c-Src tyrosine kinase co-associates with and phosphorylates signal transducer and activator of transcription 5b which mediates the proliferation of normal human B lymphocytes.

c-Src is the normal human cellular protein homologue of the viral oncogene v-src. c-Src activity was reported recently to increase in CD40-activated human B lymphocytes, suggesting its involvement in proliferation. To elucidate the exact role of c-Src in this process, we investigated the effects of c-Src over-expression on normal B lymphocyte growth. B lymphocytes purified from human peripheral blood were infected with Ad5/F35 vector encoding either a constitutively active c-Src (c-Src/dominant-positive) or a dominant-negative c-Src (c-Src/DN). Little variation of B lymphocytes expansion could be observed between control enhanced yellow fluorescent protein and c-Src/dominant-positive-infected cells. In contrast, over-expression of c-Src/DN results in a 40% inhibition of B lymphocyte expansion. These results suggest that DN c-Src may compete with endogenous c-Src, resulting in partial inhibition of a transcriptional pathway involved in B lymphocyte proliferation. We demonstrate further that c-Src can phosphorylate signal transducer and activator of transcription 5b (STAT5b) on tyrosine 699 and that c-Src and STAT5b co-associate during B lymphocyte proliferation. These results confirm an important role for c-Src in the expansion of normal human B lymphocytes in vitro, in which c-Src may regulate STAT5b in the intracellular signalling pathway important for the proliferation of normal human B lymphocytes.

When the auditory cortex turns visual.

We studied visually guided behavior and the visual response properties of single auditory cortex (A1) neurons in neonatally operated hamsters with surgically induced, permanent, ectopic retinal projections to auditory thalamic nuclei and to visual thalamic nuclei which normally receive little direct retinal input. The surgically induced retino-thalamo-cortical pathways can mediate visual guided behaviors whose normal substrate, the pathway from the retina to the primary visual cortex via the primary thalamic visual nucleus, is missing. The visually evoked response properties of A1 neurons resemble in many respects those of neurons in V1 of normal hamsters: many A1 neurons have well-defined visual receptive fields and preferences for orientation or direction of movement. In addition, some visually responsive cells in A1 are bimodal--they also respond to auditory stimuli. The visually responsive neurons in A1 probably account for the capacity of the auditory cortex to mediate visual behavior in 'rewired hamsters'.

Airbags and children: a spectrum of C-spine injuries.

Over 30 children who were improperly restrained or in rear facing safety seats have been reported killed in motor vehicle accidents (MVA) involving airbags. The authors report one minor and two major injuries in properly restrained children in the front passenger seat. In case 1, A 10-year-old seat-belted boy was involved in an MVA (40 km/h) with deployment of both airbags. Physical examination findings showed right hyphema with corneal abrasion, right cheek abrasion and minimal cervical tenderness. C-spine x-ray was normal. He was treated for whiplash and facial burns resulting from contact with hot gas released by the airbags and discharged. In case 2, a 4-year-old boy wearing a lapbelt was in a MVA (20 km/h) with airbag deployment. On arrival, his Glasgow coma score was 3 and he was hemodynamically unstable. Secondary survey after stabilization showed left neck abrasions and ecchymoses, quadraplegia, priapism, and absent rectal tone. C-spine x-ray showed atlanto-occipital dislocation with possible complete spinal cord transection at C1. Aggressive maneuvers were withheld, and the patient was pronounced dead. Autopsy findings confirmed the clinical diagnosis. In case 3, a 3-year-old boy in a forward-facing safety seat was in a MVA (60 km/h) with air bag deployment. The patient was fully awake. C-spine x-rays were normal. Because of fluctuating level of consciousness, he underwent head computed tomography (CT) scan, which demonstrated a posterior fossa subarachnoid hemorrhage and a hematoma posterior to the odontoid, suggesting a ligamentous tear. He remained asymptomatic and was discharged on day 6. A head CT scan at 1 month showed a periosteal reaction in the area of the alar ligament suggestive of partial ligamentous avulsion; this injury was the forerunner of atlanto-occipital dislocation. Airbags deploy by releasing a hot effluent at 300 km/h. Mechanisms of injury include direct contact of hot gas with facial skin and energy transmitted directly from the airbag system to the child's head and neck. These cases illustrate a spectrum of C-spine injuries caused by airbag deployment and support the recommendation that children under 12 years of age travelling in a car equipped with dual airbags be seated in the back.

