Monoaminergic control of vasopressin and VIP expression in the mouse suprachiasmatic nucleus.

We studied the effects of serotonin and noradrenaline on the expression of arginine-vasopressin (AVP) and vasoactive intestinal peptide (VIP) in the suprachiasmatic nucleus (SCN). We used transgenic Tg8 mice knockout for the MAO-A (monoamine oxidase A) gene, which are characterized by increased amounts of serotonin and noradrenaline in brain compared to wild-type mice (C3H). The MAO-A deficiency caused an increase in AVP and VIP expression (determined by immunohistochemistry, enzyme immunoassay, and in situ hybridization) compared to C3H mice. The number of peptidergic neurons was also increased. Inhibiting serotonin or noradrenaline synthesis in Tg8 mice by the administration of parachlorophenylalanine or alpha-methylparatyrosine, respectively, the amounts of AVP, VIP and their mRNAs were decreased, but not the number of peptidergic neurons. This study indicates that serotonin and noradrenaline stimulate AVP and VIP expression, and could participate in the differentiation of the neurochemical phenotype in the mouse SCN.

The Ca/P range of nanoapatitic calcium phosphate cements.

Nanoapatites are apatites consisting of nanometer size crystals. The commercial calcium phosphate cements set by the precipitation of nanoapatitic calcium phosphates in the range 1.5 < or = Ca/P < 1.8. In this study it is shown that a continuum of nanoapatites can precipitate in the range 0.8 < Ca/P< or = 1.5. In order to be formed these nanoapatites need to incorporate K+ ions. In addition they can incorporate some Na+ ions. Upon immersion in aqueous solutions these nanoapatites loose phosphate, K+ and Na+ so that in an open system they are transformed into calcium deficient hydroxyapatite Ca9(HPO4)(PO4)5OH within about 2 months.

Infrared spectrometric study of acid-degradable glasses.

The composition of glasses used in glass-ionomer cements affects their leaching behavior and hence the properties of the cement. The aim of this study was to correlate the composition and leaching behavior of these glasses with their infrared absorption characteristics. The wavenumber of the absorption band of the Si-O asymmetric stretching vibration shifts to a higher value with decreasing content of mono- and bivalent cations in the glass. This effect can be ascribed to the influence of these extraneous ions on the glass network order and connectivity. Preferential leaching of these ions induces an increase of asymmetric stretching vibration and a general modification of the band profile. The results can be correlated with the x-ray diffraction characteristics of the glass.

Altered regulation of the 5-HT system in the brain of MAO-A knock-out mice.

Genetic deficiency of monoamine oxidase-A (MAO-A) induces major alterations of mood and behaviour in human. Because serotonin (5-HT) is involved in mood regulation, and MAO-A is responsible for the catabolism of 5-HT, we investigated 5-HT mechanisms in knock-out mice (2-month-old) lacking MAO-A, using microdialysis, electrophysiological, autoradiographic and molecular biology approaches. Compared to paired wild-type mice, basal extracellular 5-HT levels were increased in ventral hippocampus (+202%), frontal cortex (+96%) and dorsal raphe nucleus (DRN, +147%) of MAO-A mutant mice. Conversely, spontaneous firing rate of 5-HT neurons in the DRN (recorded under chloral hydrate anaesthesia) was approximately 40% lower in mutants. Acute 5-HT reuptake blockade by citalopram (0.2 and 0.8 mg/kg i.v.) produced a much larger increase in extracellular 5-HT levels (by approximately 4 fold) and decrease in DRN neuronal firing (with a approximately 4.5 fold decrease in the drug's ED50) in MAO-A knock-out mice, which expressed lower levels of the 5-HT transporter throughout the brain (-13 to -34% compared to wild-type levels). The potency of the 5-HT1A agonist 8-OH-DPAT to produce hypothermia and to reduce the firing of DRN serotoninergic neurons was significantly less in the mutants, indicating a desensitization of 5-HT1A autoreceptors. This was associated with a decreased autoradiographic labelling of these receptors (-27%) in the DRN. Altogether, these data indicate that, in MAO-A knock-out mice, the enhancement of extracellular 5-HT levels induces a down-regulation of the 5-HT transporter, and a desensitization of 5-HT1A autoreceptors which allows the maintenance of tonic activity of 5-HT neurons in the DRN.

