses of 200 mg/kg and 400 mg/kg elicited a pulmonary response equivalent to that observed after treatment with an intratracheal surfactant bolus of 200 mg/kg. This bench-characterized nebulized surfactant delivery strategy is now under evaluation in Phase II clinical trial (EUDRACT No.:2016-004547-36).

Tracking the origin and dispersal of the Asian chestnut gall wasp Dryocosmus kuriphilus Yasumatsu (Hymenoptera, Cynipidae) in Europe with molecular markers.

The Asian chestnut gall wasp, Dryocosmus kuriphilus, is an invasive pest causing significant damage to chestnut trees (Castanea spp., Fagaceae). Originating from China, it has recently invaded a wide range of regions in Europe and North America. Understanding the population genetic structure of important invasive pests is very useful for improving the knowledge concerning routes of expansion and colonizing capacity. Despite its economic importance, limited attention has been given to D. kuriphilus origin and spread, or to its genetic structure. In this study, D. kuriphilus populations sampled in eight European countries were screened using both mitochondrial (cytochrome c oxidase subunit 1; COI) and nuclear (internal transcribed spacer 2; ITS2) sequences, and Amplified Fragment Length Polymorphism (AFLP) markers. The molecular markers COI and ITS2 highlighted the presence of a single haplotype in all the studied populations. The recorded mitochondrial haplotype was identical to one of the most widespread haplotypes occurring in the native area (China). AFLP results indicated that D. kuriphilus individuals belong to two genetically distinct clusters without any further geographic clustering. These results suggest that D. kuriphilus populations in Europe could be the result of a single introduction of a Chinese founder population characterized by two genetically distinct lineages that subsequently spread rapidly across Europe. However, the possibility that populations originated from multiple introductions of the same Chinese mitochondrial haplotype cannot be excluded. The reported results provide useful information concerning this invasive species, potentially facilitating integrated pest management.

Pattern of association between endemic Hawaiian fruit flies (Diptera, Tephritidae) and their symbiotic bacteria: Evidence of cospeciation events and proposal of "Candidatus Stammerula trupaneae".

Several insect lineages have evolved mutualistic association with symbiotic bacteria. This is the case of some species of mealybugs, whiteflies, weevils, tsetse flies, cockroaches, termites, carpenter ants, aphids and fruit flies. Some species of Tephritinae, the most specialized subfamily of fruit flies (Diptera: Tephritidae), harbour co-evolved vertically transmitted, bacterial symbionts in their midgut, known as "Candidatus Stammerula spp.". The 25 described endemic species of Hawaiian tephritids, plus at least three undescribed species, are taxonomically distributed among three genera: the cosmopolitan genus Trupanea (21 described spp.), the endemic genus Phaeogramma (2 spp.) and the Nearctic genus Neotephritis (2 spp.). We examined the presence of symbiotic bacteria in the endemic tephritids of the Hawaiian Islands, which represent a spectacular example of adaptive radiation, and tested the concordant evolution between host and symbiont phylogenies. We detected through PCR assays the presence of specific symbiotic bacteria, designated as "Candidatus Stammerula trupaneae", from 35 individuals of 15 species. The phylogeny of the insect host was reconstructed based on two regions of the mitochondrial DNA (16S rDNA and COI-tRNALeu-COII), while the bacterial 16S rRNA was used for the symbiont analysis. Host and symbiont phylogenies were then compared and evaluated for patterns of cophylogeny and strict cospeciation. Topological congruence between Hawaiian Tephritinae and their symbiotic bacteria phylogenies suggests a limited, but significant degree of host-symbiont cospeciation. We also explored the character reconstruction of three host traits, as island location, host lineage, and host tissue attacked, based on the symbiont phylogenies under the hypothesis of cospeciation.

First Record of Thousand Cankers Disease Fungal Pathogen Geosmithia morbida and Walnut Twig Beetle Pityophthorus juglandis on Juglans regia in Europe.