Flumazenil in cirrhotic patients in hepatic coma: a randomized double-blind placebo-controlled crossover trial.

Previous reports have suggested that "endogenous" benzodiazepines could contribute to neural inhibition in hepatic encephalopathy. RO 15-1788 (flumazenil), a specific antagonist of brain benzodiazepine receptors, could thus reverse the neurological symptoms of hepatic encephalopathy. To test this possibility, we conducted a double-blind, placebo-controlled crossover trial of the efficacy of flumazenil in cirrhotic patients in hepatic coma. Seventy-seven cirrhotic patients in hepatic coma were evaluated. Fifty-six were excluded from the trial because of multiorgan failure or because coma was precipitated by prior use of benzodiazepines, and 21 patients were randomly assigned to the flumazenil group (11 patients) or the placebo group (10 patients). Treatment was administered intravenously as a 20-ml solution (placebo or 2 mg flumazenil); seven patients were crossed over. Clinical status was assessed blindly by two observers, using a modified Glasgow scale, every 15 min for 6 hr. Electroencephalogram tracings obtained before and after drug administration were evaluated blindly by two independent observers. Serum concentrations of benzodiazepines before treatment were measured by means of a fluorescence polarization immunoassay. Improvement in neurological symptoms was observed in six patients treated with flumazenil, whereas none in the placebo group showed improvement (p < 0.05; Fisher's exact test). Improvements in electroencephalogram tracings were demonstrated in four patients treated with flumazenil, compared with two patients in the placebo group (p = NS). Benzodiazepines were found in the serum of four patients treated with flumazenil (two responders and two nonresponders); all of these patients had received pharmaceutical benzodiazepines 4 to 6 days before the trial.(ABSTRACT TRUNCATED AT 250 WORDS)

Region-selective reductions in activities of glutamine synthetase in rat brain following portacaval anastomosis.

Portacaval anastomosis in the rat results in liver atrophy, sustained hyperammonemia and mild encephalopathy. Previous studies have demonstrated region-selective alterations of glutamine and other ammonia-related amino acids in brain following portacaval anastomosis. Ammonia removal by brain relies on glutamine synthesis and the enzyme responsible, glutamine synthetase, has an almost exclusively astrocytic localization. Glutamine synthetase activities were measured using a radioenzymatic assay in homogenates of seven brain regions of rats four weeks after end-to-side portacaval anastomosis. Enzyme activities were significantly reduced in hippocampus (by 25%, p < 0.01), in cerebellum (by 29%, p < 0.01) and in cerebral cortex (by 14%, p < 0.05). Enzyme activities in other brain regions were within normal limits. Region-selective reductions of glutamine synthetase following portacaval anastomosis could result in disruption of neuron-glial metabolic interactions and in a deficit of glutamatergic synaptic regulation. Similar mechanisms could be implicated in the pathogenesis of hepatic encephalopathy accompanying chronic liver disease in humans.

Increased activities of MAOA and MAOB in autopsied brain tissue from cirrhotic patients with hepatic encephalopathy.

Using radioenzymatic assays, activities of MAOA and MAOB were measured in autopsied brain tissue from cirrhotic patients who died in hepatic coma and in material from an equal number of age-matched subjects who were free from hepatic, neurological or psychiatric disorders. Activities of both MAOA and MAOB were significantly increased in frontal cortex and caudate nucleus, two brain regions shown previously to be the site of functional and morphological alterations of astrocytes and increased concentrations of the acid metabolites of dopamine and serotonin. These findings suggest that increased monoamine metabolism and subsequent modifications of monoaminergic synaptic function could contribute to the pathogenesis of hepatic encephalopathy.

Cerebral amino acid changes in an animal model of intrauterine growth retardation.

