Giacomo Diaz

APPETITE SUPPRESSION AND WEIGHT LOSS AFTER THE CANNABINOID ANTAGONIST SR 141716

The effect of the cannabinoid CB1 receptor antagonist, SR 141716, on food intake and body weight was assessed in adult, non-obese Wistar rats. The daily administration of SR 141716 (2.5 and 10 mg/kg; i.p.) reduced dose-dependently both food intake and body weight. Tolerance to the anorectic effect developed within 5 days; in contrast, body weight in SR 141716-treated rats remained markedly below that of vehicle-treated rats throughout the entire treatment period (14 days). The results suggest that brain cannabinoid receptors are involved in the regulation of appetite and body weight.

Vitamin A deficiency produces spatial learning and memory impairment in rats

Vitamin A and its derivatives (retinoids) play important roles in many physiological processes. The recent finding of high levels of cellular retinol-binding protein type 1 immunoreactivity, cellular retinoic acid-binding protein type 1 immunoreactivity and the presence of nuclear retinoid receptors in the central nervous system of adult rodents suggests that retinoids may carry out important roles in the adult brain. In consideration of the role of the hippocampus in spatial learning and memory we evaluated the effect of vitamin A deprivation in adult rats on these functions. Following 12 weeks of vitamin A-free diet, rats were trained to acquire a radial-arm maze task. Results show that this diet induced a severe deficit in the spatial learning and memory task. The cognitive impairment was fully restored when vitamin A was replaced in the diet. We also found a significant decrease in hippocampal acetylcholine release induced by scopolamine, assessed using microdialysis technique, and a reduction in the size of hippocampal nuclei of CA1 region in vitamin-deficient rats, compared to rats fed with a vitamin A-sufficient diet. These results demonstrate that vitamin A has a critical role in the learning and memory processes linked to a proper hippocampal functioning.

Early changes in hepatitis C viral quasispecies during interferon therapy predict the therapeutic outcome

Despite recent treatment advances, the majority of patients with chronic hepatitis C fail to respond to antiviral therapy. Although the genetic basis for this resistance is unknown, accumulated evidence suggests that changes in the heterogeneous viral population (quasispecies) may be an important determinant of viral persistence and response to therapy. Sequences within hepatitis C virus (HCV) envelope 1 and envelope 2 genes, inclusive of the hypervariable region 1, were analyzed in parallel with the level of viral replication in serial serum samples obtained from 23 patients who exhibited different patterns of response to therapy and from untreated controls. Our study provides evidence that although the viral diversity before treatment does not predict the response to treatment, the early emergence and dominance of a single viral variant distinguishes patients who will have a sustained therapeutic response from those who subsequently will experience a breakthrough or relapse. A dramatic reduction in genetic diversity leading to an increasingly homogeneous viral population was a consistent feature associated with viral clearance in sustained responders and was independent of HCV genotype. The persistence of variants present before treatment in patients who fail to respond or who experience a breakthrough during therapy strongly suggests the preexistence of viral strains with inherent resistance to IFN. Thus, the study of the evolution of the HCV quasispecies provides prognostic information as early as the first 2 weeks after starting therapy and opens perspectives for elucidating the mechanisms of treatment failure in chronic hepatitis C.

Hydrophobic characterization of intracellular lipids in situ by Nile Red red/yellow emission ratio

Nile Red (9-diethylamino-5H-benzo [alpha] phenoxazine-5-one) is a fluorescent lipophilic dye characterized by a shift of emission from red to yellow according to the degree of hydrophobicity of lipids. Polar lipids (i.e., phospholipids) which are mostly present in membranes, are stained in red whereas neutral lipids (esterified cholesterol and triglycerides) which are present in lipid droplets, are stained in yellow. Besides this marked, qualitative contrast between polar and neutral lipids, small differences of the hydrophobic strength could be assessed by the quantitative ratio of red and yellow emissions, in order to extend the discrimination of lipids within the groups of neutral and polar lipids. On the other hand, ratiometric data of red and yellow emissions have not yet been evaluated in the numerous previous light microscopy investigations which used Nile Red. In this work we show that the Nile Red red/yellow ratio enables discrimination of different lipids (monooleine>oleic acid>phosphatidylcholine>free cholesterol>trioleine>oleyl cholesteryl ester). We also show changes in the Nile Red red/yellow emission ratio of lipid droplets of 3T3 mouse fibroblasts induced by drugs interfering with the cholesterol cycle.