Thousand cankers disease (TCD) is a disease complex caused by the fungus Geosmithia morbida Kolarik (Ascomycota, Hypocreales) and its vector Pityophthorus juglandis Blackman 1928 (Coleoptera, Scolytinae; walnut twig beetle, WTB). Since the mid-1990s, the disease was responsible for widespread mortality of many walnut species in the United States (4). After the first detection of TCD on black walnut (Juglans nigra L.) in Italy (3), an extensive survey was activated in cooperation with the Regional Phytosanitary Service. In May 2014, early TCD symptoms (4) were observed on English walnuts (J. regia L.). Canopies showed yellowing, wilting, and dieback of the youngest twigs, and a number of small brown cankers. Longitudinal and radial sections sampled through the cankers revealed gray to brown discoloration of both phloem and bark, and the presence of bark beetle galleries. Xylem discoloration was never observed. From one ~20-year-old European walnut growing in a garden neighboring an infected black walnut plantation (Santorso, Vicenza, 45°72' N, 11°40' E), a number of 1- to 2.5-cm-diameter twigs showing cankers up to 2 cm long surrounding bark beetle holes were collected. Whitish mycelium producing verticillate conidiophores was detected inside the insect galleries. From the necrotic margin of eight cankers previously surface-sterilized with 3% sodium hypochlorite, two 4-mm-wide chips per canker were placed on potato dextrose agar and incubated at 28 ± 1°C in the dark. Slow growing lobate, plane, yellowish-ocher colonies with hyaline mycelium appeared in 5 days. After subculturing to the same medium, growth features, mycelium, conidiophores, and conidia with morphological characteristics matching Kolarik's description of G. morbida (2) were observed. The ITS region of rDNA from the fungus strain LM14GM001-JR was amplified by using ITS1F and ITS4 primers and sequenced obtaining a 387-bp gene fragment. BLAST analysis showed 99% identity to the G. morbida strain U19 (GenBank Accession No. KF808301.1) for 384 bp, and 99% identity to the G. morbida strain LM13GM001-JN previously isolated from J. nigra in Italy (3). From the same samples, two emerging beetles were collected and identified as P. juglandis both morphologically (5) and genetically by DNA extraction following a standard salting out protocol. The barcode region of the mitochondrial gene cytochrome oxidase I was then amplified by using universal primers (1) and sequenced to obtain a 614-bp fragment of the gene. BLAST analysis showed 100% identity to P. juglandis based on comparison with KJ451422. A few other English walnuts with both the fungus and WTB were also found close to other infected black walnut plantations. To our knowledge, this is the first record of G. morbida and P. juglandis on J. regia in Europe, where the tree is cultivated for both fruit and timber production, as well as a traditional landscape tree. Voucher specimens are stored in the TeSAF herbarium and in the DAFNAE insect collection. References: (1) O. Folmer et al. Mol. Marine Biol. Biotechnol. 3:294, 1994. (2) M. Kolarik et al. Mycologia 103:325, 2011. (3) L. Montecchio and M. Faccoli. Plant Dis. 98:696, 2014. (4) S. J. Seybold et al. USDA Forest Service, NA-PR-02-10, 2013. (5) S. L. Wood. Great Basin Naturalist Memoirs 6:1123, 1982.

EIA and GC/MS analysis of 8-isoprostane in EBC of children with problematic asthma.

Asthmatic airways are characterised by enhanced oxidative stress, which can be studied by measuring biomarkers, such as 8-isoprostane. The aims of the present study were: 1) to measure the concentrations of 8-isoprostane in exhaled breath condensate (EBC) and urine of children with problematic and well-controlled asthma; 2) to compare the concentrations of 8-isoprostane measured by gas chromatographic/negative ion chemical ionisation mass spectrometry (GC/NICI-MS) and by an enzymatic immunoassay (EIA). We recruited 20 asthmatic allergic children, 13 with well-controlled asthma and seven with problematic asthma. They underwent exhaled nitric oxide measurements and spirometry, and both EBC and urine samples were collected. 8-isoprostane was measured in EBC by GC/NICI-MS and EIA. 8-isoprostane concentrations in EBC were significantly higher in children with problematic asthma than in children with well-controlled asthma (p = 0.01). An acceptable reproducibility emerged between GC/NICI-MS and EIA (coefficient of reproducibility 11.5 pg x mL(-1)). 8-isoprostane levels measured in urine did not correlate with those measured in EBC. We showed that 8-isoprostane in EBC was significantly increased in children with problematic asthma, suggesting a role for oxidative stress in this asthma phenotype. In addition we found an acceptable reproducibility of EIA compared to GC/NICI-MS, even if the latter method had higher accuracy.