As part of a series of experiments to ascertain the effects of prenatal malnutrition on brain development, we measured brain amino acids in an animal model of intrauterine growth retardation (IUGR) obtained by restriction of blood supply to the fetus in utero during the last 5 days of gestation. In the present study, amino acids were measured during development by HPLC as their O-phthaldialdehyde derivatives in cerebral cortex, cerebellum and hippocampus. In rats with IUGR, significant increase of alanine (by 20% to 50%) and taurine (by 20% to 80%) were observed prior to weaning in the cerebellum and the cerebral cortex respectively. Alanine levels were also increased in hippocampus. In control animals, at birth, activities of the GABA nerve terminal marker enzyme glutamic acid decarboxylase (GAD) were found to be 32%, 17%, and 11% of adult values in cerebellum, hippocampus and cerebral cortex respectively. Two-day-old rats with IUGR had significantly lower GAD activities in all brain regions. Thus, impairment of nutrient supply to fetal brain results in selective regional abnormalities of amino acids particularly in the cerebral cortex.

Increased densities of binding sites for the 'peripheral-type' benzodiazepine receptor ligand [3H]PK 11195 in rat brain following portacaval anastomosis.

Using quantitative receptor radioautography, binding sites for the 'peripheral-type' benzodiazepine receptor ligand [3H]PK 11195 were studied in rats 4 week after end-to-side portacaval anastomosis and in sham-operated controls. Portacaval anastomosis resulted in region-selective increases in density of [3H]PK 11195 binding sites in cerebellum, pons greater than thalamus, cerebral cortex greater than hippocampus greater than striatum. Possible mechanisms implicated in these changes include (i) the action of endogenous ligands for the mitochondrial benzodiazepine receptor such as octadecaneuropeptide and (ii) neurotoxic actions of ammonia. In view of the proposed role of these receptors as modulators of intermediary metabolism and neurosteroid biosynthesis, such changes could contribute to the neurochemical mechanisms responsible for portal-systemic encephalopathy.

Increased cerebrospinal fluid lactate reflects deterioration of neurological status in experimental portal-systemic encephalopathy.

Increased brain and CSF lactate have been described in human and experimental portal-systemic encephalopathy (PSE). Using a recently described cisterna magna catheter technique, CSF lactate was measured in relation to deterioration of neurological status in portacaval shunted rats administered ammonium acetate to precipitate severe PSE. Loss of righting reflex (precoma stage of PSE) was accompanied by 2-3 fold increased CSF lactate and onset of coma by 4-fold increases of lactate (p less than 0.001 compared to either sodium acetate treated portacaval shunted rats or sham-operated controls administered ammonium acetate). The most likely explanation for increased CSF lactate is ammonia-induced inhibition of malate-aspartate shuttle and/or inhibition of tricarboxylic acid cycle flux in brain. Similar mechanisms could be involved in the pathogenesis of PSE in patients with chronic liver disease.

Increased brain content of the endogenous benzodiazepine receptor ligand, octadecaneuropeptide (ODN), following portacaval anastomosis in the rat.

Evidence suggests that endogenous benzodiazepine receptor ligands such as diazepam binding inhibitor (DBI) and its metabolite octadecaneuropeptide (ODN) may be implicated in the pathogenesis of hepatic encephalopathy. Using an immunocytochemical technique and an antibody of high specific activity to synthetic ODN, we studied the effects of portacaval anastomosis (PCA) on ODN distribution in rat brain. Four weeks after PCA, ODN immunolabeling was increased in several brain regions including cerebral cortex, hippocampus, hypothalamus and thalamus. Increased ODN immunolabeling was confined to nonneuronal elements such as astrocytes and ependymal cells. Neuropathological evaluation of brain following PCA reveals astrocytic rather than neuronal changes. These results are consistent with a role for endogenous neuropeptide ligands for astrocytic benzodiazepine receptors in the pathogenesis of hepatic encephalopathy.

Selective loss of N-methyl-D-aspartate-sensitive L-[3H]glutamate binding sites in rat brain following portacaval anastomosis.

