Longitudinal follow-up study of adenoviral vector-mediated gene transfer of dopamine D2 receptors in the striatum in young, middle-aged, and aged rats: a positron emission tomography study.

Overexpression of dopamine D(2) receptors by adenoviral vector-mediated gene transfer in the rat striatum was evaluated by positron emission tomography in vivo and by ex vivo autoradiography in 5-, 13-, and 24-month-old Fischer 344 rats. Each rat had hemilateral gene transfer of D(2) receptors mediated by adenoviral vectors (AdCMV.DopD(2)R) in the striatum with contralateral striatal injection of control vectors (AdCMV.LacZ). At day 2 or 3 after vector injection positron emission tomography or ex vivo autoradiography was performed. The binding potential of a radiolabeled D(2) receptors ligand, [11C]raclopride, was significantly higher in the D(2) receptors gene-transferred striatum than the control side in each age group at a similar degree. The binding potential in the AdCMV.DopD(2)R-injected striatum of 24-month-old rats was similar to that in the AdCMV.LacZ-injected striatum of 5-month-old rats (0.99+/-0.14 versus 0.91+/-0.08). A significant age-associated decrease of the binding potential of [11C]raclopride was found in the control vector-injected side, and a significant increase of the binding potential in the adenoviral vector-injected side in all three age groups, suggesting no aging effect on the overexpression of D(2) receptors. A group of rats underwent follow-up assessment by positron emission tomography. The overexpression of D(2) receptors decreased with time in all three groups; however, the decrease rate of the D(2) receptors expression was significantly smaller in the 24-month-old group than in the 5-month-old group. We confirmed that the adenoviral vector-mediated gene transfer of D(2) receptors compensated the decreased density of striatal D(2) receptors in the 24-month-old rats up to the level in the control striatum of 5-month-old rats, and the decrease rate of the overexpression was significantly smaller in aged rats.

[Strategy for preventing recurrence after video-assisted thoracoscopic surgery for spontaneous pneumothorax; efficacy of talc pleurodesis and absorbable mesh covering].

Video-assisted thoracoscopic surgery (VATS) has been widely used in the treatment of spontaneous pneumothorax. The unexpectedly high increase of the recurrence rate is the most important problem. We examined the interaction between recurrence, clinical features and patient's backgrounds. The main causes of recurrence were oversight of bullae and the emphysematous changes at the periphery of the site where an autosuture device was used. 90% of the recurrence occurred in the patients with type III small diffuse bullae. Introduction of the regional talc pleurodesis and covering stapled line with absorbable mesh were extremely useful to lower the recurrence rate after VATS for spontaneous pneumothorax.

Overexpression of dopamine D2 receptors reduces alcohol self-administration.

The mechanism(s) underlying predisposition to alcohol abuse are poorly understood but may involve brain dopamine system(s). Here we used an adenoviral vector to deliver the dopamine D2 receptor (DRD2) gene into the nucleus accumbens of rats, previously trained to self-administer alcohol, and to assess if DRD2 levels regulated alcohol preference and intake. We show that increases in DRD2 (52%) were associated with marked reductions in alcohol preference (43%), and alcohol intake (64%) of ethanol preferring rats, which recovered as the DRD2, returned to baseline levels. In addition, this DRD2 overexpression similarly produced significant reductions in ethanol non-preferring rats, in both alcohol preference (16%) and alcohol intake (75%). This is the first evidence that overexpression of DRD2 reduces alcohol intake and suggests that high levels of DRD2 may be protective against alcohol abuse.

Enhanced low shear stress induced platelet aggregation by Shiga-like toxin 1 purified from Escherichia coli O157.

