Present situation and new perspectives for vaccination against Neisseria meningitidis in Tuscany, Central Italy.

BACKGROUND: In Italy one third of bacterial meningitis are caused by Neisseria meningitidis. In March 2005, the Regional Health Authority of Tuscany included the meningococcal serogroup C conjugate (MCC) vaccine in the recommended vaccination program with a schedule of three doses to all newborns at 3, 5 and 13 months of age (from 2008 amended to a single dose at 13 months) and a single catch-up dose until age 6. OBJECTIVE: To evaluate the impact of the current national and regional immunization strategies against N. meningitidis and to highlight new perspectives for meningococcal disease prevention with the existing tetravalent meningococcal vaccine (ACWY) and with the future incoming meningococcal B vaccines. METHODS: Meningitis incidence rates in Italy and in Tuscany were calculated for the period 1994-2011 and 2005-2011,respectively. Immunization coverage with MCC vaccine in Tuscany and vaccination status of meningitis cases were reported. Literature review on meningococcal conjugate vaccine use and recommendation was performed. RESULTS: A decrease in incidence rates of meningococcal meningitis was observed in all age groups involved in the immunization campaign. Immunization coverage with MCC increased progressively year by year in Tuscany. A herd immunity effect was measured in unvaccinated age groups. Since 2006 no cases of invasive meningococcal C infection in vaccinated subjects were observed in Tuscany. CONCLUSIONS: Implementation of MCC vaccination in Tuscany was effective in preventing meningococcal C disease, confirming the effectiveness of the vaccine. A new tetravalent (ACWY) conjugate vaccine is now available and its use in all Italian Regions should be considered.

Maternal antibiotic prophylaxis affects Bifidobacterium spp. counts in the human milk, during the first week after delivery.

Human milk is an important source of microorganisms for infant gut colonisation. Although the maternal antibiotic prophylaxis is an important strategy to prevent maternal/neonatal sepsis, it has to be investigated how it may affect the human milk microbiota, especially the genus Bifidobacterium, which has been associated to health benefits. Here, we investigated the impact of the maternal antibiotic prophylaxis on the human milk Bifidobacterium spp. and total bacteria counts, in the first week (short-term) and first month (medium-term) after delivery. Human milk samples were collected from 55 healthy lactating women recruited from the University Hospital of the University of São Paulo at days 7±3 and 30±4 after vaginal delivery. Twenty one volunteers had received maternal antibiotic prophylaxis (MAP group) and 34 had not received MAP (no-MAP group) during or after labour. Total DNA was isolated from milk samples, and the bacterial counts were estimated by quantitative PCR (qPCR). We found lower levels of Bifidobacterium in the MAP group in the first week after delivery (median = 2.1 vs 2.4 log of equivalent cells/ml of human milk, for MAP and no-MAP groups, respectively; P=0.01), although there were no statistical differences in total bacteria count. However, no differences were found in Bifidobacterium counts between the groups at day 30±4 (median = 2.5 vs 2.2 log of equivalent cells/ml of human milk, for MAP and no-MAP groups, respectively; P=0.50). Our results suggest that MAP has a significant impact on Bifidobacterium counts in human milk, reducing this population in the first week after delivery. However, throughout the first month after delivery, the Bifidobacterium counts tend to recover, reaching similar counts to those found in no-MAP group at day 30±4 after delivery.

Saccharomyces boulardii Tht 500101 changes gut microbiota and ameliorates hyperglycaemia, dyslipidaemia, and liver inflammation in streptozotocin-diabetic mice.

