Differential age-related changes of MAO-A and MAO-B in mouse brain and peripheral organs.

Distribution and age-related changes of MAO in BL/C57 mouse were studied by quantitative enzyme radioautography with [3H]Ro41-1049 and [3H]Ro19-6327. In the brain, MAO-A was highest in locus coeruleus and interpeduncular nucleus, and MAO-B in raphe nuclei, paraventricular thalamic nucleus, and ependyma of ventricles. Extremely high MAO-B levels were also measured in the choroid plexus in contrast to the very low MAO-B levels in rat choroid plexus. With aging, brain MAO-A showed a clear decrease between 4 and 9 weeks, followed by no change between 9 weeks and 19 months, and a slight increase between 19 and 25 months. On the other hand, all brain structures showed age-related increases in MAO-B. Peripheral organs showed different patterns of MAO age-related changes. Particularly interesting was the marked MAO-B increase in heart, parallel to the MAO-A increase in rat heart. Also of interest is the decrease of liver MAO-B in old animals, which, together with the increase of MAO-B in the brain, might underlie the high sensitivity of old BL/C57 mice to MPTP.

Biphasic and region-specific MAO-B response to aging in normal human brain.

Variations of monoamine oxidases (MAO) A and B were studied during aging in 27 human subjects (age range 17-93 years) in 18 brain structures of temporal cortex, frontal gyrus, hippocampal formation, striatum, cerebellum, and brainstem. [3H]Ro41-1049 and [3H]lazabemide were used as selective radioligands to image and quantify MAO-A and MAO-B respectively by enzyme autoradiography. Postmortem delay or time of tissue storage did not affect MAO-A or MAO-B levels. There was, moreover, no evidence of sexual dimorphism. A marked age-related increase in MAO-B was observed in most structures. This increase started at the age of 50-60 years. Before this age, MAO-B levels were constant in all structures studied. MAO-B-rich senile plaques were observed in some cortical areas but they did not significantly influence the age-related MAO-B increase. Surprisingly, no age-related MAO-B changes were observed in the substantia nigra. In contrast to MAO-B, no clear age-related changes in MAO-A were observed, indicating an independent regulation of the two isoenzymes, also suggested by the cross-correlation analysis of these data.

PU.1 expression in microglia.

The transcription factor PU.1 has a pivotal role in both the generation and function of macrophages. To determine whether PU.1 is also involved in microglial regulation, we investigated its expression following hypoxic-ischemia (HI) brain injury and in the BV-2 microglial cell line. We found that microglia constitutively expressed high levels of PU.1 protein in both their 'resting' and 'activated' states.

Neuronal injury-induced expression and release of apolipoprotein E in mixed neuron/glia co-cultures: nuclear factor kappaB inhibitors reduce basal and lesion-induced secretion of apolipoprotein E.

In order to better delineate the intracellular signaling pathways underlying glial apolipoprotein E (apoE) expression and release, we have characterized an in vitro model of induction of glial apoE production induced by neuronal death. Exposure of mixed fetal cortical neuron/glia co-cultures to the neurotoxin N-methyl-D-aspartate results in increased apoE expression and release in a time- and concentration-dependent manner. Increased expression of apoE messenger RNA precedes the increase in intracellular apoE, followed by accumulation of the holoprotein in the culture medium. Neuronal injury induced by N-methyl-D-aspartate is accompanied by a reactive astrogliosis as measured by an increase in glial fibrillary acidic protein messenger RNA and protein at 48 and 72h post-lesion, respectively. A similar microgliosis was observed using the microglial marker ED-1. Neuronal injury-induced glial apoE secretion is attenuated by the nuclear factor kappaB inhibitors, aspirin, Bay 11-7082 and MG-132, suggesting that this transcription factor is involved in both constitutive and induced glial apoE expression. The present data show that up-regulation of apoE is an early event in the glial activation triggered by neurodegeneration in vitro and that activation of nuclear factor kappaB directly or indirectly mediates the increase in apoE expression.

Long-term effects of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate and 6-nitro-7-sulphamoylbenzo(f)quinoxaline-2,3-dione in the rat basal ganglia: calcification, changes in glutamate receptors and glial reactions.