Regional serotonin metabolism in the brain of transgenic mice lacking monoamine oxidase A.

The effect of a lack of the gene encoding monoamine oxidase A (MAO A) in transgenic Tg8 mice on the activity of tryptophan hydroxylase (TPH), the rate-limiting enzyme in serotonin (5-HT) biosynthesis, and on the levels of 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) in the midbrain, hypothalamus, hippocampus, striatum, amygdala, and frontal cortex was studied. It was shown that mice with a genetic MAO A knockout differed from mice of the initial C3H/HeJ strain in having a higher level of 5-HT and a lower level of its metabolite, 5-HIAA, in all brain regions but the frontal cortex, where the changes were insignificant. Although the 5-HIAA/5-HT ratio in various brain regions differed considerably, the decrease of the 5-HT oxidative deamination index in Tg8 mice was similar in different brain regions (to 41-45% of control values), with the exception of the frontal cortex, where the decrease of the 5-HIAA/5-HT was somewhat smaller (to 54%). The presence of the remaining 45% +/- 1.9% of the control ratio value indicates rather effective oxidative deamination of 5-HT in MAO A knockout mice and explains the lack of severe behavioral and pathological consequences in MAO A genetic deficiency. An increase of TPH activity in mice lacking MAO A was found in the frontal cortex, hippocampus, and amygdala. No significant changes were found in the striatum, hypothalamus, and midbrain. The data show an effect of the MAO A gene mutation on TPH and indicate a uniform decrease of 5-HT catabolism in different brain regions except for the frontal cortex, which is somewhat more resistant to the lack of MAO A than other brain structures.

X-ray diffraction study of acid-degradable glasses.

The composition of the degradable glasses used in commercial dental glass-ionomer cements determines their leaching behavior and hence the properties of the cement. The objective of the present study was to assess if the composition and leaching in acetic acid solutions are reflected in the x-ray diffraction characteristics of these glasses. The position (2theta) of the maximum of the first sharp diffraction peak shifts to higher diffraction angles with increasing number and ionic radius of mono- and bivalent cations in the glass. Upon acid-leaching, these ions are preferentially leached out, so that (2theta) decreases. These results can be related to the decreasing Si-Si distance in the glass network with increasing modifier radius.

X-ray diffractometric determination of crystalline phase content in bioactive glasses.

A rapid routine determination of the content of crystalline CaF(2) and Al(2)O(3) inclusions in bioactive glass ceramics is performed using X-ray diffractometry with a standard addition technique. Multiple ratio analysis, even using peaks with different broadenings, indicates that differences in crystallite properties (e.g., crystal imperfection, particle size and morphology, preferred orientation) between the unknown sample and standard do not bias the result. In this respect, an exact match between their crystallographic integrities does not seem to be required for a reliable estimation of the crystalline content with a relative standard deviation of 7%.

Fluoride release from glass ionomer activated with fluoride solutions.

The mechanism of the fluoride release from glass-ionomer cements (GICs) is not yet completely understood, due to the complexity of these systems. The objective of the present study was to investigate the fluoride and alkali metal ion release from a relatively simple GIC formulation with fluoride- and alkali metal-free glass and activated with a NaF or KF solution. The set formulations were eluted during 168 days in water at 37 degrees C. The cumulative fluoride release was the result of an initial high release that ceased after some time and a prolonged but slow release, both of which increased with increasing fluoride concentrations in the activating solution, independently of the type of alkali metal. Maturation prior to elution decreased the fluoride and slowed the alkali metal release. The release of these ions was not (completely) correlated. The results suggest that the release process is due not only to a loss of relatively loosely bound fluoride in the cement matrix, but also to the release of fluoride which becomes strongly bound during the setting reaction and induces a long-term release.

Fluoride release of polyacid-modified composite resins with and without bonding agents.

