Differential effects of amphotericin B and liposomal amphotericin B on inflammatory cells in vitro.

OBJECTIVE AND DESIGN: To understand whether the pseudo-allergic reactions to amphotericin B (AmB) administration are due to AmB or to the solubilizing vehicles, a study was designed to evaluate the effects of AmB, liposomal AmB, (L-AmB), AmB-deoxycholate micellar complex (AmB-DC) and deoxycholate (DC) on the responses of rat serosal mast cells (RSMC) and of human basophils (HB), in vitro. MATERIALS AND METHODS: Serosal mast cells were obtained by density gradient centrifugations from male Wistar albino rats. Partially purified HB were obtained from healthy donors. The cells were exposed to AmB, L-AmB, AmB-DC and DC. Histamine release was measured fluorometrically, and the release of lactic dehydrogenase (LDH) was measured spectrophotometrically. HB activation was evaluated cytofluorimetrically by CD63 expression. RESULTS: AmB and L-AmB did not evoke histamine or LDH release from either RSMC or HB. CD63 expression was not induced in HB challenged with AmB and L-AMB. On the other hand, AmB-DC and DC produced a cytotoxic histamine release from both RSMC and HB, and a sustained increase of CD63 expression on HB. CONCLUSIONS: Only AmB-DC was able to induce the release of inflammatory mediators from RSMC and HB. Conceivably, the cytotoxic effect is accounted for by the micellar complexes with DC, which has been confirmed as a powerful histamine releaser, and held responsible for the pseudo-allergic reactions after AmB-DC administration. The data lend support to a better safety profile of L-AmB.

Interactions between histaminergic and cholinergic systems in learning and memory.

The aim of this review is to survey biochemical, electrophysiological and behavioral evidence of the interactions between the cholinergic and histaminergic systems and evaluate their possible involvement in cognitive processes. The cholinergic system has long been implicated in cognition, and there is a plethora of data showing that cholinergic deficits parallel cognitive impairments in animal models and those accompanying neurodegenerative diseases or normal aging in humans. Several other neurotransmitters, though, are clearly implicated in cognitive processes and interact with the cholinergic system. The neuromodulatory effect that histamine exerts on acetylcholine release is complex and multifarious. There is clear evidence indicating that histamine controls the release of central acetylcholine (ACh) locally in the cortex and amygdala, and activating cholinergic neurones in the nucleus basalis magnocellularis (NBM) and the medial septal area-diagonal band that project to the cortex and to the hippocampus, respectively. Extensive experimental evidence supports the involvement of histamine in learning and memory and the procognitive effects of H(3) receptor antagonists. However, any attempt to strictly correlate cholinergic/histaminergic interactions with behavioral outcomes without taking into account the contribution of other neurotransmitter systems is illegitimate. Our understanding of the role of histamine in learning and memory is still at its dawn, but progresses are being made to the point of suggesting potential treatment strategies that may produce beneficial effects on neurodegenerative disorders associated with impaired cholinergic function.

Cortical acetylcholine release elicited by stimulation of histamine H1 receptors in the nucleus basalis magnocellularis: a dual-probe microdialysis study in the freely moving rat.

Perfusion of the nucleus basalis magnocellularis (NBM) with histamine agonists and antagonists modulates the spontaneous release of cortical acetylcholine (ACh) in freely moving rats. Perfusion of the NBM with Ringer solution containing 100 mM K+ strongly stimulated the spontaneous release of cortical ACh in freely moving rats, whereas perfusion with 1 microM tetrodotoxin reduced cortical ACh spontaneous release by more than 50%. Administration of histamine to the NBM concentration-dependently increased the spontaneous release of cortical ACh. Administration of H1 (methylhistaprodifen) but not H2 (dimaprit) or H3 (R-alpha-methylhistamine) receptor agonists to the NBM mimicked the effect of histamine. Perfusion of the NBM with either H1 (mepyramine or triprolidine) or H2 (cimetidine) receptor antagonists failed to alter ACh spontaneous release from the cortex, however, H1 but not H2 receptor antagonists antagonized the releases of cortical ACh elicited by histamine and methylhistaprodifen. Local administration of H3 receptor antagonists (clobenpropit and thioperamide) to the NBM increased the spontaneous release of ACh from the cortex; this effect was antagonized by H1 receptor antagonism. Conversely local administration of MK-801, a noncompetitive receptor antagonist of the N-methyl-D-aspartate receptor, to the NBM failed to alter ACh spontaneous release from the cortex and to antagonize ACh release elicited by histamine. This study demonstrates that activation of histamine H1 receptors in the NBM increases ACh spontaneous release from the cortex.