Periductal Lymphocytic Infiltrate at Bilary Atresia Diagnosis Increases the Risk of Addition to the Transplant Waitlist.

PURPOSE: Biliary atresia (BA) is the main indication for pediatric liver transplantation. The aim of this study is to correlate aspects of histological examinations of diagnostic hepatic biopsies for BA with the patients' clinical progression and successful addition to the liver transplant waitlist. METHODS: This was a retrospective study of all 108 BA cases treated at the Federal University of São Paulo (1998-2015). Demographic and clinical data were correlated with histological findings. A logistic regression was used for outcome analysis, while the Kaplan-Meier method was applied for survival analysis. RESULTS: There were 108 patients with BA, 68.5% of whom underwent Kasai surgery. Patients added to the transplant waitlist tended to undergo Kasai surgery at a later time (P = .035). Periductal lymphocytic infiltrate was correlated with the addition to the transplant waitlist, with an odds ratio of 3.92 (P = .033). Patients who developed ascites after surgery were more frequently added to the transplant waitlist (P = .05). CONCLUSION: Patients added to the transplant waitlist underwent Kasai surgery later than other patients. Periductal lymphocytic infiltrate in the diagnostic hepatic biopsy and ascites after Kasai surgery were associated with an increased likelihood of addition to the transplant waitlist.

Gene transfer into neurones for the molecular analysis of behaviour: focus on herpes simplex vectors.

The use of viral vectors to transfect genes into specific brain-cell populations is a novel approach that can be used to investigate the molecular and cellular basis of brain function. Ideal vectors should be targetable and capable of regulated transgene expression. From the viral vectors developed so far, this article focuses on herpes simplex virus 1 (HSV-1)-based vectors. HSV-1 vectors can be engineered for gene transfer to the brain, which makes them suitable for neuroscience research applications. In particular, genetic manipulations of the virus can almost eliminate toxicity and allow expression of multiple transgenes simultaneously. In some instances, transfection of selected neuronal populations is also possible. Specific alterations in behaviour and in disease models have been described after the viral-vector-mediated expression of specific genes within highly localized brain regions.

Dynorphin and epilepsy.

Studies on dynorphin involvement in epilepsy are summarised in this review. Electrophysiological, biochemical and pharmacological data support the hypothesis that dynorphin is implicated in specific types of seizures. There is clear evidence that this is true for complex partial (limbic) seizures, i.e. those characteristic of temporal lobe epilepsy, because; (1) dynorphin is highly expressed in various parts of the limbic system, and particularly in the granule cells of the hippocampus; (2) dynorphin appears to be released in the hippocampus (and in other brain areas) during complex partial seizures; (3) released dynorphin inhibits excitatory neurotransmission at multiple synapses in the hippocampus via activation of kappa opioid receptors; (4) kappa opioid receptor agonists are highly effective against limbic seizures. Data on generalised tonic-clonic seizures are less straightforward. Dynorphin release appears to occur after ECS seizures and kappa agonists exert a clear anticonvulsant effect in this model. However, more uncertain biochemical data and lack of efficacy of kappa agonists in other generalised tonic-clonic seizure models argue that the involvement of dynorphin in this seizure type may not be paramount. Finally, an involvement of dynorphin in generalised absence seizures appears unlikely on the basis of available data. This may not be surprising, given the presumed origin of absence seizures in alterations of the thalamo-cortical circuit and the low representation of dynorphin in the thalamus. In conclusion, it may be suggested that dynorphin plays a role as an endogenous anticonvulsant in complex partial seizures and in some cases of tonic-clonic seizures, but most likely not in generalised absence. This pattern of effects may coincide with the antiseizure spectrum of selective kappa agonists.

The neurochemistry of morphine addiction in the neocortex.