Type 1 diabetes mellitus (T1DM) is a disorder resulting from chronic autoimmune destruction of insulin-producing pancreatic ß-cells, lack of insulin production and hyperglycaemia. The aim of this study was to evaluate the hypothesis that streptozotocin-diabetic mice treated with Saccharomyces boulardii THT 500101 strain present improvement of glucose and triglycerides metabolism, reduction of liver inflammation concomitant with a beneficial impact in the gut microbiota profile. C57BL/6 male mice were randomly assigned into three groups: Control, Diabetes, Diabetes+Probiotic, and were euthanised 8 weeks after probiotic chronic administration. Mice submitted to treatment presented reduced glycemia in comparison with the diabetic group, which was correlated with an increase in C-peptide level and in hepatic glycogen content. Fat metabolism was significantly altered in streptozotocin-induced diabetic group, and S. boulardii treatment regulated it, leading to a decrease in serum triglycerides secretion, increase in hepatic triglycerides storage and modulation of inflammatory profile. The phenotypic changes seen from chronic S. boulardii treatment were found to be broadly associated with the changes in microbioma of diabetic animals, with increased proportion in Bacteroidetes, Firmicutes and Deferribacteres, and a decreased proportion of Proteobacteria and Verrucomicrobia phylum. Thus, the data presented here show up a novel potential therapeutic role of S. boulardii for the treatment and attenuation of diabetes-induced complications.

From [11C]CO2 to [11C]amides: a rapid one-pot synthesis via the Mitsunobu reaction.

A novel amide synthesis methodology is described using amines, CO2 and Grignard reagents and Mitsunobu reagents. The method was applied to carbon-11 radiochemistry to label amides using cyclotron-produced [11C]CO2. The synthetic utility of the one-pot labelling methodology was demonstrated by producing [11C]melatonin. The incorporation of [11C]CO2 into [11C]melatonin was 36% - determined by radioHPLC 2 min post [11C]CO2 delivery.

Polyelectrolyte multilayer film coating and stability at the surfaces of oral prosthesis base polymers: an in vitro and in vivo study.

A new type of coating involving a layer-by-layer technique has been recently reported. This coating is composed of a polyelectrolyte multilayer film that confers specific properties on surfaces to which it is applied. Here, we studied the applicability of such a technique to the coating of oral prostheses, by first testing the construction of polyelectrolyte multilayer films on several polymers used in oral prosthesis bases, and, subsequently, by studying the stability of these coatings in vitro, in human saliva, and in vivo in a rat model. We demonstrated that the multilayered films are able to coat the surfaces of all tested polymers completely, thus increasing their wettability. We also showed that saliva does not degrade the film after 7 days in vitro and after 4 days in vivo. Taken together, our results establish that the layer-by-layer technique is suitable for the coating of oral devices.

Amino acids of the alpha1B-adrenergic receptor involved in agonist binding: differences in docking catecholamines to receptor subtypes.

Site-directed mutagenesis and molecular dynamics analysis of the 3-D model of the alpha1B-adrenergic receptor (AR) were combined to identify the molecular determinants of the receptor involved in catecholamine binding. Our results indicate that the three conserved serines in the fifth transmembrane domain (TMD) of the alpha1B-AR play a distinct role in catecholamine binding versus receptor activation. In addition to the amino acids D125 in TMDIII and S207 in TMDV directly involved in ligand binding, our findings identify a large number of polar residues playing an important role in the activation process of the alpha1B-AR thus providing new insights into the structure/function relationship of G protein-coupled receptors.

Synthesis, absolute configuration, and biological profile of the enantiomers of trans-[2-(2,6-dimethoxyphenoxy)ethyl] [(3-p-tolyl-2,3-dihydro-1,4-benzodioxin-2-yl)methyl]amine (mephendioxan), a potent competitive alpha 1A-adrenoreceptor antagonist.