Local GABAergic modulation of acetylcholine release from the cortex of freely moving rats.

Cortical perfusion with GABA agonists and antagonists modulates the spontaneous release of cortical acetylcholine and GABA in freely moving rats. Twenty-four hours after implantation of a dialysis fibre, cerebral cortex spontaneously released acetylcholine (3.8 +/- 0.2 pmol/10 min) and GABA (6.6 +/- 0.4 pmol/10 min) at a stable rate. Local administration of GABA (1 or 5 mM) or the GABAA agonist muscimol (25 or 50 microM) had no effect on the spontaneous release of acetylcholine. However, bicuculline (1-25 microM), a GABAA antagonist, added to the dialysis perfusate, elicited a concentration-dependent increase of acetylcholine release to approximately double that of control. This effect of bicuculline (25 microM) was completely prevented by coperfusion with muscimol (50 microM). Local administration of the GABAB receptor agonist baclofen (10 or 50 microM) elicited a concentration-dependent increase in spontaneous acetylcholine release with a maximal increase of about 60%. Intracortical administration of baclofen also decreased the spontaneous release of GABA. The GABAB receptor antagonist CGP 35348 (1 mM), administered alone for 20 min through the dialysis fibre, was without effect on spontaneous acetylcholine release; however, it completely blocked both the baclofen-induced increase in acetylcholine release and the decrease in GABA release. These results suggest that cortically released GABA exerts a tonic influence on cholinergic activity.

Central histaminergic system and cognition.

The neurotransmitter histamine is contained within neurons clustered in the tuberomammillary nuclei of the hypothalamus. These cells give rise to widespread projections extending through the basal forebrain to the cerebral cortex, as well as to the thalamus and pontomesencephalic tegmentum. These morphological features suggest that the histaminergic system acts as a regulatory center for whole-brain activity. Indeed, this amine is involved in the regulation of numerous physiological functions and behaviors, including learning and memory, as indicated by extensive research reviewed in this paper. Histamine effects on cognition might be explained by the modulation of the cholinergic system. However, interactions of histamine with any transmitter system, and/or a putative intrinsic procognitive role cannot be excluded. Furthermore, although experimental evidence indicates that attention-deficit hyperactivity disorder symptoms arise from impaired dopaminergic and noradrenergic transmission, recent research suggests that histamine is also involved. The possible relevance of histamine in disorders such as age-related memory deficits, Alzheimer's disease and attention-deficit hyperactivity disorder is worth of consideration, and awaits validation with clinical trials that will prove the beneficial effects of histaminergic drugs in the treatment of these diseases.

Effects of histamine H3 receptor agonists and antagonists on cognitive performance and scopolamine-induced amnesia.

In previous research we found that pre-training administration of histamine H3 receptor agonists such as (R)-alpha-methylhistamine and imetit impaired rat performance in object recognition and a passive avoidance response at the same doses at which they inhibited the release of cortical acetylcholine in vivo. Conversely, in the present study we report that the post-training administration of (R)-alpha-methylhistamine and imetit failed to affect rat performance in object recognition and a passive avoidance response, suggesting that H3 receptor influences the acquisition and not the recall processes. We also investigated the effects of two H3 receptor antagonists, thioperamide and clobenpropit, in the same behavioral tasks. Pre-training administration of thioperamide and clobenpropit failed to exhibit any procognitive effects in normal animals but prevented scopolamine-induced amnesia. However, also post-training administration of thioperamide prevented scopolamine-induced amnesia. Hence, the ameliorating effects of scopolamine-induced amnesia by H3 receptor antagonism are not only mediated by relieving the inhibitory action of cortical H3 receptors, but other mechanisms are also involved. Nevertheless, H3 receptor antagonists may have implications for the treatment of degenerative disorders associated with impaired cholinergic function.

Serum biochemical markers of bone turnover in healthy infants and children.

