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Top of pageAbstract Gene expression from first generation adenovirus vectors in the CNS is efficient and safe in na|[iuml]|ve animals. However, transgene expression is eliminated within 30 days in animals immunized against adenovirus. High-capacity adenovirus (HC-Adv) provide an advantage in that they sustain transgene expression in the brain even in animals that have been preimmunized against adenovirus, preceding the administration of HC-Adv. In the present experiment we analyzed the long term expression of a HC-Adv in the brain of mice preimmunized with a first generation adenovirus. C57Bl/6 mice were immunized ip with 1|[times]|108 i.u. of RAdHPRT, and 30 days later they were injected in the striatum with 1|[times]|106 bfu of HC-Adv-STkmCMV-LacZ. Mice were perfused-fixed at different time points after the intrastriatal injection (14, 30, 60 90 and 180 days). Brains were then removed and cut in 40mm thick serial sections. Immunohistochemistry for |[beta]|-galactosidade was performed and expressing cells and fibers were counted by quantitative unbiased stereology. After 14 days the expression in the striatum was high covering the entire caudate-putamen complex. Fibers and neurons were observed in the motor cortex, globus pallidus, entopeduncular nucleus, amygdaloid complex and Substantia nigra pars compacta. 30 days after the injection, the expression of the transgene in the striatum was diminished 30% in non immunized animals, while in immunized animals it was reduced by more than 50%. However after 60 days the expression was stable in both groups, and this expression lasted for up to 6 months. These results confirm the stable expression of the transgene in the brain with HC-Adv even in animals immunized systemically before the delivery of HC-Adv into the brain, and demonstrate the usefulness of HC-Adv as powerful tool for gene therapy for the treatment of neurological disorders such as Parkinson's and Huntington's disease.

'Dept of Anatomy & Physiology, University of Dundee. Dundee, DDl 4HN, UK. @Dept. of Molecular & Life Science, Dundee Institute of Technology, Dundee DDl lHG, UK. 'Institute of Virology. MRC Virology Unit, Univesity of Glasgow, Glasgow G11 SJR, UK. Fig. 1. The distribution of MAP-2 (a) and svnaptophvsin (b) in neocortical cultures after 23 da s i n vitro. In spite of their importance, little is known about how Neurons were p rocesse~ for double label l ing

High affinity binding of [3H]dihydroergocryptine (DHE) to crude membrane preparations of bovine and rat pineal glands was examined by a rapid filtration procedure through Whatman GFB paper. Scatchard analysis of bovine pineal membranes revealed a single population of binding sites with Kd = (4.8 +/- 1.9) X 10(-8) M (mean +/- S.E.M., n = 3) and binding site concentration = 834 +/- 103 fmol/mg of protein. When various alpha- and beta-adrenergic agonists or antagonists were tested for their ability to inhibit [3H]DHE binding the following Ki values (microM) were obtained: DHE (0.039), phentolamine (0.102), phenoxybenzamine (0.262), methysergide (0.670), L-epinephrine (2.27), serotonin (3.09), L-norepinephrine (3.20), octopamine (30.3), L-propranolol (1710), L-isoproterenol (greater than 2000). In order to obtain information on the pre- or postsynaptic location of pineal DHE binding sites, pineal 900 g supernatants were prepared from rats subjected to bilateral superior cervical ganglionectomy or sham-operation 7 days earlier. Ganglionectomy caused a 53% increase in the number of binding sites without changing significantly their Kd (5.2 X 10(-8) M and 5.5 X 10(-8) M in ganglionectomized and control rats, respectively). These results are compatible with the view that postsynaptic alpha-adrenoceptors are present in the pineal gland.

In Alzheimer's disease (AD), there is a loss of presynaptic cholinergic markers in the cerebral cortex, but the nature of cholinergic receptor changes is unclear. In this study, [3H]acetylcholine and [3H]nicotine were used to label nicotinic cholinergic binding sites in cerebral cortical tissues obtained at autopsy from patients with AD and from matched controls. A consistent and severe loss of nicotinic receptors was found in AD.