The effect of Shiga-like toxin 1 (Stx1) produced by Escherichia coli O157 on platelets was studied with an argon laser light-assisted shear-induced platelet aggregometer and with binding assays. Stx1 markedly enhanced the platelet aggregation under low shear stress but did not affect it under high shear stress. Minimal concentration of Stx1 required for the enhancement was 0.25 ng/ml, and almost maximal enhancement was observed at a final concentration of > or =2.5 ng/ml. This enhanced platelet aggregation disappeared after leukocyte depletion from normal platelet-rich plasma with a specific filter. In contrast, a standard platelet aggregometer was unable to detect this enhanced platelet aggregation in either the presence or the absence of ADP. 125I-labeled purified Stx1 did not specifically bind to normal washed platelets depleted of leukocytes, and thin-layer chromatographic analysis of glycolipids extracted from normal platelet lysates also confirmed that leukocyte-depleted normal platelets lack Stx1-specific receptor globotriaosylceramide (Gb3). Supernatant from the monocyte suspension stimulated with Stx1 exhibited the enhanced low shear stress induced platelet aggregation, but that from the polymorphonuclear cell suspension did not. Several cytokines produced from monocytes reproduced this event in vitro. Further, plasmas from six out of seven patients with hemolytic uremic syndrome (HUS) had activity similar to the purified Stx1. This activity was almost totally impaired after treatment of HUS plasmas with Gb3 in accord with reduction of plasma Stx1 levels. Taken together, these results indicate that platelets lack Gb3, and Stx1 appears to modulate platelet aggregation in an indirect fashion, presumably by the release of cytokines or chemical compounds from the target tissues.

Involvement of dopamine D(2) receptors in complex maze learning and acetylcholine release in ventral hippocampus of rats.

In the current study we focus on the involvement of dopamine D(2) receptors in the ventral hippocampus in memory performance and acetylcholine release. Using the aversively motivated 14-unit T-maze (Stone maze) the injection of raclopride, a D(2) receptor antagonist, into the ventral hippocampus (8 microg/kg) was found to impair memory performance. Co-injection of quinpirole, a D(2) receptor agonist (8 microg/kg), overcame the impairment in performance. Microdialysis study revealed that quinpirole infusion (10-500 microM) into the ventral hippocampus stimulated acetylcholine release in a dose-dependent manner, and systemic injection of quinpirole (0.5 mg/kg, i.p.) also stimulated acetylcholine release in the ventral hippocampus. Infusion of eticlopride, another D(2) receptor antagonist, into the ventral hippocampus suppressed acetylcholine release in the hippocampus induced by systemic injection of quinpirole. Taken together, we suggest that D(2) receptors in the ventral hippocampus are involved in memory performance, possibly through the regulation of acetylcholine.

Dietary restriction of choline reduces hippocampal acetylcholine release in rats: in vivo microdialysis study.

We fed rats with a diet deficient in choline for 12 weeks and studied how dietary choline deficiency affected their behavior and their ability to release acetylcholine in discrete regions of rat brain using step-through passive avoidance task and in vivo microdialysis. In comparison with the control, rats fed the choline-deficient diet showed poorer retention of nociceptive memory in the passive avoidance task. Average choline level in cerebrospinal fluid in the choline-deficient group was significantly less (33.1%) than that of control rats. In vivo microdialysis showed no difference in the pattern of acetylcholine release enhanced by intraperitoneal administration of scopolamine hydrochloride (2 mg/kg) in the striatum between the two groups, whereas in the hippocampus, the maximum and subsequent increase of acetylcholine from the baseline by scopolamine injection was significantly lower in the choline-deficient group than in the control. From the results of our study, we speculate that long-term dietary restriction of choline can affect extra- and intracellular sources of substrates required for acetylcholine synthesis, and eventually limit the ability to release acetylcholine in the hippocampus. Reduced capacity to release acetylcholine in the hippocampus implies that the mechanism, maintaining acetylcholine synthesis on increased neuronal demand, may vary in discrete regions of the brain in response to dietary manipulation. The vulnerability of the mechanism in the hippocampus to dietary choline restriction is indicated by impaired mnemonic performance we observed.

