The clinical significance of ZAP-70 and CD38 expression in B-cell chronic lymphocytic leukaemia.

BACKGROUND: B-cell chronic lymphocytic leukaemia (B-CLL) is a disease with a highly variable clinical course; some patients never need treatment, while others require intensive treatment early after diagnosis. Recently, some new prognostic factors, such as IgVH mutational status, ZAP-70 and the expression of CD38 in leukaemic cells were introduced to identify attenuated versus progressive types of CLL bearing the potential to facilitate risk-adapted treatment strategies. PATIENTS AND METHODS: To evaluate the clinical value of ZAP-70 and CD38 as predictors of disease progression we assessed the expression of these markers by the flow cytometry method in 156 B-CLL patients. RESULTS AND CONCLUSIONS: Both ZAP-70 and CD38 expression were shown to predict the clinical course of the disease, while ZAP-70 expression appeared to be more predictive than CD38 expression and more relevant in defining the cases of B-CLL responsive or refractory to first line chemotherapy. A simultaneous evaluation of ZAP-70 and CD38 expression allowed distinguishing the patients groups with the most favourable prognosis as well as those with the worst. Taken together we recommend assessing both ZAP-70 and CD38 protein expression for the definition of prognostic subgroups in patients with B-CLL.

The analysis system COGITAT for the study of cognitive deficiencies in rodents.

COGITAT is an automated hole board system that, following minimal experimental interventions, makes it possible to measure a variety of parameters associated with learning, memory, relearning, cognition, and cognitive shifts, but also changes in exploratory and sensorimotor performance in rodent models. The individual parameters--that is, overall exploratory activity, number of visits (deep in the hole) into or inspections of (at the upper surface) holes, number of baited, unbaited, or previously baited holes visited or inspected, reinspections of or revisits into any holes, number of pellets eaten, time to find pellets, serial order collection (without intermediate inspections or visits), and reference and working memory errors (visits, inspections, or total)--are obtained simultaneously, and the results are immediately available after the end of each experiment. The system appears to be well suited to neurophysiological, neuropharmacological, and gene-technological investigations in rodent models.

Cerebral oligaemia episode triggers free radical formation and late cognitive deficiencies.

Sixty minutes of cerebral oligaemic hypoxia, induced by bilateral clamping of the carotid arteries (BCCA) in pentobarbital-anaesthetized normotensive rats, induces a late progressive cognitive decline when compared with sham-operated controls. Analysis at BCCA of hippocampal metabolism using microdialysis showed increased release of glutamate, aspartate and gamma-aminobutyric acid, followed by a progressive rise in the formation of hydroxyl free radicals measured as 2,3-dihydroxybenzoic acid (2,3-DHBA), their reaction product with salicylate, though only in the re-perfusion phase. In the striatum increased dopamine release occurred during BCCA, whereas glutamate and aspartate showed an increase only during the late re-perfusion phase. gamma-Aminobutyric acid (GABA) concentration increased during BCCA and early re-perfusion. An increase in 2,3-DHBA was seen during BCCA, and persisted over 2 h of re-perfusion. Six and 13 months after surgery, though not as early as 3 months, BCCA-treated rats perform worse than sham-operated controls in a water-maze, where decreased swimming speed reveals striatal dysfunction, while hippocampal dysfunction manifested as diminished spatial bias. These results show that cerebral oligaemia, similarly to cerebral ischaemia, leads to increased extracellular dopamine, aspartate and glutamate, and the production of hydroxyl radicals in structures associated with learning and memory processes. Unlike cerebral ischaemia, in cerebral oligaemia the appearance of spatial memory deficits is delayed.

Injection of a minuscule dose of FeCl3 within the ventrolateral striatum causes a chronic disturbance of the integrative function within the limbic part of the ventral striatum.

The present study shows that low amounts of applied iron have a potent effect on the ventrolateral striatum. This is reflected by an influence on spontaneous night activity, cognitive behaviour during the water maze navigation task, exploratory activity and in response to postsynaptic apomorphine stimulation. Such functional disturbances could be observed up to months after a single application of either 0.3 microgram or 1.5 micrograms FeCl3. The low dose of iron stimulates while 1.5 micrograms inhibits the spontaneous dopaminedependent locomotor night and explorative activity. The low concentration of ionic iron injected intrastriatally also increases lipid peroxidation in striatal and hippocampal tissues. These results suggest that the functional integrity of the ventral striatum and the regulation of the iron metabolism are critical for the sensorimotor performance.