Excitatory amino acids have been implicated in the pathogenesis of hepatic encephalopathy. In the present study, kainate, quisqualate and N-methyl-D-aspartate (NMDA) subclasses of L-glutamate receptors were measured in adult rat brain by quantitative receptor autoradiography following surgical construction of an end-to-side portacaval anastomosis (PCA). PCA resulted in sustained hyperammonemia and decreased binding of L-glutamate to the NMDA receptor when compared to sham-operated controls. Decreases in binding ranged from 17 to 39% in several regions of cerebral cortex, hippocampus, striatum, and thalamus. Binding to quisqualate and kainate receptor subtypes was not altered. PCA leads to astrocytic changes in brain but does not result in any measurable loss of neuronal integrity. It is therefore proposed that decreased glutamate binding to the NMDA receptor following PCA results from increased extracellular glutamate caused by decreased reuptake into perineuronal astrocytes and a compensatory down-regulation of these receptors. Such changes could be of pathophysiological significance in hepatic encephalopathy.

Pharmacokinetics of benzodiazepine antagonist Ro 15-1788 in cirrhotic patients with moderate or severe liver dysfunction.

Ro 15-1788, a benzodiazepine antagonist, has been advocated as a new treatment for hepatic encephalopathy. This drug is extensively metabolized by the liver in normal subjects. In the present study, we examined Ro 15-1788 disposition in eight healthy controls (Group I), eight cirrhotic patients with moderately impaired liver function (Pugh score less than 10, Group II) and eight patients with severe liver dysfunction (Pugh score greater than 10, Group III). The subjects of each group were age and sex matched. After an intravenous infusion of 2 mg Ro 15-1788 over 5 min, blood samples were taken at fixed intervals up to 7 hr after the infusion. Plasma levels of the drug were determined by capillary gas chromatography. In controls, Ro 15-1788 had a high plasma clearance [16.3 +/- 2.6 ml per min per kg (mean +/- S.D.)], a short half-life (45.7 +/- 8.5 min), a large volume of distribution (0.62 +/- 0.09 liter per kg) and a low plasma protein binding (45 +/- 6%). Plasma clearance was reduced markedly in both groups of cirrhotic patients (-57 and -74%, respectively); the volume of distribution was unchanged in Group II and moderately increased in Group III (+37%). The elimination half-life was markedly prolonged in Groups II and III (+66 and +210%, respectively). Plasma clearance and Pugh score were highly correlated in cirrhotic patients (r = 0.830, p less than 0.001). The plasma protein binding of Ro 15-1788 was lower in cirrhotics, resulting in a significant increase in the free fraction of the drug (+16% in Group II; +44% in Group III).(ABSTRACT TRUNCATED AT 250 WORDS)

Affinities and densities of high-affinity [3H]muscimol (GABA-A) binding sites and of central benzodiazepine receptors are unchanged in autopsied brain tissue from cirrhotic patients with hepatic encephalopathy.

The integrity of GABA-A receptors and of central benzodiazepine receptors was evaluated in membrane preparations from prefrontal cortex and caudate nuclei obtained at autopsy from nine cirrhotic patients who died in hepatic coma and an equal number of age-matched control subjects. Histopathological studies revealed Alzheimer Type II astrocytosis in all cases in the cirrhotic group; controls were free from neurological, psychiatric or hepatic diseases. Binding to GABA-A receptors was studied using [3H]muscimol as radioligand. The integrity of central benzodiazepine receptors was evaluated using [3H]flunitrazepam and [3H]Ro15-1788. Data from saturation binding assays was analyzed by Scatchard plot. No modifications of either affinities (Kd) or densities (Bmax) of [3H]muscimol of central benzodiazepine binding sites were observed. These findings do not support recent suggestions that alterations of either high-affinity GABA or benzodiazepine receptors play a significant role in the pathogenesis of hepatic encephalopathy.

Regional differences in the capacity for ammonia removal by brain following portocaval anastomosis.