Plasma cortisol levels in elderly female subjects with Alzheimer's disease: a cross-sectional and longitudinal study.

We investigated the plasma cortisol levels at a fasting state in elderly female Alzheimer's disease (AD), vascular dementia (VD), and non-demented subjects (n=66, 28 and 21, respectively). Twenty-eight AD subjects were followed for 40 months. The plasma cortisol levels in AD and VD subjects were significantly higher than those of non-demented subjects at baseline. In AD subjects in relatively early stages of the disease [Mini-Mental State Examination (MMSE)], at baseline, high plasma cortisol led to rapid declines in MMSE scores over a 40-month period.

Development of an adenoviral vector for intracerebral delivery of the dopamine D(2) receptor.

The age-related loss of striatal dopamine D(2) receptors (D(2)R) has been observed in numerous species, including rodents, monkeys, and man, and is partly responsible for impaired motor function in aged mammals. We have developed an adenoviral vector designed for intracerebral transfer of cDNA for D(2)R. Results of in vitro studies demonstrated that the vector produced abundant message for D(2)R and that the vector was membrane bound and capable of binding appropriate ligand. Results of in vivo studies provided clear evidence of D(2)R production when injected into the striatum of rats. The D(2)R produced were capable of binding appropriate ligand. In addition, evidence of functional receptors was produced by demonstrating apomorphine-induced rotational behavior in rats receiving a unilateral injection of the vector. Despite these successes, we have been unable to demonstrate improvement in the motor behavior of aged rats receiving bilateral injections of the vector. A major problem with this vector as with similar adenoviral vectors is the loss of expression beginning 3-5 days after injection to undetectable levels at 21 days. Because of the lack of motor functional effects in aged rats and the loss of expression of the vector, other strategies for development of the vector are being pursued. Regarding functional effects, we have examined the feasibility of manipulating hippocampal acetylcholine (ACh) release through D(2)R manipulation to improve memory performance. Using microdialysis, we have demonstrated in vivo in rats that treatment with a D(2)R agonist increases hippocampal ACh release while treatment with a D(2)R antagonist attentuates this effect as well as impairs performance in a complex maze task. In addition, a D(2)R null mutant mouse is being used to examine possible therapeutic effects of the vector. These mice show specific motor deficits. Recent studies using positron emission tomography have also demonstrated the feasibility of in vivo imaging of the vector. Thus, use of adenoviral vectors specific for neurotransmitter receptors can provide a highly useful research tool for examining age-related alterations in behavioral function and a possible strategy for therapeutic intervention.

In vivo imaging of adenovirus-mediated over-expression of dopamine D2 receptors in rat striatum by positron emission tomography.

PET was used to provide in vivo imaging of the over-expression of dopamine D2 receptor (D2R) induced by adenovirus vector-mediated gene transfer in rat striatum. The uptake of three kinds of D2R-specific ligands, [11C]raclopride, [11C]nemonapride and [11C]N-methylspiperone, measured by PET was higher in the striatum injected with the vectors for D2R than the contralateral striatum injected with a control vector 2-3 days after injection. However, the uptake of [11C]SCH 23390, a dopamine D1 receptor specific ligand, or [11C]beta-CIT-FP, a dopamine transporter specific tracer, was not different between bilateral striata. Co-injection of excess unlabeled raclopride inhibited the uptake of [11C]raclopride. At day 16 the increased uptake of [11C]raclopride declined to basal level, consistent with past in vitro assessment of this vector. In vivo imaging of D2R will permit longitudinal assessment of the efficiency of this and similar vectors in rat brain that can be related to functional changes being observed.

Low plasma epinephrine in elderly female subjects of dementia of Alzheimer type.