The role of calcium in the regulation of sodium-dependent high-affinity uptake of choline was assessed in vitro in slices of the rat brain, by measuring the specific binding of [3H]hemicholinium-3 ([3H]HCh-3) and the uptake of [3H]choline. Depolarization with potassium of slices of hippocampus, cortex, or striatum significantly increased the specific binding of [3H]HCh-3 when compared to control slices. However, the observed potentiation of specific binding of [3H]HCh-3 was markedly inhibited by the removal of calcium from the incubation medium in cortex or hippocampus, but not in slices of striatum. Alterations in the uptake of [3H]choline directly paralleled the observed changes in the specific binding of [3H]HCh-3 in striatum of the rat and were unaffected by the reduction of calcium in the incubation medium. The inorganic calcium channel antagonists, cadmium and cobalt, but not magnesium, zinc, manganese or lanthanum, significantly inhibited the 40 mM potassium chloride-induced stimulation of the binding of [3H]HCh-3 in the striatum. Finally, the calcium ionophore A23187 significantly increased the binding of [3H]HCh-3 in slices of striatum, either in the presence or absence of calcium in the bathing medium. This study demonstrates regional differences in the role of extracellular calcium in the regulation of the uptake of choline and suggests the involvement of intracellular release of calcium in the in vitro regulation of the sodium-dependent high-affinity uptake of choline in the striatum.

The effects of various drugs known to affect the sodium-dependent high-affinity choline-uptake system (SDHACU) in the brain were examined for their action upon the [3H]hemicholinium-3 [( 3H]HCh-3) binding site, which is associated with the choline carrier. The [3H]HCh-3 binding sites are affected in a similar way to the SDHACU system. Thus, alterations in the velocity of choline-uptake are mediated through changes in the apparent number of available transport sites at cholinergic terminals.

In a previous work, pinealectomy was found to depress benzodiazepine (BZP) receptor binding in cerebral cortex membranes of rats killed at noon. In order to assess the effect of pineal removal on diurnal variations of BZP binding site concentration and affinity, groups of intact, pinealectomized, or sham-pinealectomized rats (subjected to surgery 2 wk earlier) were killed at six different time intervals during the 24-h cycle. BZP binding was assessed by Scatchard analysis of 3H-flunitrazepam high-affinity binding to cerebral cortex membranes. In intact and sham-pinealectomized rats, a maximum in BZP receptor concentration was found at midnight. Pinealectomy blunted the nocturnal peak of receptor concentration and caused a significant depression of binding site number at noon. No changes in the affinity of the binding sites for the radioligand were detected as a function of time of day or following surgery. In a dose-response experiment for melatonin ability to restore the depressed BZP receptor concentration of cerebral cortex membranes of pinealectomized rats killed at noon, a minimal effective dose of 25 micrograms/kg body weight was obtained. These results further support a link between pineal activity and brain BZP receptors in rats.

As a part of a study aiming to characterize the physiological and pharmacological significance of the high affinity pineal benzodiazepine (BZP) binding sites reported previously, we examined the uptake of the BZP derivative 3H-flunitrazepam (FNZP) by rat pineal glands in vitro. At 37 degrees C, 3H-radioactivity was taken up by tissue up to a pineal/medium concentration of about 12, while at 0 degrees C the uptake amounted to only one-third that at 37 degrees C. Reciprocal of uptake analyzed by Lineweaver-Burk plots indicated apparent Km's of 1.74 and 1.45 microM, and Vmax's of 1.32 and 1.04 pmol per min per mg tissue, for control and superior cervical ganglionectomized rats, respectively, suggesting that the neural compartment does not participate significantly in 3H-FNZP uptake. Cerebral cortex explants of similar size and weight as the pineal ones took up 3H-FNZP to a maximum tissue/medium concentration of about 2. Neither pineal nor cerebral cortex 3H-radioactivity uptake exhibited significant changes as a function of time of day. A number of agents, including several BZP analogues, cocaine, desipramine, melatonin, fluoxetine, nomifensine, and dipiridamol, as well as changes in the ionic environment or metabolic inhibitors, did not affect 3H-FNZP uptake significantly. Other tissues, such as liver, muscle, kidney, adrenal gland, or anterior pituitary, took up 3H-radioactivity to tissue concentrations slightly lower than those of the cerebral cortex, suggesting that drug liposolubility accounted only to a limited extent for the high in vitro uptake detected in incubated pineals.

Lesions of basal forebrain cholinergic nuclei projecting to neocortex have recently been employed as an animal model for the cholinergic deficits in Alzheimer's desease. However, unlike Alzheimer's patients, whose deterioration appears to be progressive and irreversible, basalis lesioned rats usually recover both behaviorally and neurochemically within several months after the lesion. We now demonstrate that this recovery may be a function of the age of the rat and that cholinergic deficits re-occur in the aged rat. Choline acetyltransferase (ChAT) activity and [ 3 H]hemicholinium-3 ([ 3 H]HCh-3) binding are reduced in cortex ipsilateral to ibotenic acid lesions in the 12-month postlesion rat following an initial recovery to normal levels by about 3 months postlesion. The recurrence of decrease of cholinergic markers is not a consequence of a non-specific age-related decline since the activity of glutamic acid decarboxylase remains constant between 3 and 12 months postlesion.