Serum osteocalcin (OC), bone alkaline phosphatase (BAP), carboxyterminal propeptide of type I procollagen (PICP), carboxyterminal telopeptide of type I collagen (ICTP), parathyroid hormone (PTH) and 1,25 dihydroxyvitamin D [1,25(OH)2D] were measured in 241 normal infants and children (134 males and 107 females aged 1.9 months-14 years, 1.8 months-12 years, respectively). Regarding the analysis of data for children above 2 yrs, we chose data with the following normalization: data/body surface x standard body surface, to eliminate biological variations not exclusively related to chronological age. The increase in serum OC occurred at the expected age of growth spurts in both sexes: in the first year of life OC values (mean +/- SD) were 82.6 +/- 34.3 and 60.2 +/- 32.9 OC ng/ml in males and females, respectively; during puberty, peak values occurred at the age of 10-12 yrs in girls (76.6 +/- 25.8) and at the age of 12-14 yrs in boys (113 +/- 48.3). Furthermore, significant positive correlations with age were found for males from 2 to 14 yrs (p < 0.00001) and for females from 2 to 12 yrs (p < 0.001). Elevated levels of BAP occurred in the first year, 70.4 +/- 28.2 and 71.8 +/- 28.5, and in the second year, 69.4 +/- 26.7 and 67.4 +/- 33.8 ng/ml, for males and females, respectively. For children older than 2 yrs, a positive correlation with age (p < 0.01) was found for females only, with a peak value of 67.2 +/- 13.9 at the age of 10-12 yrs. For ages 2-14 yrs the reference values (mean +/- 2SD) were 15.5 - 90.3 and 17.2 - 95.2 ng/ml for males and females, respectively. The highest PICP levels (1354 +/- 680 ng/ml in males and 1041 +/- 766 in females) were observed in infants less than 1 year of age, decreasing by about 60% at the age of 2. There was no significant change in serum PICP for children older than 2 yrs with values covering a range (mean +/- 2SD) of 52 - 544 and 18 - 546 ng/ml in males and females, respectively. Similarly, the highest ICTP values were seen in infants younger than 1 year (29.7 +/- 11.7 and 29.5 +/- 20.1 ng/ml in males and females, respectively). In the ages from 2 to 14 yrs there did not seem to be any systematic age-correlated changes, with values covering a range (mean +/- 2SD) of 6.06 - 24.5 in boys and 6.84 - 22.9 ng/ml in girls. Serum PTH concentrations (mean +/- SD) in infancy were 27.2 +/- 19.3 pg/ml for males and 25.8 +/- 10.8 for females. Normal ranges (mean +/- 2SD) in the older group were 5.77 - 53.1 and 6.71 - 57.3 pg/ml for males and females, respectively. Serum 1,25(OH)2D presented values of 47.3 +/- 28.1 and 38.7 +/- 18.2 pg/ml under 2 yrs for males and females, respectively. The ranges (mean +/- 2SD) in children above 2 yrs were 9.5 - 101 pg/ml in boys and 10.9 - 88.4 in girls. The results of this study contribute to the establishment of reference values in normal children for these biochemical assays; these reference values are needed when the above biological markers will be applied in the monitoring of metabolic bone diseases.

Quantitative comparison between 99mTc-HMPAO and 99mTc-ECD: measurement of arterial input and brain retention.

This report describes a comparative study between technetium-99m ethyl cysteinate dimer (ECD) and 99mTc-hexamethylpropylene amine oxime (HMPAO) in five neurological patients. The conversion kinetics of the tracers in the blood from forms capable of diffusion across the blood-brain barrier to non-diffusible forms were studied by arterial sampling and rapid octanol extraction. We observed that HMPAO has a faster conversion rate in the blood but that the fraction of the injected dose available for brain extraction is higher than in the case of ECD. Regional brain concentrations of the tracers were measured with single-photon emission tomography (SPET) 35 min and 60 min after the injection and remained stable within this interval. On the basis of the measurements of the arterial input and of SPET brain concentrations of the tracers, the regional steady-state influx constants (Ki in ml/min/g) were determined for several brain regions. In the grey matter the Ki values were (mean +/- SD) 0.32 +/- 0.03 and 0.35 +/- 0.04 for HMPAO and ECD, respectively; in the white matter the values were 0.23 +/- 0.01 and 0.23 +/- 0.02, respectively. The Ki values of the two tracers in corresponding regions were closely correlated (P < 0.001). The correspondence of the Ki values of ECD and HMPAO demonstrates that ECD can also be considered a tracer that may be used for quantitative measurements of brain perfusion.