One of the robust features of brain pathologies of dementia of Alzheimer type (DAT) is the impairment of the hippocampus, especially the cholinergic system. Several animal studies have suggested that the cholinergic system in the hippocampus is involved in the control of the plasma level of catecholamines and glucose. The stimulation of the hippocampal cholinergic system has resulted in the elevation of plasma catecholamines and glucose in rats. In the present study, we measured the plasma level of epinephrine, norepinephrine, dopamine, glucose, and insulin during a fasting state in the morning in hospitalized DAT (n=66), vascular dementia (VD) (n=28), or non-demented (ND) (n=21) females (mean age DAT=82. 49+/-4.98, VD=82.86+/-5.86, ND=82.95+/-7.77, respectively). Statistical analysis showed that the plasma level of epinephrine during a fasting state in DAT subjects was significantly lower than that of ND subjects; however, in VD subjects the level of epinephrine was not different from that of ND subjects. Other values did not differ significantly among the groups.

[Gene therapy to treat Parkinson's disease].

One recent strategy of gene therapy is to have cells express the lacking substances. Decline in dopamine D2 receptors (D2R) is observed in late-stage Parkinson's disease. We have constructed a replication-deficient adenovirus vector to transfer rat D2R cDNA (AdCMV.DopD2R) to the brain as a possible therapeutic strategy and a replication-deficient adenovirus vector to express nothing (AdCMV.Null) as a control. Using tissue culture cells infected with this vector, we detected D2 R cDNA by Northern analysis and receptor protein in membrane preparations as specific binding of the D2R ligand, [3H] spiperone. In vivo demonstration involved autoradiographic analysis of [3H] spiperone binding in rat striatum, D2R expression was amplified above normal concentrations in the injection site. We investigated the expression and functionality of the adenoviral vector. Comparative analysis of the autoradiographic images from the striatum injected with AdCMV.DopD2R and the contralateral striatum injected with a control vector, AdCMV. Null, in male rats indicated that D2R binding was increased by 40-60% on days 3 and 5 after injection, but then declined to baseline levels by day 21. When injected with apomorphine on days 3 and 7 after vector injection, experimental groups that had received unilateral striatal injections of AdCMV. DopD2R exhibited a distinct and significant laterality in rotational behavior. These results provide the first demonstration of an adenovirally mediated, intracerebral delivery of a functional neurotransmitter receptor.

Angiomyolipoma of the lung.

Extrarenal angiomyolipomas are rare tumors that have been reported in several organs, including the liver, reproductive organs, skin, and retroperitoneum. In this report, we present the clinicopathologic findings of an angiomyolipoma of the lung in a 67-year-old man with no history of tuberous sclerosis or lymphangioleiomyomatosis. To our knowledge, this is the second reported case of pulmonary angiomyolipoma. The tumor was coincidentally found in the left lower lung in association with a hemothorax. Our case was histologically identical to angiomyolipoma occurring in the kidney, but immunohistologic staining for HMB-45 was negative, similar to the first reported case.

Swimming stress that causes hyperglycemia increases in vivo release of noradrenaline, but not acetylcholine, from the hypothalamus of conscious rats.

The effects of acute swimming stress (10 min) on noradrenaline release from the medial basal hypothalamus (MBH; consisting of the ventromedial and dorsomedial hypothalamus) and acetylcholine release from the lateral hypothalamic area (LHA) were investigated in freely moving rats by using in vivo microdialysis techniques. Serum glucose, noradrenaline and adrenaline concentrations were also determined. Acute swimming stress produced significant hyperglycemia, with increases in serum noradrenaline and adrenaline concentrations. The release of noradrenaline from the MBH was significantly stimulated during the swimming stress. On the other hand, the swimming stress has no significant effect on the release of acetylcholine from the LHA. These findings support the idea that hypothalamic noradrenergic neurons play an important role in the sympathoadrenal hyperglycemic response to stressful stimuli. Moreover, it is suggested that hypothalamic cholinergic neurons are not involved in the responses of serum glucose, noradrenaline and adrenaline concentrations to swimming stress.