Portocaval anastomosis (PCA) in the rat leads, within 4 weeks, to severe liver atrophy, sustained hyperammonemia, and increased brain ammonia. Because brain is not equipped with an effective urea cycle, removal of ammonia involves glutamine synthesis and PCA results in significantly increased brain glutamine. Glutamine synthetase activities, however, are decreased by 15% in cerebral cortex and are unchanged in brainstem of shunted rats. Administration of ammonium acetate to rats following PCA results in severe encephalopathy (loss of righting reflex and, ultimately, coma). Glutamine concentrations in brainstem of comatose rats are increased a further two-fold, whereas those of cerebral cortex are unchanged. Consequently, ammonia levels in cerebral cortex reach disproportionately high levels (of the order of 5 mM). These findings suggest a limitation in the capacity of cerebral cortex to remove additional blood-borne ammonia by glutamine formation following PCA. Such mechanisms may explain the hypersensitivity of rats with PCA and of patients with portal-systemic shunting to small increases of blood ammonia. Disproportionately high levels of brain ammonia in certain regions, such as cerebral cortex, may then result in alterations of inhibitory neurotransmission and, ultimately, loss of cellular (astrocytic) integrity.

Activities of neuronal and astrocytic marker enzymes in autopsied brain tissue from patients with hepatic encephalopathy.

Activities of the gamma-aminobutyric acid (GABA) and cholinergic nerve-terminal marker enzymes glutamic acid decarboxylase (GAD) and choline acetyltransferase (CAT) as well as the astrocytic enzyme glutamine synthetase (GS) were measured in homogenates of dissected brain tissue obtained at autopsy from nine cirrhotic patients dying in hepatic encephalopathy and an equal number of control subjects matched for age, agonal status, and time interval from death to freezing of autopsied material. GAD activities varied as a function of agonal status in control samples, confirming a previous report, but were unchanged in brain tissue from cirrhotic patients, suggesting no loss of integrity of presynaptic GABA nerve terminals in this disease. On the other hand, GS activities were selectively decreased by 25% (P less than 0.01) in caudate nuclei of cirrhotic patients, reflecting, no doubt, the severe astrocytosis consistently observed in this brain structure. CAT activities, expressed per milligram of protein, were found to be increased by 30% (P less than 0.01) in the prefrontal cortex of cirrhotic patients. Whether such changes result from a relative increase in CAT as a consequence of losses of astrocytic protein or reflect altered cholinergic function in hepatic encephalopathy associated with chronic liver disease awaits further study.

Amino acid changes in autopsied brain tissue from cirrhotic patients with hepatic encephalopathy.

Brain tissue was obtained at autopsy from nine cirrhotic patients dying in hepatic coma and from an equal number of controls, free from neurological, psychiatric, or hepatic diseases, matched for age and time interval from death to freezing of dissected brain samples. Glutamine, glutamate, aspartate, and gamma-aminobutyric acid (GABA) levels were measured in homogenates of cerebral cortex (prefrontal and frontal), caudate nuclei, hypothalamus, cerebellum (cortex and vermis), and medulla oblongata as their o-phthalaldehyde derivatives by HPLC using fluorescence detection. Glutamine concentrations were found to be elevated two- to fourfold in all brain structures, the largest increases being observed in prefrontal cortex and medulla oblongata. Glutamate levels were selectively decreased in prefrontal cortex (by 20%), caudate nuclei (by 27%), and cerebellar vermis (by 17%) from cirrhotic patients. On the other hand, GABA content of autopsied brain tissue from these patients was found to be within normal limits in all brain structures. It is suggested that such region-selective reductions of glutamate may reflect loss of the amino acid from the releasable (neurotransmitter) pool. These findings may be of significance in the pathogenesis of hepatic encephalopathy resulting from chronic liver disease.

Activities of thiamine-dependent enzymes in two experimental models of thiamine deficiency encephalopathy: 3. Transketolase.

Chronic thiamine deprivation in the rat leads to ataxia, loss of righting reflex and neuropathological damage to lateral vestibular nucleus. Before onset of neurological symptoms, transketolase (TK) activities were found to be selectively reduced by 25% in lateral vestibular nucleus and surrounding pons. Further progression of thiamine deprivation resulted in a generalized reduction in TK activity. Measurement of enzyme activity in the presence of added TPP cofactor in vitro did not lead to normalisation of enzyme activities suggesting loss of apoenzyme. Administration of thiamine to symptomatic thiamine-deprived rats resulted in reversal of neurological symptoms and to normalisation of defective TK activities in less vulnerable structures such as cerebral cortex, striatum and hippocampus; reduction of TK activity, however, persisted in brainstem and cerebellar regions. Pyrithiamine treatment results, within 3 weeks, in loss of righting reflex, convulsions and more widespread neuropathological damage compared to that observed following thiamine deprivation. TK activity was found to be significantly decreased before the onset of neurological symptoms in all brain regions and appearance of symptoms was accompanied by more severe reductions of TK. In contrast to chronic thiamine deprivation, TK activities following pyrithiamine treatment were: equally reduced in magnitude in vulnerable and non-vulnerable brain structures, unchanged following reversal of neurological abnormalities by thiamine administration.