Different strategies have been used in an attempt to understand the neurobiology of opioid addiction. Here, Michéle Simonato initially discusses the identification of key anatomical areas involved in the phenomenon and purposes an explanation of opioid addiction based on the theory of complexity. The variable importance of direct and indirect effects in phenotypically different neuronal populations can imply differences in the adaptive changes that occur with chronic morphine exposure. Opioid addiction is therefore proposed as a complex multicellular event, where individual neurones differentially adapt both on the basis of the signals they receive and of their second messengers and genetic programmes.

Targeting kinin receptors for the treatment of neurological diseases.

Kinins (bradykinin, kallidin and their active metabolites) are peptide autacoids with established functions in cardiovascular homeostasis, contraction and relaxation of smooth muscles, inflammation and nociception. They are believed to play a role in disease states like asthma, allergies, rheumatoid arthritis, cancer, diabetes, endotoxic and pancreatic shock, and to contribute to the therapeutic effects of ACE inhibitors in cardiovascular diseases. Although kinins are also neuromediators in the central nervous system, their involvement in neurological diseases has not been intensively investigated thus far. This review analyzes the potential of central kinin receptors as therapeutic targets for neurological disorders. Initial data highlight potential roles for B(1) receptor antagonists as antiepileptic agents, and for B(2) receptor antagonists (and/or B(1) agonists) in the treatment of stroke. Functional B(1) receptors located on T-lymphocytes and on the blood brain-barrier are also putative targets for the management of multiple sclerosis. However, successful elucidation of the therapeutic value of these new pharmacological approaches will require refinement of our knowledge on the physiology and cellular localization of central kinin receptors.

Induction of B1 bradykinin receptors in the kindled hippocampus increases extracellular glutamate levels: a microdialysis study.

A link between temporal lobe epilepsy (the most common epileptic syndrome in adults) and neuropeptides has been established. Among neuropeptides, the possible involvement of bradykinin has recently received attention. An autoradiographic analysis has shown that B1 receptors, which are physiologically absent, are expressed at high levels in the rat brain after completion of kindling, a model of temporal lobe epilepsy. Thus, the present work aimed at investigating the functional implications of this observation, by studying the effect of B1 receptor activation on extracellular glutamate levels in the kindled hippocampus. Microdialysis experiments have been performed in two groups of rats, control and kindled. Glutamate outflow has been measured under basal conditions and after chemical stimulation with high K+ (100 mM in the dialysis solution). Basal glutamate outflow in kindled animals was significantly higher than in controls. High K+-evoked glutamate outflow was also more pronounced in kindled animals, consistent with the latent hyperexcitability of the epileptic tissue. The B1 receptor agonist Lys-des-Arg9-BK induced an increase of basal and high K+-evoked glutamate outflow in kindled but not in control rats, and the selective B1 receptor antagonist R-715 prevented both these effects. Furthermore, R-715 significantly reduced high K+-evoked glutamate outflow when applied alone. These data suggest that the bradykinin system contributes to the modulation of epileptic neuronal excitability through B1 receptors.

Increased extracellular levels of glutamate in the hippocampus of chronically epileptic rats.

An increase in the release of excitatory amino acids has consistently been observed in the hippocampus during seizures, both in humans and animals. However, very little or nothing is known about the extracellular levels of glutamate and aspartate during epileptogenesis and in the interictal chronic period of established epilepsy. The aim of this study was to systematically evaluate the relationship between seizure activity and changes in hippocampal glutamate and aspartate extracellular levels under basal and high K(+)-evoked conditions, at various time-points in the natural history of experimental temporal lobe epilepsy, using in vivo microdialysis. Hippocampal extracellular glutamate and aspartate levels were evaluated: 24h after pilocarpine-induced status epilepticus (SE); during the latency period preceding spontaneous seizures; immediately after the first spontaneous seizure; in the chronic (epileptic) period. We found that (i) basal (spontaneous) glutamate outflow is increased in the interictal phases of the chronic period, whereas basal aspartate outflow remains stable for the entire course of the disease; (ii) high K(+) perfusion increased glutamate and aspartate outflow in both control and pilocarpine-treated animals, and the overflow of glutamate was clearly increased in the chronic group. Our data suggest that the glutamatergic signaling is preserved and even potentiated in the hippocampus of epileptic rats, and thus may favor the occurrence of spontaneous recurrent seizures. Together with an impairment of GABA signaling (Soukupova et al., 2014), these data suggest that a shift toward excitation occurs in the excitation/inhibition balance in the chronic epileptic state.