Application of gene therapy to treat age-related loss of dopamine D2 receptor.

We have investigated the feasibility of using gene therapy to attenuate the age-related decline in striatal dopamine D2 receptors (D2R) associated with reduced motor control. To this end, we have constructed an adenoviral vector containing the cDNA for the rat D2R. When injected into HeLa and HS24 cells in vitro, the vector induced an abundant message for D2R, as demonstrated by Northern analysis, and produced a membrane-bound protein capable of binding a D2R ligand, [3H]spiperone. When injected into rat striatum in vivo, the vector produced a marked increase in D2R near the site of injection, as evidenced by increased [3H]spiperone binding as well as by another more specific ligand, [125I]iodosulpride. The D2R produced in the striatum were functional, as evidenced by rotational behavior induced by a subcutaneous injection of the dopamine agonist, apomorphine. However, we did not observe any significant improvement in motor performance during preliminary experiments in which aged rats received bilateral striatal injections of the vector. In young rats, vector-induced expression of D2R in striatum was increased markedly three to five days after infection, but then declined to baseline levels by day 21. Loss of expression in aged rats proceeded at a somewhat lower rate. Because of the loss of expression and lack of significant performance enhancement in aged rats following vector injection into the striatum, we are now pursuing other strategies. These include functional assessment of the current vector in D2R null mutant mice as well as construction of new vectors that may yield more long-term expression.

[A case of postoperative recurrence of hepatocellular carcinoma successfully treated with arterial infusion chemotherapy using cisplatin and 5-fluorouracil].

We reported a 71-year-old male patient with multiple liver recurrence of hepatocellular carcinoma, who responded to arterial infusion chemotherapy using CDDP and 5-FU. The patient was administered 10mg/body CDDP and 250 mg/body 5-FU on day 1-5./1 course. As a result, the tumor decreased and AFP was decreased to 50.8 ng/ml (from 93099 ng/ml). The patient is alive 13 months after the beginning of therapy in a condition of partial response (PR).

Pharmacokinetic study of aniracetam in elderly patients with cerebrovascular disease.

The clinical pharmacokinetics of the cognitive enhancer, aniracetam (200 mg), was studied in elderly patients with cerebrovascular disease (CVD) and compared with those of young healthy volunteers. Six female hospitalized patients (mean age 84.5 years) were used in this study. The serum level of anisic acid and p-methoxyhippuric acid, major metabolites of aniracetam, reached a peak at 2 h after oral administration, and returned to basal level by 6 h. Mean creatinine clearance was 20-30 ml/min. The t1/2 of metabolites was increased by 4- to 7-fold in the elderly patients compared with young volunteers. This study showed that tmax, t1/2, and AUC were enlarged in the elderly; however, no clinical side effects were observed.

Rotational behavior produced by adenovirus-mediated gene transfer of dopamine D2 receptor into rat striatum.

We investigated the expression and functionality of a previously developed adenoviral vector carrying the rat cDNA for the dopamine D2 receptor (D2R), AdCMV.DopD2R. Comparative analysis of the autoradiographic images from the striatum injected with AdCMV.DopD2R and the contralateral striatum injected with a control vector, AdCMV.Null, in male rats indicated that D2R binding was increased by 40-60% on days 3 and 5 after injection, but then declined to baseline levels by day 21. When injected with apomorphine on days 3 and 7 after vector injection, experimental groups that had received unilateral striatal injections of AdCMV.DopD2R exhibited a distinct and significant laterality in rotational behavior. These results provide the first demonstration of an adenovirally mediated, intracerebral delivery of a functional neurotransmitter receptor.

[Hypersensitivity in the pupil dilation response to a cholinergic antagonist in patients with Alzheimer's disease and Down's syndrome].