Nanoparticles from Lipid-Based Liquid Crystals: Emulsifier Influence on Morphology and Cytotoxicity

Here, monoolein-based nanoparticles (NPs), obtained through fragmentation of bulk liquid crystalline phases, and stabilized by two different emulsifiers, namely, Pluronic F127 (PF127) and lauroylcholine chloride (LCh), are investigated for structural features and for short-term in vitro cytotoxicity. Depending on the emulsifiers, different morphologies of the lipid NPs (cubosomes and liposomes) are obtained, as demonstrated by cryo-TEM images. Although NPs offer many advantages in medical applications and various chemicals used for their preparation are under investigation, so far there are no standardized procedures to evaluate cell biocompatibility. Two different protocols to evaluate the impact of these lipid NPs on biological systems are presented. Results show that nanoparticles stabilized by PF127 (cubosomes) display a relevant toxicity toward different cell lines, whereas those stabilized by LCh (liposomes) affect cell viability at a much lesser extent.

Chronic morphine and naltrexone fail to modify μ-opioid receptor mRNA levels in the rat brain

Previous radioligand-binding studies have reported conflicting results concerning the effect of chronic morphine administration on the regulation of mu-opioid receptor (MOR) density. On the other hand, chronic administration of an opioid antagonist, such as naltrexone, has been shown to increase the density of the MOR. In order to determine if the changes in the MOR are associated with alterations in receptor mRNA levels, we investigated MOR gene expression following chronic treatment with morphine and/or naltrexone. MOR mRNA levels, determined by the ribonuclease protection assay (RPA), were unchanged with respect to control during chronic morphine treatment and morphine withdrawal in each of the analysed brain areas. Furthermore, chronic administration of naltrexone did not result in changes of MOR mRNA levels in rat striatum of naive and morphine-dependent rats, suggesting that the up-regulation of the MOR density, at least in this tissue, is not regulated at transcriptional level.

B cell gene signature with massive intrahepatic production of antibodies to hepatitis B core antigen in hepatitis B virus–associated acute liver failure

Hepatitis B virus (HBV)-associated acute liver failure (ALF) is a dramatic clinical syndrome due to a sudden loss of hepatic cells leading to multiorgan failure. The mechanisms whereby HBV induces ALF are unknown. Here, we show that liver tissue collected at the time of liver transplantation in two patients with HBV-associated ALF is characterized by an overwhelming B cell response apparently centered in the liver with massive accumulation of plasma cells secreting IgG and IgM, accompanied by complement deposition. We demonstrate that the molecular target of these antibodies is the hepatitis B core antigen (HBcAg); that these anti-bodies display a restricted variable heavy chain (VH) repertoire and lack somatic mutations; and that these two unrelated individuals with ALF use an identical predominant VH gene with unmutated variable domain (IGHV1-3) for both IgG and IgM anti-HBc antibodies, indicating that HBcAg is the target of a germline human VH gene. These data suggest that humoral immunity may exert a primary role in the pathogenesis of HBV-associated ALF.

Calcitonin gene-related peptide in the human trigeminal sensory system at developmental and adult life stages: immunohistochemistry, neuronal morphometry and coexistence with substance P.