OBJECTIVES: The aim of this study was to investigate the influence of the proprietary bonding agents Hytac OSB (OSB) (Espe), Prime&Bond 2.1 (PB) (Dentsply DeTrey) and Syntac Single Component (SSC) (Vivadent) on the fluoride release of the corresponding polyacid-modified composite resins Hytac (HTC), Dyract AP (DAP) and Compoglass F (CGF), respectively. METHODS: Ten cylindrical specimens (6mm diameter and 3mm thick) of each polyacid-modified composite were prepared according to the manufacturers' instructions: five with bonding agent applied and five without bonding agent as a control. The specimens were immersed individually in 10ml ultra-pure water at 37 degrees C immediately after light-curing of the polyacid-modified composite resins. Over 140 days, the water was regularly renewed and the fluoride concentration eluted during each period was determined with a combined fluoride ion selective electrode. RESULTS: The fluoride release decreases according to the sequence: CGF>DAP>HTC. The bonding agent significantly reduces the fluoride released by DAP and CGF, respectively, by a factor 2-3 and +/-1.4. For HTC, the bonding agent reduces the fluoride released initially by a factor of +/-2, but the difference between the fluoride release with and without bonding agent becomes insignificant after approximately 3 weeks. SIGNIFICANCE: It can be concluded that the use of bonding agent can significantly reduce fluoride release of polyacid-modified composite resins in the long-term, and especially in the short-term. The decrease in fluoride release might reduce the material's potential to prevent recurrent caries.

Expression of human and macaque type I IFN transgenes interferes with HSV-1 replication at the transcriptional and translational levels: IFN-beta is more potent than IFN-alpha 2.

A study was undertaken to compare the efficacy of plasmid constructs encoding human IFN-alpha 2 and IFN-beta and macaque IFN-beta against herpes simplex virus type 1 in transfected cells. All type I IFN transgenes significantly reduced viral titers in transfected cells by 3 logs. Human IFN-alpha 2-transfected cells produced significantly more IFN (2274 pg/ml) in comparison to IFN-beta-transfected cells (134-165 pg/ml). Viral lytic gene transcript and viral protein levels were lower in IFN-beta- versus IFN-alpha 2-transfected cells, which coincided with elevated PKR and OAS transcript levels and increased total STAT1 and phosphorylated STAT1 (Y701) protein levels in the IFN-beta-transfected cells. Although comparable viral titers were recovered in IFN-alpha 2 and IFN-beta plasmid-transfected cells, IFN-alpha 2 plasmid-transfected cells exhibited significantly more cytopathic effect compared to the IFN-beta transgene-transfected cells. In addition, IFN-alpha 2 transgene-transfected, infected cells displayed a cell cycle profile similar to that of vector-transfected, infected cells, whereas IFN-beta plasmid-transfected cells displayed a profile similar to uninfected control. Collectively, the results indicate that human IFN-beta is superior to IFN-alpha 2 in antagonizing herpes simplex virus type 1 infection.

Analysis of the pharmacological and molecular heterogeneity of I(2)-imidazoline-binding proteins using monoamine oxidase-deficient mouse models.

The I(2) subgroup of imidazoline-binding sites was identified as monoamine oxidases (MAOs), but it is unclear whether there are I(2)-binding sites located on proteins distinct from MAOs. To address this issue, we characterized I(2)-binding proteins in liver and brain of wild-type and MAO A- and MAO B-deficient mice. I(2)-binding sites were identified using [(3)H]idazoxan and the photoaffinity adduct 2-[3-azido-4-[(125)I]iodophenoxyl]methylimidazoline ([(125)I]AZIPI). [(3)H]Idazoxan labeled binding sites with ligand recognition properties typical of I(2) sites in both brain and liver of wild-type mice. High-affinity, specific [(3)H]idazoxan binding were not altered in MAO A knockout (KO) mice. In contrast, [(3)H]idazoxan binding was completely abolished in both liver and brain of MAO B KO mice. In wild-type mice, [(125)I]AZIPI photolabeled three proteins with apparent molecular masses of approximately 28 (liver), approximately 61 (brain), and approximately 55 kDa (liver and brain). The photolabeling of each protein was blocked by the imidazoline cirazoline (10 microM). Photolabeling of the approximately 61- and approximately 55-kDa proteins was not observed in MAO A and B KO mice, respectively. In contrast, photolabeling of the liver approximately 28-kDa protein was still observed in MAO-deficient mice, indicating that this protein is unrelated to MAOs. These data indicate that I(2) imidazoline-binding sites identified by [(3)H]idazoxan reside solely on MAO B. The binding sites on MAO A and the liver approximately 28-kDa protein may represent additional subtypes of the family of the imidazoline-binding sites.

Inhibition of human immunodeficiency virus transmission to CD4+ T cells after gene transfer of constitutively expressed interferon beta to dendritic cells.