The developmental distribution of the sodium-dependent high affinity choline uptake (SDHACU) system has been studied in the caudate and putamen of the baboon and of the human by in vitro autoradiography with the ligand, [3H]hemicholinium-3 ([3H]HCh3). Our results show that [3H]HCh3 binding sites in the newborn baboon and fetal human neostriatum are localized to patches, and then adopt a ‘striosome-like’ distribution in the juvenile baboon brain. These findings indicate a reorganization of [3H]HCh3 binding site distribution during the ontogeny of the primate neostriatum.

Testosterone administration to orchidectomized rats brought about a significant, 55% decrease of beta-adrenoceptor sites in the pineal gland, assessed from the specific binding of radioactive dihydroalprenolol (DHA). The changes in density of binding sites were not accompanied by significant modifications of the Kd. FSH or LH treatment of acutely castrated animals did not affect pineal beta-adrenoceptor binding. The depressive effects of testosterone in beta-adrenergic receptors were also observed in crude membrane fractions of medial basal hypothalamus and cerebral cortex. Sympathetic denervation of the pineal gland by superior cervical ganglionectomy did not abolish the changes in pineal beta-adrenoceptor density caused by testosterone. Hormone effects did not depend on a direct effect of the hormone on beta-adrenoceptor sites because testosterone did not compete with [3H]-DHA for the binding sites, in vitro. These results suggest that testosterone depresses pineal beta-adrenergic sites by acting mainly on postsynaptic sites.

Pinealectomy of rats resulted in significant depression of benzodiazepine receptors (assessed by [ 3 H]flunitrazepam binding) in cerebral cortex 3–14 days after surgery without affecting their affinity significantly. A single s.c. injection of melatonin (800 μg/kg body wt) restored the depressed brain benzodiazepine receptor sites. Single melatonin injections (up to 1600 μg/kg) to intact rats did not affect brain benzodiazepine binding when injected at either morning or evening hours. Daily melatonin treatment to intact rats for 5 days augmented benzodiazepine receptor density in brain (morning injections) or its dissociation constant (evening injections). Melatonin added in vitro to rat cerebral cortex membranes only slightly depressed [ 3 H]flunitrazepam binding at 100 μM concentrations. These results point out a link between pineal activity and benzodiazepine receptor function in rats. They also indicate that pharmacological doses of melatonin affect benzodiazepine binding sites in rat cerebral cortex.

The mammalian pineal gland fulfills the criteria of a “neuroendocrine” transducer.1 It translates a neural language provided by norepinephrine (NE) released at the synaptic biophase to a hormone language,melatonin and perhaps endocrine active peptides. The pinealocytes are also “endocrineendocrine” transducers inasmuch as they convert an endocrine language, e.g. estradiol attaining the gland via the general circulation, to a different endocrine signal like melatonin. Additionally “endocrine-neural” transducer events occur in the pineal gland, as revealed by the significant modifications of the activity of the innery ting sympathetic pathway after several hormone treatments.1,2

In the fetal and young primate neostriatum, cholinergic and dopaminergic markers show patches of high density surrounded by a lower-density matrix. In the adult, the same markers display the opposite pattern, a lower density in striosomes, surrounded by a higher-density matrix. In order to understand the developmental sequences leading to the adult compartmental organization of the primate neostriatum, a quantitative technique was used to study the ontogeny of pre- and postsynaptic components of cholinergic and dopaminergic neurons in baboon caudate nucleus and putamen. The development of specific uptake mechanisms for choline and dopamine and receptors was studied by means of quantitative autoradiography of the specific binding of [3H]-hemicholinium-3 [( 3H]-HC3) and [3H]-mazindol [( 3H]-MAZ) to the choline and dopamine uptake systems, respectively. [3H]-pirenzepine [( 3H]-PIR) was used to label M1 muscarinic receptors and [3H]-spiroperidol [( 3H]-SPI) was used to label striatal dopamine D2 receptors. Serial sections were used for each ligand to determine the precise anatomical relationships between the binding patterns of the different markers. Our aim was to determine whether the adult striosomal distribution of the binding sites studied was due to 1) a selective decrease in patch/striosomal binding density or 2) a selective increase in matrix binding density. Our studies show that a postnatal decrease in the density of [3H]-HC3 sites in the patch/striosomes and an increase in the matrix density of [3H]-MAZ sites are the primary, but not the sole, changes in the compartmental distribution of these sites leading to the adult striosomal organization of the striatal cholinergic and dopaminergic innervation. D2 receptors follow the general developmental pattern of [3H]-MAZ and [3H]-HC3, changing their density of distribution in both compartments during the developmental period examined. In addition, M1 muscarinic receptors already display their adult pattern in the newborn baboon striatum, and therefore represent one of the first neurochemical makers to adopt its mature organization.