The enantiomers of trans-[2-(2,6-dimethoxyphenoxy)ethyl] [(3-p-tolyl-2,3-dihydro-1,4-benzodioxin-2-yl) methyl]amine (mephendioxan, 2) were synthesized from the chiral trans-3-p-tolyl-2,3-dihydro-1,4-benzodioxin-2-carboxylic acids [(+)-3 and (-)-3] which in turn were obtained through the resolution of the racemic acid with (R)- and (S)-alpha-methylbenzylamine. Comparison of CD spectra of the enantiomers of 2 with that of (2S,3S)-3-methyl-2-phenyl-1,4-benzodioxane allowed the assignment of the 2S,3S configuration to the (-)-enantiomer of 2 and of the 2R,3R configuration to the other enantiomer. The binding profile of the enantiomers of 2 was assessed at alpha 1, alpha 2, D2, and 5-HT1A receptors, in comparison to WB 4101 (1), 5-methylurapidil, and (+)-niguldipine. In addition, the two enantiomers were investigated at native and cloned alpha 1-adrenoreceptor subtypes. (-)-2 was 10-30 times as potent as the (+)-enantiomer at alpha 1-adrenoreceptor subtypes in both functional and binding assays. It was 36-fold selective for the alpha 1A- versus alpha 1B-adrenoreceptor and 60- and 20-fold selective in binding to the alpha 1a-adrenoreceptor relative to alpha 1b and alpha 1d subtypes, respectively. Furthermore, the enantiomer (-)-2 displayed selectivities of 12000-, 2500-, and 250-fold in binding to alpha 1a-adrenoreceptors relative to alpha 2-adrenoreceptors and 5-HT1A and D2 receptors. These results indicate that (-)-2 may be a valuable tool in the characterization of alpha 1-adrenoreceptor subtypes.

Influence of age on outcome after percutaneous transluminal coronary angioplasty.

This study estimates the influence of age on outcomes (mainly survival) of 21,516 patients who underwent percutaneous transluminal coronary angioplasty (PTCA) between 1980 and 1996. We prospectively analyzed the patients in 5 age groups: <50, 50 to 59, 60 to 69, 70 to 79, and > or =80 years old. During the in-hospital period after PTCA, mortality increased from 0.28% in patients aged <50 to 3.45% in patients aged > or =80; Q-wave myocardial infarction was not significantly associated with age, and the 2 older groups were referred less often to coronary artery bypass graft surgery. During follow-up, lasting up to 10 years, the hazard of death was significantly influenced by age; Q-wave myocardial infarction was influenced by age, although the magnitude of the effect was relatively small and of questionable clinical significance; and coronary artery bypass graft surgery was performed less often in the 2 older age groups. Additional PTCA was similarly performed among the age groups. Age, diabetes mellitus, systemic hypertension, heart failure class, angioplasty in graft vessel, number of coronary vessels narrowed, and previous myocardial infarction were predictors of death over the 10-year follow-up. Age was the most important correlate of death after PTCA, with a 65% increase in the hazard of death for each 10-year increase in age. Age has an independent effect on early and late survival after PTCA.

Mediation of noradrenaline-induced contractions of rat aorta by the alpha 1B-adrenoceptor subtype.

1. The subtypes of alpha 1-adrenoceptor mediating contractions to exogenous noradrenaline (NA) in rat aorta have been examined in both biochemical and functional studies. 2. Incubation of rat aortic membranes with the irreversible alpha 1B-adrenoceptor antagonist, chloroethylclonidine (CEC: 10 microM) did not change the KD of [3H]-prazosin binding in comparison to untreated membranes, but reduced by 88% the total number of binding sites (Bmax). 3. Contractions of rat aortic strips to NA after CEC (50 microM for 30 min) incubation followed by repetitive washing, showed a marked shift in the potency of NA and a partial reduction in the maximum response. The residual contractions to NA after CEC incubation were not affected by prazosin (10 nM). 4. The competitive antagonists prazosin, terazosin, (R)-YM-12617, phentolamine, 5-methylurapidil and spiperone inhibited contractions to NA with estimated pA2 values of 9.85, 8.54, 9.34, 7.71, 7.64 and 8.41, respectively. 5. The affinity of the same antagonists for the alpha 1A- and alpha 1B- adrenoceptors was evaluated by utilizing membranes from rat hippocampus pretreated with CEC, and rat liver, respectively. 5-Methylurapidil and phentolamine were confirmed as selective for the alpha 1A-adrenoceptors, whereas spiperone was alpha 1B-selective. 6. A significant correlation was found between the pA2 values of the alpha 1-adrenoceptor antagonists tested and their affinity for the alpha 1B-adrenoceptor subtype, but not for the alpha 1A-subtype. 7. In conclusion, these findings indicate that in rat aorta most of the contraction is mediated by alpha 1B-adrenoceptors, and that the potency (pA2) of an antagonist in this tissue should be related to its antagonistic effect on this subtype of the alpha 1-adrenoceptor population.