CD34(+)-derived dendritic cells (DCs) can be infected by the T cell-tropic HIVLAI strain, but are poorly permissive for efficient virus production. However, HIVLAI-infected DCs are able to transmit a vigorous cytopathic infection to activated CD4(+) T cells. We show that DCs differentiated from CD34(+) cells can be efficiently transduced by a retroviral vector carrying the IFN-beta coding sequence. This results in resistance to infection by HIV as shown by a threefold reduction in the HIV DNA copy number per cell, and by inhibition of HIV transmission from DCs to CD4(+) T cells. Moreover, constitutive IFN-beta production by DCs increases the synthesis of IL-12 and IFN-gamma Th1-type cytokines and of the beta-chemokines MIP-1alpha, MIP-1beta, and RANTES. This indicates that IFN-beta transduction of DCs blocks HIV infection and viral transmission to CD4(+) T cells, and could favor cellular immune responses in HIV-infected patients.

Effect of a neutral citrate solution on the fluoride release of resin-modified glass ionomer and polyacid-modified composite resin cements.

The effect of 0.01 mol/l citrate solution at pH = 7 on the fluoride release is compared for the resin-modified glass ionomer cements (RM-GIC) GC Lining LC, PhotacBond, Vitremer and Vitrebond and for the polyacid-modified composite resins (PAM-C) Variglass and Dyract by means of the six-month fluoride release profiles at 37 degrees C. The fluoride release of both RM-GIC and PAM-C increases in the neutral citrate solution as compared to water, which can be explained by the ability of citrate to complex metal ions and hence to degrade the glass as well as the polysalt matrix of the cement. Although RM-GIC release more fluoride than PAM-C in water as well as in citrate solution, the relative increase in fluoride release upon immersion in citrate solution is most pronounced for PAM-C. Whereas for the latter citrate affects both the short-term and long-term fluoride release, for RM-GIC only the long-term fluoride release is affected. This suggests that the action of citrate increases with decreasing importance of the polysalt formation in the hardening of the material. This could be explained on the basis of the difference in the chemical properties of the cement matrix.

Conversion of octacalcium phosphate in calcium phosphate cements.

This study investigated the in vitro conversion reaction in calcium phosphate cements (CPCs) containing octacalcium phosphate (OCP) as one of the reagents. OCP is known to be a precursor for apatite formation in vivo. The reaction products were characterized using infrared spectroscopy and X-ray diffraction. Although the conversion of OCP into hydroxyapatite is thermodynamically favorable, OCP only yields apatite formation in CPC provided it is combined with a highly soluble Ca(2+) and OH(-) releasing reaction partner. In this respect, tetracalcium phosphate is a promising compound. Adding small amounts of monocalcium phosphate monohydrate can stimulate the setting through intermediate brushite formation. The preparation method of OCP might drastically affect the performance of the cement. The reaction path of the setting of these CPC probably does not conform to the singular point principle described in the literature, and an in situ hydrolysis of OCP to apatite is conceivable. Simulation of apatite formation using OCP as the precursor and/or seed in CPC might be beneficial for some biomedical applications.

Effect of maturation on the fluoride release of resin-modified glass ionomer and polyacid-modified composite resin cements.

The effect of an early water contact on the fluoride release is studied for the resin-modified glass ionomer cements (RM-GIC) GC Lining LC, PhotacBond, Vitremer and Vitrebond and for the polyacid-modified composite resins (PAM-C) Variglass and Dyract. Six months fluoride release profiles were determined in regularly renewed water (37 degrees C), for the products directly after light curing and after 24 h maturation in a humid atmosphere (85% RH). ANOVA shows that both the short-term and the long-term fluoride release of a RM-GIC are influenced by this maturation. This indicates that direct water contact for this material should be avoided. For the RM-GIC a correlation is found between the initial fluoride release process and the long-term process. For the PAM-C materials, no differences in the fluoride release are found as a function of maturation, indicating that early water contact has no effect. The amounts of fluoride released by PAM-C are low compared to RM-GIC, which can affect their caries preventive potential. The results are explained on the basis of the setting reaction of both types of materials.

Abnormal phrenic motoneuron activity and morphology in neonatal monoamine oxidase A-deficient transgenic mice: possible role of a serotonin excess.