[3H]Flunitrazepam (FNZP) binds to a single class of high affinity binding sites in bovine pineal membranes (KD 2.8 nM). FNZP binding increased by 25% after exposure to gamma-aminobutyric acid and exhibits a high degree of specificity for drugs acting on the central type of benzodiazepine receptor. High affinity binding of [3H]ethyl-3-carboline ester to pineal membranes has a KD 0.8 nM and about 60% of the maximal number of FNZP binding sites.

High affinity binding of 3H-flunitrazepam (FNZP) to crude membrane preparations of bovine pineal membranes was examined by a rapid filtration procedure through Whatman GFB paper. At 0 degrees C binding reached equilibrium in about 20 min. Scatchard analysis of data at equilibrium revealed a single population of binding sites with dissociation constant (Kd) = 3.14 +/- 0.45 nM and binding site concentration (Bmax) = 55.6 +/- 5.6 fmol/mg protein. Kinetic analysis of the association and dissociation curves indicated a kinetic Kd = 1.13 nM, in reasonable agreement to that obtained at equilibrium. When various benzodiazepine (BZP) analogues were tested for their ability to inhibit 3H-FNZP binding, the following Ki (nM) were obtained: clonazepam (0.22), Ro 15-1788 (0.48), FNZP (0.95), Ro 5-4864 (greater than 10,000). When the beta-carboline derivative 3H-ethyl-beta-carboline-3-carboxylate ester (E beta CEE) was used as a radioligand, Kd at equilibrium (0.98 nM), kinetic Kd (1.69 nM) and affinity order for analogues were in close agreement to those found for FNZP binding; however, Bmax was about 60% that observed for 3H-FNZP binding. Addition of GABA or pentobarbital (100 microM) to pineal membranes increased 3H-FNZP binding by 55 and 72%. These results suggest the existence of a mixed population of type 1 and type 2 central BZP receptor subclass in bovine pineal gland.

The possible existence of pineal progesterone (P) receptors was assessed in 105,000 g supernatants of bovine pineal glands incubated with 3H-P in the presence of 10 microM cortisol. By charcoal adsorption analysis specific binding (defined as the difference between 3H-P binding in the absence and presence of 1,000-fold excess of unlabeled P) attained its maximum after 60 min at 37 degrees C. Binding was abolished by heating the cytosol or by preincubating it with trypsin. A single population of binding sites with a Kd = 1.64 nM and binding site concentration = 34 fmol/mg of cytosol protein was detectable by Scatchard analysis. In competition experiments the B50 progesterone values (nM) were: P (2), 20 alpha-dihydroprogesterone (12), 20 beta-dihydroprogesterone (25), estradiol (158), testosterone (675) and corticosterone (greater than 1,000). After sucrose gradient centrifugation of pineal cytosol incubated with 3H-P a discrete peak of radioactivity in the 7-8 S region of the gradient was observed, and disappeared following incubation with excess unlabeled P. These results indicate that P receptors are present in the bovine pineal gland.

The intraperitoneal injection of the methylxanthine derivative pentoxifylline [3,7-dimethyl-1-(5-oxo-hexyl)-xanthine] brought about, 3 hr later, a significant depression of α- and β-adrenoceptor sites in the cerebral cortex, and of β-adrenoceptor sites in medial basal hypothalamus and pineal gland, (assessed from the specific binding of radioactive dihydroergocryptine and dihydroalprenolol respectively). The changes in the density of binding sites were not accompanied by significant modifications of the K d ′s. Sympathetic denervation of the pineal gland by superior cervical ganglionectomy (SCGx) abolished the changes of β-adrenoceptor number in the pineal caused by pentoxifylline. The increase of α-adrenoceptor sites in the hypothalamus brought about by ganglionectomy was not affected by injection of pentoxifylline. Pentoxifylline did not compete in vitro for radioligand binding to brain membranes. These results suggest that methylxanthines depress brain adrenoceptor sites acutely, probably by down-regulation of receptors following the increase in catecholamine release caused by injection of the drug.