Immunolocalization of cytoskeletal proteins in the previtellogenic ovarian follicle of the lizard Podarcis sicula

We report the immunolocalization of intermediate filament proteins (vimentin and cytokeratins) in the previtellogenic ovarian follicle of the lizard Podarcis sicula. Vimentin is present, in accordance with their mesenchymal origin, in all the cells of the polymorphic follicular epithelium (small, intermediate and pyriform). Cytokeratin, absent from the small follicle cells, is present in those cells (intermediate and pyriform) connected to the oocyte by an intercellular bridge. In particular, this protein surrounds the nucleus in the pyriform cells and is mainly localized at the apex adjacent to the oocyte surface; it forms a network, that crosses the zona pellucida through the intercellular bridge and appears to be continuous with a cortical ring of cytokeratin in the oocyte. The presence of a cytokeratin cytoskeleton in the pyriform cells in continuity with that of the oocyte lends support to the previously reported hypothesis that, during oogenesis in Podarcis sicula, somatic cells constitute an integral system with the germ cell and provide for its growth. Preliminary observations indicating the presence of actin and tubulin inside the cytoplasmic bridges connecting the pyriform cells to the oocyte are in agreement with an involvement of these connections in the transfer of cytoplasmic materials.

Autoradiographic localization of [(3)H]paroxetine specific binding in the rat brain.

Specific binding of [(3)H]paroxetine, a selective serotonin uptake inhibitor, has recently been described. We used the autoradiographic technique to establish the regional distribution of these binding sites in the brain of control and 5,7-dihydroxytryptamine (5,7-DHT) lesioned rats. Binding levels were highest in the dorsal raphe, followed by superior colliculus, thalamus, mammillary nuclei, ventral tegmental area and substantia nigra. Values were intermediate in hippocampus, hypothalamus, caudate nucleus and median raphe and lowest in the cerebral cortex and cerebellum. The degeneration of serotonergic terminals by 5,7-DHT completely prevented specific binding in the majority of brain regions analyzed, with the exceptions of raphe nuclei, ventral tegmental area and caudate nucleus. These data indicate that [(3)H]paroxetine binding sites are mainly localized on serotonin nerve terminals even if it seems that they cannot always be directly correlated to 5-HT uptake.

Characterization of high affinity and stereospecific [ (3)H ]d- fenfluramine binding to rat brain.

Specific binding of [(3)H]d- fenfluramine , an anorectic agent acting on serotonergic system, was characterized in rat total brain minus cerebellum. The binding was reversible, stereospecific and with high affinity (K(d) about 80 nM). The receptor density in total brain was about 32 pmol/g tissue. Subcellular distribution showed a preferential localization in the microsomal and membrane fractions. The pharmacological characterization of [(3)H]d- fenfluramine specific binding showed that the d-normetabolite and the serotonin-uptake inhibitors are the most active compounds; d-amphetamine was inactive in inhibiting [(3)H]d- fenfluramine binding; NaC1 and GTP decreased [(3)H]d- fenfluramine binding. Although the results are not fully conclusive in clarifying the meaning of [(3)H]d- fenfluramine binding to rat brain membranes, the implication of these sites in mediating the anorectic activity of the drug is proposed.

Acetylcholinesterase in neuroblastoma and neuroblastoma x glioma hybrid cells: cellular localization and molecular forms.

The cellular localization of acetylcholinesterase (AChE) was investigated at the electron microscope (E.M.) in a neuroblastoma and neuroblastoma x glioma hybrid line, which differ for their ability to establish synaptic contacts. Only cells of the latter line show association of AChE to the plasmamembrane, while in the former the activity is mainly intracellular. Sucrose sedimentation analysis of AChE molecular forms has shown no significant differences in the distribution of the two forms, G2 and G4, between the two cell lines. On the contrary a marked difference is observed in the ability of the cell to release the enzyme in the culture medium. In fact the cells lacking AChE on their surface release in the medium a much higher proportion of their enzyme, than the cells showing AChE association to their plamamembrane. The possible role of two alternative fates for AChE, secretion or membrane insertion, in determining the observed differences of enzyme localization is discussed.