Cerebral oligemic hypoxia and iron toxicity in the mesolimbic system of rats.

60 min of bilateral clamping of the carotid arteries (BCCA) in pentobarbital anaesthetized adult Wistar rats increases the lipid peroxidation in hippocampal tissue as estimated four weeks later. 1.5 micrograms FeCl3 in 2 microliters buffer injected unilaterally in the ventrolateral striatum enforced the effect when applicated one week after the BCCA treatment. During ageing, the explorative and locomotor behaviour of BCCA rats decreased earlier than the behaviour activities of the controls. These activities show no more changes 9 months after surgery but the BCCA treated rats were more sensitive to 2 mg/kg apomorphine s.c., demonstrated by the distance travelled during 1 hour of habituation in a new environment. BCCA treated rats rotate in response to apomorphine after an intrastriatal and ventrolateral injection of 0.3 microgram FeCl3 15 and 14 weeks, respectively, after BCCA and iron instead of 1.5 micrograms after iron injection alone. Transient BCCA leads to a dramatical increase of released DA. We assume, therefore, that during the autooxidation of released DA, free radicals trigger the increase of lipid peroxidation. Predamaged tissue due to increased lipid peroxidation reacts to very small amounts of iron and iron seems to be more toxic in such cerebral tissue.

Effect of transient reduction of cerebral blood flow on membrane anisotropy and lipid peroxidation in different rat brain areas.

Light-microscopical studies revealed that oligemic hypoxia for 24 and 60 min as produced by bilateral clamping of the carotid arteries (BCCA) in normotension does not produce neuronal cell necrosis in the vast majority of rat brain. Less than 5% of cases showed a pattern of mild selective neuronal necrosis as would be expected in ischemia. However, significant changes in both lipid peroxidation (as measured by MDA formation) and membrane anisotropy (measured by DPH or TMA-DPH, respectively, as a fluorescence probe) in cortical and striatal, but not in hippocampal, membrane fractions could be measured in ex vivo studies. Twenty-four and 60 min of BCCA without reperfusion decreased lipid peroxidation in the cerebral cortex but not in the striatum. BCCA, either for 24 or 60 min, and 60 min of reperfusion produced no changes in lipid peroxidation in either structure. However, 24 and 60 min of BCCA followed by 14 days of reperfusion led to a significant increase in MDA formation in the striatum, while lipid peroxidation in the cortex was only increased after 60 min of BCCA. Cortical as well as striatal membrane anisotropy increased significantly 14 days later in rats submitted to BCCA for 24 or 60 min. The study shows an increased lipid peroxidation 2 weeks after a transient reduction in cerebral blood flow although no neuronal necrosis could be observed in general.

Levemopamil injection after cerebral oligemia reduces spatial memory deficits in rats.

Transient reduction of cerebral blood flow to oligemic levels as produced by bilateral clamping of carotid arteries (BCCA) in pentobarbital anesthetized Wistar rats leads to spatial orientation deficiencies in a water maze test 8-10 days after surgery. These deficiencies are more pronounced in 4-month-old than in 6-week-old animals. Levemopamil [(2S)-2-isopropyl-5-(methylphenethylamino)-2-phenylvaleronitril e hydrochloride], a Ca2+ channel blocker and 5-HT2 antagonist, prevents the deficiencies in groups of animals of both ages, even when administered 24 h after the transient vessel occlusion. Levemopamil did not influence the maze performance of sham-operated control rats. Levemopamil, therefore, appears to modulate mechanisms that are altered specifically either by, or as a consequence of, the BCCA procedure. Levemopamil did not influence the altered GABA or ACh content in different vulnerable brain structures following BCCA, showing that the substance acts via additional mechanisms affected by the BCCA procedure.

Influence of short-lasting bilateral clamping of carotid arteries (BCCA) on GABA turnover in rat brain structures.