Preclinical evaluation of CHF3381 as a novel antiepileptic agent.

CHF3381 [n-(2-indanyl)-glycinamide hydrochloride] has been selected on the basis of a screening program as the compound displaying the highest anticonvulsant activity in the maximal electroshock seizure (MES) test and the best therapeutic index with reference to the rotarod test in mice and rats. In this study, the antiepileptic activity and the behavioural toxicity of CHF3381 were characterised in multiple model systems. CHF3381 effectively prevented MES-induced convulsions when administered i.p. (ED50, 24 mg/kg and 7.5 mg/kg) or p.o. (ED50, 21 mg/kg and 21 mg/kg) in both mice and rats, respectively. The time course of oral anti-MES activity in the rat was related to the brain concentration profile of unchanged CHF3381. Interestingly, the brain drug levels were about 4-5 times higher than in plasma. CHF3381 was very effective in mice against picrotoxin-, and i.c.v. N-methyl-D-aspartate (NMDA)-induced hind limb tonic extension (ED50 Approximately/=10 mg/kg), but was a weaker antagonist of 4-amynopyridine- and bicuculline-induced tonic seizures (ED50 approximately/=100 mg/kg), and ineffective against pentylentetrazole- and picrotoxin-induced clonic seizures. CHF3381 antagonised the behavioural effects and lethality of i.p. administered NMDA (ED50 = 57 mg/kg p.o.), indicating that the compound may act as a functional NMDA antagonist. In keeping with this idea, CHF3381 weakly displaced [(3)H]-TCP from binding to NMDA receptor channels (Ki, 8.8 microM). In the rat amygdala kindling model, CHF3381 was more efficient against kindling development than against kindled seizures (minimally active dose = 80 vs. 120 mg/kg i.p). Furthermore, it significantly increased the seizure threshold in kindled rats at relatively low doses (40 mg/kg i.p.). In contrast with MK-801-induced hyperactivity, CHF3381 moderately reduced the spontaneous locomotor activity in mice at anticonvulsant doses. Toxic effects on motor performance (rotarod test) were found at high doses only (TD50 approximately/= 300 mg/kg p.o., congruent with 100 mg/kg i.p. in both mice and rats). Furthermore, CHF3381 did not impair passive avoidance and Morris water maze responding in the therapeutic range of doses. Finally, the development of tolerance after repeated doses was negligible. These data indicate that CHF3381 exerts anticonvulsant and antiepileptogenic effects in various seizure models and possesses good therapeutic window, with scarce propensity to cause neurological side-effects.

Decreased levels of neuropeptide Y(5) receptor binding sites in two experimental models of epilepsy.

It has been suggested that the anticonvulsant effects of neuropeptide Y (NPY) could be mediated by the activation of Y(2) and/or Y(5) receptors. NPY Y(1) receptor levels are known to decrease and Y(2) to increase in rat models of epilepsy. By using an autoradiographic approach, we investigated whether epilepsy models (kainic acid and kindling) are also associated with changes in Y(5) receptors. Compared with naive controls, [125I][Leu(31), Pro(34)]PYY/BIBP3226-insensitive (Y(5)) binding sites in the hippocampus (strata oriens and radiatum of CA3 and CA1) and in the neocortex (superficial layers) were unchanged in sham-stimulated rats, but reduced by approximately 50% in kindled rats (seven days after the last stimulus evokes seizure), and further reduced (to approximately -90%) 1h after a kindled seizure. Additionally, Y(5) receptor binding sites in the hippocampus and in the neocortex were unchanged 6h after kainic acid injection, but were highly reduced at 12 and 24h. No changes in Y(5) binding levels were found in the dentate gyrus and the pyramidal cell layer of the hippocampus. The present data suggest that changes in Y(5) receptor levels occur in epilepsy models. These changes may play a role in seizure expression and/or in the maintenance of kindling hyperexcitability.