Receptor binding profile of cyclazosin, a new alpha 1B-adrenoceptor antagonist.

The binding profile of cyclazosin, a new prazosin-related alpha 1-adrenoceptor antagonist, at alpha 1-, alpha 2-adrenoceptors, dopamine D2 and 5-HT1A receptors was compared to that of 5-methylurapidil, spiperone, risperidone and other prazosin-related ligands. In addition, cyclazosin was investigated at native and cloned alpha 1-adrenoceptor subtypes. Cyclazosin showed high specificity for alpha 1-adrenoceptors and a 10-15-fold selectivity for alpha 1B (alpha 1b)-adrenoceptors with respect to the alpha 1A (alpha 1a) subtype (pKi values of 9.23-9.57 and 8.18-8.41, respectively). However, it failed to discriminate between cloned alpha 1b and alpha 1d-adrenoceptors (pKi values of 9.23 and 9.28, respectively).

Acute noise stress reduces [3H]5-hydroxytryptamine uptake in rat brain synaptosomes: protective effects of buspirone and tianeptine.

Acute noise stress decreased [3H]5-hydroxytryptamine ([3H]5-HT) uptake in synaptosomes from rat hypothalamus, hippocampus and cerebral cortex. The decrease was due to the maximum rate of [3H]5-HT uptake, which peaked 30 min after stress and partly returned to resting values within 4 h, with no changes in affinity (Km values). No changes in [3H]paroxetine binding and basal [3H]5-HT release were found in stressed rats. Tianeptine, given at the dose of 10 mg/kg 1 h before stress, counteracted the noise-induced decrease of 5-HT uptake, since it increased [3H]5-HT uptake in both resting and stressed animals, but did not prevent the rise in plasma corticosterone of stressed rats. Buspirone pretreatment had no effect on [3H]5-HT uptake in resting rats but prevented the noise-induced decrease in [3H]-HT uptake. Diazepam did not modify either the basal or the noise-induced reduction in [3H]5-HT uptake. The evidence that treatments reducing extrasynaptic 5-HT, by increasing its reuptake (tianeptine) or reducing its release (buspirone) in innervated regions are able to modify the stress-induced decrease in 5-HT uptake, further confirms the importance of serotonin in the mechanisms mediating neurochemical responses to stress.

Characterization of alpha 1-adrenoceptor subtypes in prostate and prostatic urethra of rat, rabbit, dog and man.

The alpha 1-adrenoceptor subtypes present in the smooth muscle of urethra and prostate of different animal species, including man, were characterized by using receptor binding techniques. In prostatic urethra and prostate membranes, [3H]prazosin labelled a single population of alpha 1-adrenoceptors (Hill coefficient not different from unity) with a high affinity in the range 0.21-0.51 nM. The number of specific [3H]prazosin binding sites was partially affected by chloroethylclonidine only in human and rat prostate membranes, whereas this agent proved practically devoid of activity in rabbit and dog prostate membranes as well as in the prostatic urethra membranes of all the animal species examined. These findings indicate that in prostatic and urethral membranes the alpha 1-adrenoceptors mainly belong to the alpha 1A subtype. The binding results were confirmed by in vitro functional studies on noradrenaline-induced contractions of rabbit and dog urethral preparations. The agonist-induced contractions were practically unaffected by preincubation of both tissues with chloroethylclonidine, but were sensitive to nifedipine. We found, moreover, a good correlation between the potency of different selective and non-selective alpha 1-adrenoceptor antagonists (WB-4101, 5-methylurapidil, phentolamine, spiperone, prazosin and urapidil) tested against the noradrenaline-induced contractions of rabbit urethra and their affinity for the alpha 1A-adrenoceptor subtype, no correlation with the affinity for the alpha 1B subtype, and a lower correlation with the affinity for the alpha 1C-adrenoceptor subtype.

Comparative studies on the anorectic activity of d-fenfluramine in mice, rats, and guinea pigs.