We have previously shown that short-lasting reduction of cerebral blood flow by bilateral clamping of carotid arteries (BCCA) results in long-lasting increase in regional GABA concentration and decrease in seizure susceptibility in rats. In the present experiments, the effect of BCCA on GABA turnover and the enzymes involved in GABA synthesis and degradation were studied in rats. Regional GABA turnover was measured by means of GABA accumulation induced by the GABA-transaminase (GABA-T) inhibitor aminooxyacetic acid (AOAA). Fourteen days after BCCA, GABA turnover was significantly increased in hippocampus, substantia nigra and cortex, but not different from sham-operated controls in several other brain regions, including striatum, hypothalamus and cerebellum. The activity of glutamate decarboxylase (GAD) measured ex vivo did not show any changes in investigated structures, while the activity of GABA-T was slightly increased in hippocampus. The increased GABA turnover in some brain regions may explain our previous findings of increased GABA content in these brain regions and decreased sensitivity of BCCA treated animals to the GABAA-receptor antagonist bicuculline.

Metabolic changes during and after transient clamping of carotid arteries in normotensive rats.

In the present experiments changes of local metabolism in the striatum, hippocampus, and frontal cortex during and after transient clamping of carotid arteries in normotensive rats (BCCA) were studied by continuous measurement of local cerebral temperature, partial oxygen tension (PO2), and extracellular levels of lactate. Local temperature in the striatum, hippocampus, and frontal cortex fell between 1.7 and 2.3 degrees C upon occlusion and quickly returned to preocclusion values after free flow had been established. Local PO2 was reduced in the striatum, hippocampus, and frontal cortex to values between 70 and 30% during BCCA. Immediately after termination of BCCA the PO2 showed a tendency to recover in the striatum and frontal cortex, whereas in the hippocampus, this process started later. Extracellular levels of lactate within these three structures increased during BCCA and went down to preocclusion values within the observed period of reperfusion. The results suggest that BCCA induces a transient anaerobic metabolism that seems to be sufficient to evoke functional changes without neuronal damage.

Transient reduction of cerebral blood flow leads to longlasting increase in GABA content in vulnerable structures and decreased susceptibility to bicuculline induced seizures.

Rats were exposed for 24 min to bilateral clamping of the common carotid arteries (BCCA) in pentobarbital anaesthesia. The GABA content was measured 24 hours, 48 hours, 4 days, 14 days and 3 months after BCCA. In other groups of rats seizures were elicited by i.p. injection of (+)-bicuculline (3 mg/kg) 24 hours, 48 hours, 4 days, 14 days and 3 months after BCCA. Analysis of the GABA content revealed significant increase compared with controls in the hippocampus, frontal cortex and substantia nigra from 24 hours up to 3 months. Bicuculline treatment induced tonic/clonic seizures and status epilepticus in sham operated animals; these effects were drastically diminished at various time points after BCCA. The present results suggest that BCCA produces a longlasting increase in GABA content and as a consequence protection from bicuculline-induced seizures.

Quinolinate-like neurotoxicity produced by aminooxyacetic acid in rat striatum.

The endogenous tryptophan metabolite quinolinic acid elicits in rodent brain a pattern of neuronal degeneration which resembles that caused by L-glutamate. Its qualities as a neurotoxic agent raised the hypothesis that quinolinic acid might be involved in the pathogenesis of human neurodegenerative disorders. Kynurenic acid, another endogenous tryptophan metabolite and preferential N-methyl-D-aspartate (NMDA) antagonist, has been shown to block quinolinic acid neurotoxicity. Here we report that microinjections of aminooxyacetic acid (AOAA), an inhibitor of kynurenine transaminase and of other pyridoxal phosphate-dependent enzymes, into the rat striatum produce neuronal damage resembling that caused by quinolinic acid. AOAA-induced striatal lesions can be prevented by kynurenic acid and the selective NMDA antagonist 2-amino-7-phosphonoheptanoic acid. These results suggest that AOAA produces excitotoxic lesions by depleting brain concentrations of kynurenic acid (inhibition of synthetic enzyme) or due to impairment of intracellular energy metabolism (depletion of cell energy resources). The concept of deficient neuroprotection due to metabolic defects might help to clarify the pathogenesis of human neurodegenerative disorders and to develop strategies that may be useful in their treatment.

Aminooxyacetic acid produces excitotoxic lesions in the rat striatum.