Changes in hippocampal and cortical B1 bradykinin receptor biological activity in two experimental models of epilepsy.

An increased response to the activation of receptors mediating excitatory effects may be involved in some forms of epilepsy. In this study, it has been tested whether B1 bradykinin receptors (which mediate excitatory effects in the peripheral nervous system and have little constitutional expression in the central nervous system) may be proposed in this role. Two experimental models of epilepsy (kindling and kainate) have been employed, and glutamate outflow experiments have been performed in hippocampal and cortical slices taken from control, kindled and kainate-treated rats. The endogenous B1 receptor agonist Lys-des-Arg9-bradykinin (10(-7) M) did not affect electrically-evoked glutamate overflow in control animals, but concentration-dependently increased it in kindled rats (maximal effect +40 to + 50%) and, to a lesser extent (+20%), in kainate-treated rats. These effects were fully prevented by the selective B1 receptor antagonist R-715 (10(-6) M), but not by the selective B2 receptor antagonist Hoe 140 (10(-6) M). The observed changes in B1 bradykinin receptor biological activity may play a role in epileptic hyperexcitability.

Changes in [3H]-UK 14304 binding to alpha 2-adrenoceptors in morphine-dependent guinea-pigs.

1. The aim of this study was to investigate the effect of a noradrenergic input in the cortex of morphine-dependent animals. Binding of the alpha 1-adrenoceptor ligand [3H]-prazosin did not change in cortical membranes taken from morphine-dependent as compared to control guinea-pigs. However, binding of the alpha 2-adrenoceptor ligand [3H]-UK 14304 showed decreased KD (-30%) in the absence of significant changes in Bmax, either in cortical membranes or in synaptosomes. 2. Several characteristics of this phenomenon were identified. First, it occurs in a time-dependent fashion, in that it takes 5 days of chronic morphine treatment to start developing. Second, it can be observed after acute administration of high doses of morphine (100 mg kg-1). Third, it does not require a connection with the locus coeruleus or with other subcortical structures, in that it can be reproduced in vitro in isolated cortical slices. Fourth, it requires the integrity of cortical structures, since it cannot be reproduced in vitro in cortical synaptosomes. 3. Release studies were run to attempt identification of a functional correlate of the above observations. No changes were observed in the ability of the alpha 2-adrenoceptor agonist UK 14304 to inhibit 35 mM K(+)-evoked [3H]-noradrenaline outflow from cortical synaptosomes taken from morphine-dependent as compared to control guinea-pigs. However, a large decrease in the IC50 of UK 14304 for the inhibition of 35 mM K(+)-evoked [3H]-gamma-aminobutyric acid ([3H]-GABA) outflow (41 vs. 501 nM) was observed in morphine-dependent as compared to control animals. 4. These data suggest that, in the guinea-pig, chronic morphine treatment is associated with a shift from a low to high affinity agonist state in alpha 2-adrenoceptors on cortical GABA terminals.

Cortical acetylcholine release is increased and gamma-aminobutyric acid outflow is reduced during morphine withdrawal.

The effects of naloxone on acetylcholine (ACh) and gamma-aminobutyric acid (GABA) outflow from the cerebral cortex of freely moving, morphine-dependent guinea-pigs was studied. The cortical efflux of ACh in chronically-treated guinea-pigs was about half of that of normal animals. GABA efflux was unaffected. During opioid withdrawal precipitated by naloxone (0.1-10 mg kg-1, i.p.) the guinea-pigs showed jumping, hyperactivity and wet dog shakes, the intensity of which was directly related to the dose of naloxone. The withdrawal syndrome was accompanied by a dose-dependent increase in ACh release and reduction in GABA outflow; ACh release was increased by naloxone at doses lower (0.1-3 mg kg-1) than those acting on GABA efflux (1-10 mg kg-1). Atropine (10 mg kg-1) and diazepam (5 mg kg-1) did not prevent GABA and ACh changes.

5-Hydroxytryptamine-mediated effects of nicotine on endogenous GABA efflux from guinea-pig cortical slices.