The present study compares the anorectic activity of d-fenfluramine and its metabolite d-norfenfluramine in three animal species. d-Fenfluramine and d-norfenfluramine show anorectic activity at increasing doses (ED50) in rats, guinea pigs, and mice, d-norfenfluramine being more active than d-fenfluramine in all three species. Equiactive anorectic activities are reached with different brain levels of d-fenfluramine and d-norfenfluramine, guinea pigs being the most sensitive species, followed by rats then mice. The metabolite most probably plays a major role in the anorectic effect of d-fenfluramine in guinea pigs, contributes to the anorectic activity in rats, but adds little to the action of the parent drug in mice. The different sensitivity to d-fenfluramine and d-norfenfluramine in these three species does not appear to be explained by a number of biochemical parameters, including serotonin uptake or release, receptor subtypes, or 3H-d-fenfluramine binding and uptake.

Anorectic effect and brain concentrations of D-fenfluramine in the marmoset: relationship to the in vivo and in vitro effects on serotonergic mechanisms.

The present study investigated the anorectic activity of d-fenfluramine (d-F) and the relationship with brain levels of unchanged drug and its metabolite d-norfenfluramine (d-NF) in marmosets, relating them to neurochemical effects on the serotoninergic system. d-F and d-NF were equally active in reducing food intake (ED50 about 3 mg/kg, p.o.). However, the brain concentrations of the metabolite required to reduce food intake after synthetic d-NF were more than twice those after d-F, indicating that d-NF contributes to but does not completely explain the anorectic effect of d-F. At this dose d-F did not appreciably modify the serotonin (5-HT) and 5-hydroxyindoleacetic (5-HIAA) contents of the brain regions examined, except for a slight enhancement of 5-HIAA in hippocampus. In vitro in brain cortical synaptosomes d-F inhibited [3H]5-HT uptake more potently than d-NF, as in other species. d-F and d-NF showed similar potency in stimulating [3H]5-HT release, in a Ca++ dependent manner. The tritium released by d-F and d-NF appeared to be mainly unmetabolized [3H]5-HT. Like in other species the marmoset too has saturable and specific [3H]d-F binding sites, for which d-NF has lower affinity. d-F and d-NF have low affinities for 5-HT receptor subtypes, except that d-NF has appreciable affinity for 5-HT1C and 5-HT1D receptors. Unlike in rodents but similarly to primates in the striatum the pharmacology of 5-HT receptors seems to correspond to the 5-HT1D subtype.(ABSTRACT TRUNCATED AT 250 WORDS)

Different components of 3H-imipramine binding in rat brain membranes: relation to serotonin uptake sites.

In the present paper we confirm and extend previous studies showing heterogeneous 3H-imipramine (3H-IMI) binding sites. Inhibition curves of various drugs (serotonin, imipramine, desmethyl-imipramine, d-fenfluramine, d-norfenfluramine and indalpine, a potent serotonin uptake inhibitor) obtained using 2 nM 3H-IMI and in presence of 120 mM NaCl, confirmed the presence of at least three 3H-IMI binding sites: two of these (high and low affinities) were serotonin-insensitive while the third one was selectively inhibited by serotonin and indalpine with nanomolar affinities. Moreover, this last component was found to be selectively modulated by chronic imipramine treatment thus suggesting a closer relation to serotonin uptake mechanism. These data indicate that the use of a more selective inhibitors of the serotonin-sensitive component (like indalpine or serotonin itself) to define non specific 3H-IMI, may be of help in understanding its relation with serotonin uptake system.

Affinity of different alpha 1-agonists and antagonists for the alpha 1-adrenoceptors of rabbit and rat liver membranes.

In membranes prepared from rabbit liver, competition with [3H]prazosin by different alpha 1-agonists and antagonists revealed different affinities in comparison to the results obtained on rat liver membranes, and showed a good correlation with the affinity of the same compounds for the cloned alpha 1c-adrenoceptor subtype. The potencies observed on rat liver membranes were well correlated with the affinity observed for the cloned alpha 1b-adrenoceptors. These results confirm that rabbit and rat liver membranes preparations can be utilized to evaluate the affinity of compounds for these alpha 1-adrenergic subtypes.