In rodent neonates, the neurotransmitter serotonin (5-HT) modulates the activity of both the medullary respiratory rhythm generator and the cervical phrenic motoneurons. To determine whether 5-HT also contributes to the maturation of the respiratory network, experiments were conducted in vitro on the brainstem-spinal cord preparation of neonatal mice originating from the control strain (C3H) and the monoamine oxidase A-deficient strain, which has a brain perinatal 5-HT excess (Tg8). At birth, the Tg8 respiratory network is unable to generate a respiratory pattern as stable as that produced by the C3H network, and the modulation by 5-HT of the network activity present in C3H neonates is lacking in Tg8 neonates. In addition, the morphology of the phrenic motoneurons is altered in Tg8 neonates; the motoneuron dendritic tree loses the C3H bipolar aspect but exhibits an increased number of spines and varicosities. These abnormalities were prevented in Tg8 neonates by treating pregnant Tg8 dams with the 5-HT synthesis inhibitor p-chlorophenylalanine or a 5-HT(2A) receptor antagonist but were induced in wild-type neonates by treating C3H dams with a 5-HT(2A) receptor agonist. We conclude that 5-HT contributes, probably via 5-HT(2A) receptors, to the normal maturation of the respiratory network but alters it when present in excess. Disorders affecting 5-HT metabolism during gestation may therefore have deleterious effects on newborns.

Fluoride release profiles of mature restorative glass ionomer cements after fluoride application.

This study investigates the fluoridation of four conventional glass ionomer cements (GIC) (ChemFil Superior encapsulated, Fuji Cap II, Ketac-Fil and Hi Dense) and three resin-modified GIC (RM-GIC) (Fuji II LC encapsulated, Photac-Fil and Vitremer). The fluoride release of matured restorative GIC was measured as a function of time, after four repeated fluoridations in a 2% NaF aqueous solution for 1 h. This release was corrected for the intrinsic release as determined with a control group. It was demonstrated that application of fluoride is capable of recharging GIC but the subsequent high fluoride release only lasts for one or a few days. Moreover, the fluoride release behaviour depends on the cement formulation. Comparable to the intrinsic release, the net fluoride release after fluoridation is composed of a short- and a long-term process, the former being predominant after fluoridation. The total amount of fluoride released according to the short-term process increases with consecutive fluoridations. This is especially pronounced for the RM-GIC, who exhibit a relatively slow release after fluoridation as compared to the conventional GIC. An explanation for these results is suggested on the basis of the physicochemistry of the setting reaction of the cements and of the fluoridation process.

Retrovirally mediated IFN-beta transduction of macrophages induces resistance to HIV, correlated with up-regulation of RANTES production and down-regulation of C-C chemokine receptor-5 expression.

Constitutive expression of IFN-beta by HIV target cells may be an alternative or complementary therapeutic approach for the treatment of AIDS. We show that macrophages derived from CD34+ cells from umbilical cord blood can be efficiently transduced by a retroviral vector carrying the IFN-beta coding sequence. This results in resistance to infection by a macrophage-tropic HIV type 1, as shown by the drastic reduction in the HIV DNA copy number per cell and in p24 release. Moreover, IFN-beta transduction totally blocked secretion of proinflammatory cytokines after HIV infection. The constitutive IFN-beta production also resulted in an increased production of IL-12 and IFN-gamma Th1-type cytokines and of the beta-chemokines macrophage-inflammatory protein-1alpha, macrophage-inflammatory protein-1beta, and RANTES. RANTES was found to be involved in the HIV resistance observed, and this was correlated with a down-regulation of the CCR-5 HIV entry coreceptor. These results demonstrate the feasibility and the efficacy of such IFN-beta-mediated gene therapy. In addition to inhibiting HIV replication, IFN-beta transduction could have beneficial immune effects in HIV-infected patients by favoring cellular immune responses.

Effect of temperature and immersion on the setting of some calcium phosphate cements.

Calcium phosphate cements based on powders containing alpha-Ca3(PO4)2 and aqueous solutions containing Na2HPO4 as accelerator were used to determine the effects of accelerator concentration, temperature and immersion on the setting time. Increases in accelerator concentration and temperature increased the rate of setting, but immersion had a retarding effect. These results were used to design a method whereby a syringe filled with cement paste can be kept ready for injection of the paste into the implantation site for a long time, whereas setting of the cement paste in the body takes place in a short time.