1. The effect of nicotine on endogenous basal GABA outflow was studied in guinea-pig cerebral cortex slices. 2. Nicotine 1.86-18.6 mumol l-1 significantly decreased the basal, tetrodotoxin-sensitive GABA efflux, whereas at higher concentrations (186-620 mumol l-1) nicotine increased it. The inhibition was prevented by mecamylamine while the facilitation was blocked by mecamylamine, (+)-tubocurarine and tetrodotoxin. 3. The effect of nicotine was due to an indirect 5-hydroxytryptaminergic action. In fact, MDL 72222 (1 mumol l-1) completely prevented the alkaloid inhibition and methysergide (1 mumol l-1) reversed the facilitation into inhibition; concomitant treatment with methysergide and MDL 72222 antagonized the effect of nicotine at 186 mumol l-1 4. Lower concentrations of 5-HT (3-10 mumol l-1) decreased, whereas higher concentrations (30-100 mumol l-1) increased, spontaneous GABA outflow. The inhibition of GABA efflux was prevented by MDL 72222 whereas the facilitation was reversed by methysergide (1 mumol l-1) into inhibition, and prevented by MDL 72222 1 mumol l-11. 5. These results suggest that, by activating nicotinic receptors present on 5-hydroxytryptaminergic terminals, nicotine releases 5-HT which, in turn, inhibits or increases the secretory activity of cortical GABA interneurones via 5-HT3 and methysergide-sensitive receptors, respectively.

Region-specific changes in prodynorphin mRNA and ir-dynorphin A levels after kindled seizures.

The opioid peptide dynorphin is thought to be implicated in specific types of seizures. In particular, complex partial seizures have been shown to cause release of dynorphin, activation of prodynorphin gene expression, and new peptide synthesis in the hippocampus. In this study, the kinetics of the seizure-induced changes in prodynorphin mRNA and ir-dynorphin A levels in the hippocampus have been compared with those induced in the temporal and frontal cortex, i.e., in other regions involved in the pathophysiology of complex partial seizures. Experiments have been run using kindling, one of the most valuable models of partial epilepsy. In the hippocampus (1) prodynorphin mRNA levels transiently increase (threefold) 1 h after kindled seizures, and return to baseline by 2 h, and (2) dynorphin A levels are slightly decreased at 1 h, but increase (twofold) at 2 h and return to baseline by 6 h. In the temporal and in the frontal cortex, a late (beginning at 2 h) and prolonged (up to 24 h) decrease in both prodynorphin mRNA and ir-dynorphin A levels have been observed. These data suggest that differential changes in dynorphin metabolism occur in different brain areas after seizures. The mechanisms and functional implications of this observation remain to be investigated.

Risk of hospitalization for upper gastrointestinal tract bleeding.

OBJECTIVE: This study evaluates the hospitalization risk for upper gastrointestinal bleeding (UGIB) with reference to the clinical characteristics of patients and drugs taken before admission. METHODS: This study is based on the GIFA (Italian Group for the Pharmacosurveillance in the Elderly) database. Cases with an ICD-9 code of esophagus, stomach or duodenum bleeding, or acute esophago-gastroduodenal disease associated with anemia have been classified as UGIB. Sex, age, year of observation, drugs taken at home, comorbidity, smoking, alcohol, and use of gastroprotectants have been also taken into account. Statistical analysis has been conducted using multivariate logistic regression models. RESULTS: 32,388 patients have been enrolled, 940 of which presented UGIB. Age, comorbidity, use of smoke and alcohol, hospitalization duration, and mortality during hospitalization were significantly higher in UGIB than nonUGIB patients. Increased UGIB risk has been found in patients taking NSAIDs (both when aspirin was included or excluded), acetaminophen, constipating agents, iron, ethacrynic acid, propranolol. Reduced UGIB risk has been found in patients taking nitrates. CONCLUSIONS: UGIB risk appears to correlate with clinical characteristics of the patient: it increases with age, comorbidity, and smoke and alcohol consumption. Among drugs, NSAIDs are associated with the highest UGIB risk, while nitrates with a reduction of risk.