Quantitation of brain perfusion with 99mTc-bicisate and single SPECT scan: comparison with microsphere measurements.

This study describes and validates in a preliminary manner a method to measure the steady-state influx constant (Ki) of 99mTc-bicisate with one single photon emission computed tomography (SPECT) scan. The method is based on the analysis of the arterial concentration of the radioactivity. The results of this quantitation procedure were compared with regional CBF (rCBF) measurements made using 99mTc-microspheres (MI). Two quantitative indexes of perfusion, fractional brain uptake (FBU) and normalized (with cerebellum) brain uptake (NBU), were also evaluated. Two SPECT studies were performed on seven cardiovascular patients who had no signs of neurological disease. In the first of these, 99mTc-bicisate was used, while in the other, which was performed 2 days later, MI were injected into the left heart ventricle. The values of the FBU, NBU, and Ki of 99mTc-bicisate were calculated in several gray and white matter brain regions of interest (ROIs) and compared with the rCBF values measured with MI in coupled ROIs. Mean FBU values were 0.00008 +/- 0.00002 and 0.00004 +/- 0.00001 in the gray and the white matter, respectively. Mean NBU values were 0.99 +/- 0.04 and 0.54 +/- 0.05, mean Ki values were 0.36 +/- 0.06 and 0.19 +/- 0.03 ml g-1 min-1 and mean rCBF values were 0.51 +/- 0.04 and 0.27 +/- 0.04 ml g-1 min-1 in gray and white matter, respectively. Analysis of variance of the regression gave different F values for the regressions with rCBF of FBU (F = 19, n = 126), NBU (F = 289, n = 112), and Ki (F = 117, n = 126).(ABSTRACT TRUNCATED AT 250 WORDS)

Preliminary evaluation in humans of Tc-99m-Q3, a new tracer for myocardial perfusion imaging.

Trans[99m-Tc-acac2en(TMPP)2]+, where acac2en is N,N'-ethylenebis(acetylacetone iminate) and TMPP is tris-(3 methoxy propyl) phosphine, (shortened Q3) is a new lipophilic Technetium-99m-labelled compound developed for myocardial perfusion imaging. Encouraging data were obtained in the experimental animal. Aim of this study was to perform a preliminary evaluation of Q3 imaging in humans and to compare it with a reference coronary flow tracer such as Tc-99m microspheres. Eight coronary artery disease patients (males, age 58.5 +/- 10 years) were studied. They were injected with 740 MBq of Q3 and single photon emission computed tomography (SPECT) was performed 90 minutes later. After 3 days 740 MBq of Tc-99m microspheres were injected through a pig-tail catheter in the left ventricle (LV), during heart catheterization, and SPECT was acquired 60 minutes later. Q3 images showed a good target/background activity ratio:LV wall/LV cavity = 4.16 +/- 1.2; myocardium/lung = 3.95 +/- 0.52; the related values with microspheres were 6.36 +/- 2.48 (N.S.) and 4.57 +/- 1.07 (N.S.), respectively. Q3 was cleared by the liver and at the moment of SPECT collection the myocardium/liver activity ratio was 1.54 +/- 0.32. The Q3 LV lateral wall/septum activity ratio showed a good correlation with the corresponding microspheres ratio: Q3 ratio = 0.027 + 0.95 microspheres ratio, r = 0.89, SEE = 1.12, p < 0.005.

An analysis of the arterial input curve for technetium-99m-HMPAO: quantification of rCBF using single-photon emission computed tomography.

Arterial radioactivity content after the intravenous administration of HMPAO in seven human subjects was analyzed. Arterial sampling of 99mTc-HMPAO was performed on each subject over a 25-min period postinjection. The lipophilic fraction of the tracer present in the blood was rapidly extracted with octanol. An analysis of the time course of the extracted and nonextracted octanol fractions was performed in order to calculate the arterial input of the tracer available for brain extraction. HMPAO net regional brain clearances were then calculated and compared with rCBF values obtained in the same patients using 99mTc-microspheres injected into the left ventricle of the heart. HMPAO brain clearances were 0.41 +/- 0.01 and 0.27 +/- 0.01 ml/min/g for grey and white matter, respectively. Linear regression analysis was performed and the following result was obtained: clearance (HMPAO) = 0.07 + 0.43 . rCBF with a high significance (p less than 0.001). This equation can be used for the transformation of HMPAO clearances into rCBF values. Our study demonstrates that by using HMPAO and SPECT it is possible to obtain a quantitative estimate of rCBF in humans.