The neuropathological, biochemical, and behavioral effects of intrastriatal injection of aminooxyacetic acid (AOAA), a non-specific transaminase inhibitor, were examined in rats. AOAA, 0.1-1 mumol, produced neuronal damage when injected into the striatum of adult rats but failed to damage the striatum of 6-d-old or decorticated rats. AOAA-induced (0.25 mumol-1 mumol) striatal lesions in adult rats displayed excitotoxic characteristics and could be prevented by the N-methyl-D-aspartate (NMDA) receptor antagonists (-)-2-amino-7-phosphono-heptanoate (AP7; 0.25 mumol) or kynurenate (KYNA; 0.5 mumol), but not by the non-NMDA antagonist 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline (NBQX; 0.25 mumol). AOAA produced a dose-dependent reduction in striatal L-glutamate decarboxylase activity, as measured 14 d following intrastriatal injection, which could also be prevented by AP7 or KYNA, but not by NBQX. These findings suggest that AOAA-induced lesions are preferentially mediated by activation of the NMDA subtype of excitatory amino acid receptors. Behavioral studies revealed that the cataleptic response to haloperidol, 2 mg/kg, was decreased whereas the cataleptic response to arecoline, 15 mg/kg, and morphine, 15 mg/kg, was potentiated in AOAA lesioned animals 14 d following bilateral intrastriatal injections of AOAA, 0.25 and 1 mumol. In rats which received unilateral intrastriatal injection of AOAA, 0.1-1 mumol, apomorphine, 0.5 mg/kg, induced circling towards the lesioned side. Rats which received AP7, 0.25 mumol, or KYNA, 0.5 mumol, coadministered with AOAA, 0.25 mumol, behaved as vehicle-treated controls, while those which received NBQX, 0.25 mumol, and AOAA, 0.25 mumol, had behavioral patterns similar to those subjected to AOAA alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of bilateral clamping of carotid arteries on hippocampal kindling in rats.

Moderate reduction of cerebral blood flow by bilateral clamping of carotid arteries (BCCA) in pentobarbital anaesthetized Wistar rats induces decreased PO2 and temperature values in vulnerable brain structures such as hippocampus and frontal cortex during the acute phase of clamping. Up to two weeks chronic increased GABA contents in hippocampus, substantia nigra and frontal cortex could be observed. During this time the development of hippocampal kindling was difficult. These results demonstrated an acute and chronic influence of GABA on most vulnerable brain structures which, however, do not lead to ischemic impacts as histological analyses demonstrate.

Decreased susceptibility to seizures induced by bicuculline after transient bilateral clamping of the carotid arteries in rats.

Rats were exposed for 24 min to bilateral clamping of the common carotid arteries (BCCA) in pentobarbital anaesthesia. 14 days later the animals were subjected to subcutaneous injection of (+)-bicuculline (3 or 4 mg/kg). A significantly decreased susceptibility to bicuculline-induced seizures could be observed in BCCA treated rats compared with sham operated controls. It is suggested that BCCA treatment protects animals against status epilepticus and lethal toxicity produced by bicuculline. Electrographic recordings of the BCCA animals revealed no ictal activity within 1 h after bicuculline injection. An analysis of the GABA content showed a significant increase in the hippocampus (HPC), frontal cortex (FCX), parietal cortex and substantia nigra in BCCA animals compared with controls. It is therefore possible that an increase in GABA content postsynaptically counteracts the GABAA antagonistic effect of bicuculline in BCCA animals thus preventing the normal seizure inducing effect of this substance.

Transient occlusion of rat carotid arteries increases formation of inositol phosphate. Evidence for a specific effect on ?(1)-receptors.

Rats which had both carotid arteries occluded (BCCA) for 24 min did not suffer histological damage to hippocampal tissue. However, BCCA treatment followed by transient normobaric hypoxia (reduced oxygen) for 30 min caused damage to the pyrimidal cells in the hippocampus. Intraventricular injection of [(3)H]inositol followed by BCCA + hypoxia treatment caused an increase in the release of [(3)H]inositol-mono-phosphate ([(3)H]Ins-1-P) and [(3)H]inositol-bis-phosphate ([(3)H]Ins-2-P) in both groups (BCCA and BCCA + hypoxia) but not in sham controls. These results suggest that BCCA or oxygen reduction stimulates phosphoinositide metabolism, causing a release of [(3)H]inositol triphosphate ([(3)H]Ins-3-P). Agonist-induced stimulation of inositol phosphates (InsPs) was also analysed in BCCA animals 3 and 14 days after their operations. Noradrenaline produced an increase in Ins-Ps in the hippocampus (but not in the frontal cortex) 14 days after BCCA. This effect did not occur for sham controls nor for carbachol and quisqualate stimulation. The results show a delayed effect of the ?(1)-receptors in the hippocampus of BCCA animals.