Ammonia: key factor in the pathogenesis of hepatic encephalopathy.

There is substantial clinical and experimental evidence to suggest that ammonia toxicity is a major factor in the pathogenesis of hepatic encephalopathy associated with subacute and chronic liver disease. Ammonia levels in patients with severe liver disease are frequently found to be elevated both in blood and cerebrospinal fluid (csf). Hepatic encephalopathy results in neuropathological damage of a similar nature (Alzheimer type II astrocytosis) to that found in patients with congenital hyperammonemia resulting from inherited defects of urea cycle enzymes. Following portocaval anastomosis in the rat, blood ammonia concentration is increased 2-fold, and brain ammonia is found to be increased 2-3-fold. Administration of ammonia salts or resins to rats with a portocaval anastomosis results in coma and in Alzheimer type II astrocytosis. Since the CNS is devoid of effective urea cycle activity, ammonia removal by brain relies on glutamine formation. Cerebrospinal fluid and brain glutamine are found to be significantly elevated in cirrhotic patients with encephalopathy and in rats following portocaval anastomosis. In both cases, glutamine is found to be elevated in a region-dependent manner. Several mechanisms have been proposed to explain the neurotoxic action of ammonia. Such mechanisms include: Modification of blood-brain barrier transport; alterations of cerebral energy metabolism; direct actions on the neuronal membrane; and decreased synthesis of releasable glutamate, resulting in impaired glutamatergic neurotransmission.

Cerebral GABA-ergic and glutamatergic function in hepatic encephalopathy.

Measurement of amino acids in brain tissue obtained at autopsy from cirrhotic patients dying in hepatic coma revealed a threefold increase in glutamine and a concomitant decrease in brain glutamate. The GABA levels were found to be unaltered. Studies using an animal model of portal-systemic encephalopathy gave similar results. Glutamic acid decarboxylase (GAD) activities were within normal limits, both in the brains of cirrhotic patients and portocaval-shunted rats. A previous study reported normal [3H]GABA binding to synaptic membrane preparations from cerebral cortex in these animals. Taken together, these findings suggest that cerebral GABA function is not impaired in hepatic encephalopathy associated with chronic liver disease and portal-systemic shunting. On the other hand, there is evidence to suggest that the releasable pool of glutamate may be depleted in brain in hepatic encephalopathy. Data consistent with this hypothesis include: Reduction in the evoked release of endogenous glutamate by superfusion of hippocampal slices with pathophysiological levels of ammonia; ammonia-induced reduction of glutamatergic neurotransmission; and an increase in the number of [3H]glutamate binding sites in synaptic membrane preparations from hyperammonemia rats and from rats with portocaval shunts. Such neurochemical changes may be of pathophysiological significance in hepatic encephalopathy.

Cerebral aminoacids in portal-systemic encephalopathy: lack of evidence for altered gamma-aminobutyric acid (GABA) function.

Construction of an end-to-side portocaval anastomosis in the rat resulted, 4 weeks later, in sustained hyperammonemia and two- to threefold increases in brain ammonia. Measurement of cerebral amino acids using a sensitive double-isotope dansyl microtechnique revealed substantial increases in the glutamine content of cerebral cortex and brain stem. Glutamate levels were found to be concomitantly reduced in both brain regions compared to those of sham-operated controls. The gamma-aminobutyric acid (GABA) content of cerebral cortex and brain stem was unaffected by portocaval shunting, as were activities of the GABA nerve-terminal marker enzyme glutamic acid decarboxylase (GAD). These findings suggest that impaired GABA function may not play a major role in the pathogenesis of hepatic encephalopathy associated with portocaval shunts. Preliminary evidence suggests that decreased cerebral glutamate may reflect its loss from the releasable (neurotransmitter) pool.