Evaluation of technetium 99m cyclobutylpropylene amine oxime as a potential brain perfusion imaging agent for SPET.

Technetium 99m d,l-cyclobutylpropylene amine oxime (99mTc-CBPAO) has been developed as a brain-imaging agent for single photon emission tomography (SPET). 99mTc-CBPAO can be prepared using a simple labelling procedure suitable for routine clinical use. It has a high in vitro stability, as has been demonstrated by high-pressure liquid chromatography (HPLC) analysis. This shows that 3 h after labelling, less than 5% of the primary lipophilic complex which is capable of crossing the blood-brain barrier (BBB) converts to a secondary hydrophilic complex. Brain uptake (% dose/g wet tissue) of 99mTc-CBPAO, determined at 5 and 30 min after injection in two groups of six adult male Sprague-Dawley rats, was found to be 0.74 +/- 0.06 and 0.73 +/- 0.13 (mean +/- SD), respectively. These values are not significantly different from those obtained repeating the experiment with 99mTc-labelled hexamethylpropylene amine oxime (99mTc-HMPAO) (0.72 +/- 0.15 at 5 min and 0.88 +/- 0.24 at 30 min after injection). Since the rat brain uptake of 99mTc-CBPAO remained unchanged for a period of time suitable for tomographic study, the comparison of the two tracers was extended to two groups of ten patients. The latter were affected by neurological and psychiatric disorders and were studied with SPET. Human brain uptake (% dose/cc cortical grey matter) of 99mTc-CBPAO and 99mTc-HMPAO were 3.04 +/- 0.57 and 4.22 +/- 0.46 (mean x 10(-3) +/- SD x 10(-3), respectively, with a 32% significant difference. In two other groups of five patients, the first transit time-activity curves of the two tracers were compared.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphine withdrawal in cortical slices: suppression by Ca2+-channel inhibitors of abstinence-induced [3H]-noradrenaline release.

1. The effects of morphine withdrawal were evaluated in vitro by monitoring the actions of naloxone on the depolarization-induced release of [3H]-noradrenaline (NA) in cortical slices taken from naïve or dependent rats. The effects of dihydropyridine molecules acting on Ca2+-channels (nimodipine and Bay K 8644) were also studied in this model. 2. Naloxone (10(-8)-10(-5) M) dose-dependently enhanced the K+ induced release of [3H]-NA in slices taken from dependent rats, but failed to modify the [3H]-NA release from 'naïve' slices. 3. The naloxone-induced potentiation of release was significantly reversed by nimodipine (10(-8)-10(-6) M). These doses of nimodipine did not change [3H]-NA release (both basal and K+ induced) in preparations obtained from naïve rats. 4. Bay K 8644 potentiated the K+-induced [3H]-NA release from cortical slices taken from naïve rats to a similar extent as that of naloxone in dependent rats. 5. These results suggest that the naloxone potentiation of the depolarization-induced [3H]-NA release in slices taken from dependent rats may be considered a model of morphine withdrawal in vitro. In this model dihydropyridine Ca2+-channel antagonists suppress morphine-withdrawal effects in a similar manner to observations made in vivo.

Biochemical and behavioural studies on indole-pyruvic acid: a keto-analogue of tryptophan.

The effects of acute or repeated administration of indole-pyruvic acid (IPA), a keto-analogue of tryptophan (TRP), were studied in various brain areas of rats by measuring the changes of 5-hydroxytryptamine (5-HT) and of norepinephrine (NE) content and metabolism. The analgesic and sedative properties of the molecule were evaluated by measuring the tail-flick latency, the spontaneous activity and the potentiation of the barbiturate-induced sleep. Acute or repeated administrations of IPA (20 or 50 mg/kg) increased the utilization of 5-HT in the cortex, hippocampus, diencephalon and brain-stem of rats fed a standard laboratory diet. IPA, however, did not substitute TRP in rats fed a TRP-free diet. The administration of this keto-analogue resulted also in a decreased content of 3-methoxy-4-hydroxy-phenylglycol (MHPG) in the cortex and in the brain-stem, thus suggesting a decreased utilization of NE in these areas. Furthermore, IPA administration decreased the rats' spontaneous activity, increased the duration of barbiturate-induced sleep and increased the tail-flick time, thus indicating that it has sedative and analgesic properties.