We used hepatitis B virus (HBV) transgenic mice as recipients of cytotoxic T cells (CTLs) specific for the immunodominant Env28–39 epitope (Env28) of HbsAg, and C57BL/6J mice acutely infected with a recombinant replication-deficient adenovirus expressing lacZ (RAd35) to show that platelets accumulate in necroinflammatory foci of the liver during the course of a disease similar to acute hepatitis in humans. Liver disease was monitored by quantifying the activity of serum alanine aminotransferase (sALT), a hepatocellular enzyme that is released into the circulation by necrotic hepatocytes, and by evaluating histological changes at the time of autopsy. Livers from mice sacrificed 2 days after CTL transfer were stained with a mixture of rat monoclonal antibodies (α-PLT) against mouse glycoprotein (GP) Ibα, a receptor exclusively expressed on platelets and megakaryocytes. Livers from mice injected with saline (0.9% NaCl) alone served as controls. Platelets were detectable only inside vessels and hepatic sinusoids in saline-injected controls, while they accumulated alongside apoptotic hepatocytes and inflammatory cells within necroinflammatory foci of the liver in CTL-injected or RAd35-infected animals. The injection of α-PLT caused a >97.5% decrease in the number of circulating platelets (less than 2x104 platelets/μl of blood) within 30 min, and similar low counts were maintained for up to 6 days. Mice that received either saline or an irrelevant antibody (α-Irr) exhibited stable platelet counts (8–10 x 105 platelets/μl of blood). Previous platelet depletion in CTL-injected or RAd35-infected animals reduced the intrahepatic accumulation of virus-specific CTLs and the resulting liver damage, as evidenced by an 80% reduction in sALT activity (from 1600 to 300 U/L) and a reduction the size of necroinflammatory foci (from 6724 μm2 to 1245 μm2) as compared to mice that received α-Irr. Infusion of washed mouse platelets expressing human GP Ibα (thus, insensitive to the depleting effect of α-Plt) within hours after CTL transfer or RAd35 infection restored CTL accumulation and liver disease severity, but not when the infused platelets were pretreated with the activation inhibitor, prostaglandin E1. Accordingly, platelet activation was required to promote CTL/platelet interactions under flow conditions in vitro. Fibrin deposition was abundant within hepatic necroinflammatory foci of CTL-injected or RAd35-infected animals, and markedly decreased in animals that were previously made thrombocytopenic. The platelet-dependent intrahepatic deposition of fibrin could be selectively prevented by anticoagulant treatment of the mice, which had no effect on the platelet count, but this did not ameliorate liver injury. Our findings indicate that activated platelets contribute to CTL-induced liver immunopathology by facilitating the accumulation of CTLs at the site of inflammation, independently of procoagulant function.

Gene therapy using Fas ligand (FasL) for treatment of tumours and protection of transplant rejection is hampered because of the systemic toxicity of FasL. In the present study, recombinant replication-defective adenovirus vectors (RAds) encoding FasL under the control of either the neuronal-specific neuronal-specific enolase (NSE) promoter or the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter have been constructed. The cell type-specific expression of FasL in both neurons and glial cells in primary cultures, and in neuronal and glial cell lines is demonstrated. Furthermore, transgene expression driven by the neuronal and glial promoter was not detected in fibroblastic or epithelial cell lines. Expression of FasL driven by a major immediate early human cytomegalovirus promoter (MIEhCMV) was, however, achieved in all cells tested. As a final test of the stringency of transgene-specific expression, the RAds were injected directly into the bloodstream of mice. The RAds encoding FasL under the control of the non-cell type-specific MIEhCMV promoter induced acute generalized liver haemorrhage with hepatocyte apoptosis, while the RAds containing the NSE or GFAP promoter sequences were completely non-toxic. This demonstrates the specificity of transgene expression, enhanced safety during systemic administration, and tightly regulated control of transgene expression of highly cytotoxic gene products, encoded within transcriptionally targeted RAds.

The high affinity binding of [3H]flunitrazepam (FNZP) to crude membrane preparations was examined in human pineal glands. Scatchard analysis of the data at equilibrium revealed a single population of binding sites with dissociation constant = 2.36-2.53 nM and binding site concentration = 59-108 fmol/mg protein. When various benzodiazepine (BZP) analogues were tested for their ability to inhibit [3H]FNZP binding the following Ki (nM) were found: clonazepam (0.13), RO 15-1788 (0.60), FNZP (2.14), diazepam (13.5), Ro 5-4864 (greater than 10 000). In both human pineal gland and cerebral cortex 10-100 microM gamma-aminobutyric acid (GABA) increased BZP binding by about 30%, an effect inhibited by the GABA receptor blocker bicuculline. The stimulatory effect of GABA on [3H]FNZP binding in rat cerebral cortex (about 60%) decreased as a function of time elapsed postmortem at room temperature to reach values similar to those observed in human brains. These results suggest the existence of central type BZP receptors in the human pineal glands.