The distribution of calcitonin gene-related peptide (CGRP) has been examined by the indirect immunofluorescence technique in the Gasserian ganglion and spinal nucleus of the human trigeminal nerve. In the ganglion CGRP is present in almost 50% of primary sensory neurons, in varicose and non-varicose nerve fibres and in pericellular basket-like plexuses around non-immunoreactive ganglionic perikarya. Morphometric analysis reveals that the CGRP-positive neuronal population is heterogeneous in cell size. Observation of specimens from subjects at fetal, perinatal and adult life stages reveals that the percentage of CGRP-immunoreactive cells reaches a maximum at perinatal stages and then remains constant, declining only in old age. Pericellular basket-like nerve fibres are detectable only in fetal and pre-term and full-term newborn tissue. Coexistence between CGRP and substance P (SP) occurs, SP being present in about one quarter of the CGRP-immunoreactive neurons and CGRP being localized in a little more than half of the SP-immunoreactive neurons. However, perikarya, nerve fibres and pericellular fibres containing only one or other peptide are also present. Bundles of immunoreactive fibres and dot-like nerve terminals occur in the spinal tract and superficial and deep regions of the spinal trigeminal nucleus. A particularly dense plexus is present in the peripheral nuclear layers. Double immunostaining shows a similar regional distribution for SP. However, in inner substantia gelatinosa the density of CGRP-immunoreactive fibres is much higher than that of SP-immunoreactive ones. The results obtained add information to our knowledge of the organization of neurochemically identified neurons in the human trigeminal sensory system.

Oral self-administration of γ-hydroxybutyric acid in the rat

Abstract The present study describes the induction of γ-hydroxybutyric acid (GHB) preference over water in rats. GHB solution (1% w / v in water) was initially offered as the sole fluid available for 14 consecutive days. Subsequently, rats were given a free choice of GHB solution and tap water for 20 consecutive weeks. Under the free-choice regimen, all rats showed periods of preference for GHB solution over water and periods of voluntary abstinence from GHB. On GHB-preference days, GHB was ingested at pharmacologically relevant doses. GHB intake occurred in 2–3 discrete episodes during the nocturnal phase. The development of an animal model of GHB self-administration may constitute a useful tool in the investigation of the neurobiological substrates of GHB-reinforcing properties.

Cannabinoid CB1 receptors in the paraventricular nucleus and central control of penile erection: immunocytochemistry, autoradiography and behavioral studies.

[N-(piperidin-1-yl)-5-(4-chlorophenyl)-4-methyl-1H-pyrazole-3-carboxyamide] (SR 141716A), a selective cannabinoid CB1 receptor antagonist, injected into the paraventricular nucleus of the hypothalamus (PVN) of male rats, induces penile erection. This effect is mediated by the release of glutamic acid, which in turn activates central oxytocinergic neurons mediating penile erection. Double immunofluorescence studies with selective antibodies against CB1 receptors, glutamic acid transporters (vesicular glutamate transporters 1 and 2 (VGlut1 and VGlut2), glutamic acid decarboxylase-67 (GAD67) and oxytocin itself, have shown that CB1 receptors in the PVN are located mainly in GABAergic terminals and fibers surrounding oxytocinergic cell bodies. As GABAergic synapses in the PVN impinge directly on oxytocinergic neurons or on excitatory glutamatergic synapses, which also impinge on oxytocinergic neurons, these results suggest that the blockade of CB1 receptors decreases GABA release in the PVN, increasing in turn glutamatergic neurotransmission to activate oxytocinergic neurons mediating penile erection. Autoradiography studies with [(3)H](-)-CP 55,940 show that chronic treatment with SR 141716A for 15 days twice daily (1 mg/kg i.p.) significantly increases the density of CB1 receptors in the PVN. This increase occurs concomitantly with an almost twofold increase in the pro-erectile effect of SR 141716A injected into the PVN as compared with control rats. The present findings confirm that PVN CB1 receptors, localized mainly in GABAergic synapses that control in an inhibitory fashion excitatory synapses, exert an inhibitory control on penile erection, demonstrating for the first time that chronic blockade of CB1 receptors by SR 141716A increases the density of these receptors in the PVN. This increase is related to an enhanced pro-erectile effect of SR 141716A, which is still present 3 days after the end of the chronic treatment.