In 1994, Scinto et al. reported hypersensitivity in the pupil-dilation response to topical application of a cholinergic antagonist, tropicamide, in patients with Alzheimer-type dementia. Similar tests on Japanese subjects showed significant differences in pupil response between subjects with Alzheimer-type dementia (under age 70 years) and age-matched controls. The present study included 24 patients with early-onset Alzheimer-type dementia, 29 patients with late-onset Alzheimer-type dementia, 15 healthy controls (all spouse of patients with Alzheimer-type dementia), 9 patients with vascular dementia and 5 patients with Down's syndrome. After adapting to semi-darkness (10 lux), a solution of tropicamide (Midrin-P, Santen Co, Ltd) was instilled into the right eye. Pupil diameters were measured every 5 to 10 min, and the maximum pupil diameter for each eye was used in the data analysis. The solution was diluted until the tropicamide concentration reached 0.01%. Pupil diameters were measured, and differences between baseline and maximum pupil diameter were computed. Among 18 patients with early-onset Alzheimer-type dementia (mean age 63.9 years) the pupils dilated by 1.12 +/- 0.52 mm (mean +/- SD) as compared with 0.20 +/- 0.75 mm in 7 age-matched controls (mean age 57.8 years); this difference was significant (T-test, p < 0.05). Correlations between the degrees of pupillary dilation and the scores on the revised version of Hasegawa's Dementia Scale were not significant. These data suggest that in subjects under 70 years of age, the diagnostic methods described by Scinto et al. can be used to distinguish those with Alzheimer-type dementia from those without dementia, but those methods may not be effective in subjects over 70 years of age.

Cognitive enhancement. New strategies for stimulating cholinergic, glutamatergic, and nitric oxide systems.

The development of treatments for AD is being pursued along many diverse lines. While the ACh hypothesis has generated abundant development efforts, little clinical progress has been achieved to date. Recent efforts aimed at developing more potent, more specific, and safer ChE inhibitors appear to offer greater potential for therapeutic success than achieved to date. Treatments aimed at the NMDA Glu system lag much further behind in their development. Progress in this area must be tempered by the potential for glutamate excitotoxicity mediated through this neurotransmitter system. Development of indirect agonists operating at the glycine and polyamine modulatory sites on the NMDA receptor might offer the safest alternative to applying more direct agonists. While a great degree of interest had been generated by the reports of NO involvement in signal transduction through the NMDA system, this area of research has been complicated by conflicting reports regarding NO involvement in learning and LTP. Moreover, the interaction of drugs acting on NOS with the vascular effects mediated by eNOS has also complicated development of drugs that act specifically on the neural actions of NO. This area will continue to receive extensive research attention; but similar to the development of Glu agonists, attention must be given to the potential neurotoxic effects of overstimulating this system. Perhaps targeting other presynaptic mechanisms that effect glutamate release might be a safer strategy to pursue. Considerable progress has been made over the last two decades in identifying the genetic and neural mechanisms involved in AD. Progress in developing treatments will remain highly correlated with this effort, and with basic research geared to comprehending how memories are formed and why neurons degenerate and regenerate.

Immobilization stress-induced increase of hippocampal acetylcholine and of plasma epinephrine, norepinephrine and glucose in rats.

We investigated the role of the hippocampal cholinergic neurons during immobilization stress in rats using a microdialysis technique. Blood levels of glucose, epinephrine and norepinephrine during immobilization stress were also determined. Acetylcholine release was initially increased by immobilization stress, then gradually decreased. Plasma level of epinephrine increased gradually and reached significance at 30 min after the start of immobilization and remained at the elevated level during immobilization. Plasma level of norepinephrine initially increased and reached significance at 30 min after the start of immobilization and remained at the elevated level during immobilization. Plasma level of glucose increased gradually and reached maximum and significance 45 min after the start of immobilization, then decreased. Fifteen min after immobilization, acetylcholine release increased again, while concentrations of epinephrine and norepinephrine were still elevated. Thus the response of acetylcholine and the other responses to immobilization stress were not parallel.