Some pharmacological properties and subacute and chronic toxicity of tryptamide.

Studies on Albino Swiss mice and Wistar rats have demonstrated that tryptamide is less ulcerogenic than phenylbutazone, and markedly inhibits intestinal peristalsis. Both compounds have a similar tendency to accumulate in the body. Tryptamide produces a smaller hypotension and stimulates the respiratory amplitude to a lesser extent than phenylbutazone in a vivisectional experiment. Studies on the subacute and chronic toxicity have demonstrated that tryptamide administered orally (po) and intraperitioneally (ip) for 3 weeks, and orally for 3 months neither affects the body weight gain or the mass of internal organs, nor changes the locomotor activity; only in rats it disturbs the motor coordination. After ip administration tryptamide shows a moderate depressant effect on the bone marrow, evidenced by a decline in the blood hemoglobin content and the number of erythrocytes and blood platelets. As those changes were more pronounced after a 3-week than a 3-month administration, the observed effects are apparently reversible.

Frontal decortication and adaptive changes in striatal cholinergic neurons in the rat.

Interruption of the corticostriatal pathway by undercutting the cortex resulted in a reduction of glutamate uptake by 55% and in a depression of acetylcholine (ACh) synthesis by 30% in striatum after two postlesion weeks without affecting the content of ACh and choline, the specific binding of [3H]dexetimide to muscarinic receptors, the activity of choline acetyltransferase and the levels of noradrenaline, serotonin, dopamine and 3,4-dihydroxyphenylacetic acid. The influence of this excitatory pathway on striatal cholinergic neuropharmacology was investigated. It was found that the effect of a number of agonists (R-apomorphine, bromocriptine, lisuride, quinpirole, JL-14389, 2-chloroadenosine, oxotremorine and methadone), capable of depressing cholinergic activity in the striatum through receptor-mediated responses--reflected as an increase in ACh content--is operative only when the corticostriatal pathway is intact. By contrast, antagonists capable of decreasing ACh content, i.e. the typical neuroleptics pimozide, haloperidol and the atypical ones clozapine, L-sulpiride, as well as the anti-muscarinic agent scopolamine, were not influenced by the lesion. The possibility that the lesion non-specifically damaged striatal cells on which the agonists, but not the antagonists acted was excluded by results showing, firstly, that the increase in striatal ACh elicited by the ACh precursor, choline, was not blocked by decortication, and secondly, that the degeneration of the corticostriatal neurons did not prevent the ACh-increasing effect of bromocriptine, a long-acting ergot alkaloid, when sufficient time was allowed for the drug to act. It was furthermore possible to restore the inhibitory action of apomorphine on cholinergic neurons either by short-term chemical lesion of the nigrostriatal dopaminergic input or by the administration of choline.(ABSTRACT TRUNCATED AT 250 WORDS)

The influence of phentolamine on the central activity of 5-hydroxytryptamine in rat.

PHENT (20 and 100 mug ivc) affects the central action of 5-HT (0-1, 25 and 250 mug) in rats which shows itself in potentiation of 5-HT-induced inhibition of spontaneous locomotor and exploratory activities and in the prolongation of hexobarbital sleeping time. In case of postamphetamine hyperactivity and body temperature, 5-HT acts antagonistically to a lower PHENT dose and synergistically to a higher dose of this compound. No affect of 5-HT on a slight antistereotypy action of PHENT has been stated. Neither compound affects electrical seizures.

Influence of fluorostigmine on some parameters of the catecholaminergic and serotoninergic systems in the mouse brain.

Fluorostigmine (DFP) injected i.p. in doses of 2-45 and 5-0 mg/kg in mice distinctly lowered levels of noradrenaline in the brain, raised levels of dopamine slightly, and very distinctly raised levels of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid. The same dose also weakened MAO activity and diminished oxygen uptake by brain tissue. Drugs which antagonized the above-mentioned changes in the levels of biogenic amine (L-DOPA, PCPA, reserpine) or blocked receptors (phentolamine) only slightly diminished acute toxicity of DFP. On the other hand, a-methyl-p-tyrosine, d,l-tryptophane and nialamide had virtually no effect on acute toxicity.