Previous data from our laboratory indicate that 25 mM ibotenic acid induces intracellular calcifications in the rat basal forebrain. Because of the lack of specificity of ibotenic acid for a glutamate receptor subtype, a dose-response study with alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate was undertaken and calcified areas (identified with Alizarin Red staining) as well as astro- and microglial reactions (by autoradiography with [3H]lazabemide and [3H]Ro 5-4864) were quantified at one month post-lesion. alpha-Amino-3-hydroxy-5-methyl-4-isoxazole propionate administered into the globus pallidus induced, in a dose-dependent manner, the formation of calcium deposits and the activation of both glial cells, the microglial reaction being particularly robust. From this study, a dose of 5.4 mM alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate was selected for further experiments. [3H]alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate, [3H]dizocilpine maleate and [3H]PN 200-110 binding in vitro were performed to assess autoradiographically whether the tissue damage was associated with changes in glutamate receptors and calcium channel binding sites. In the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate-treated animals, the specific binding of [3H]alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate was significantly reduced by 28% in the lesioned ventral pallidum, whereas it was unchanged in the globus pallidus and substantia innominata. In these three nuclei, calcifications developed and an increase in both glial markers was measured. In contrast, the binding of [3H]PN 200-110 and [3H]dizocilpine maleate were unaffected. Co-injection of 15 mM 6-nitro-7-sulphamoylbenzo(f)quinoxaline-2,3-dione, a selective alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate/kainate receptor antagonist, prevented the formation of calcium concretions, the microglial reaction and the decrease in [3H]alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate binding but it failed to inhibit totally the astroglial reaction induced by alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate. This may suggest that the microglial reaction and calcification take place through different mechanisms from the astrogliosis associated with the neuronal loss. In conclusion, acute administration of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate in the rat globus pallidus elicits a dose-dependent calcification process associated with a chronic reaction of astrocytes and microglia. alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate-induced injury is accompanied by a slight reduction of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors in the ventral pallidum, whereas the binding of N-methyl-D-aspartate and L-type calcium channels receptors remains unchanged in any lesioned nucleus.

Increased monoamine oxidase B activity in plaque-associated astrocytes of Alzheimer brains revealed by quantitative enzyme radioautography.

The aetiology and pathogenesis of Alzheimer's disease are currently poorly understood, but symptomatic disease is associated with amyloid plaques, neurofibrillary tangles, neuronal loss and numerous alterations of neurotransmitter systems in the CNS. Monoamine oxidase type B is known to be increased in Alzheimer diseased brains. The distribution and abundance of catalytic sites for monoamine oxidases A and B in post mortem human brains of 11 Alzheimer disease cases and five age-matched controls were investigated by quantitative enzyme radioautography. Using tritiated monoamine oxidase inhibitors (Ro41-1049 and lazabemide)--as high affinity substrates selective for monoamine oxidases A and B, respectively--it was found that monoamine oxidase B activity increased up to three-fold exclusively in temporal, parietal and frontal cortices of Alzheimer disease cases compared with controls. This increase was restricted to discrete patches (approximately 185 microns in diameter) which occupied approximately 12% of the cortical areas examined. In other brain regions (hippocampal formation >> caudate-putamen > cerebellum), patches of [3H]lazabemide-enriched binding were less abundant. [3H]Ro41-1049 binding (i.e. monoamine oxidase A) was unchanged in all tissues of diseased versus control brains. The monoamine oxidase B-enriched patches in all cortical regions correlated, in their distribution and frequency, with glial fibrillary acidic protein-immunoreactive clusters of astrocytes. Diffuse and mature beta-amyloid-immunoreactive senile plaques as well as patches of high density binding of [3H]PK-11195--a high-affinity ligand for peripheral-type (mitochondrial) benzodiazepine binding sites in microglia/macrophages--were found throughout Alzheimer diseased cortices. The up-regulation of monoamine oxidase B in plaque-associated astrocytes in Alzheimer's disease--in analogy to its proposed role in neurodegenerative disorders such as Parkinson's disease--might, indirectly, be a potential source of cytotoxic free radicals. Lazabemide, a selective reversible monoamine oxidase B inhibitor, is currently under clinical evaluation for the treatment of Parkinson's and Alzheimer's diseases. We conclude that enzyme radioautography with [3H]lazabemide is a reliable high resolution assay for plaque-associated astroglioses in Alzheimer's disease. Its clinical diagnostic utility for positron emission tomography or single photon emission computer tomography studies is being investigated.

Molecular neuroanatomy of human monoamine oxidases A and B revealed by quantitative enzyme radioautography and in situ hybridization histochemistry.

Monoamine oxidases are key enzymes in the metabolism of amine neurotransmitters and neuromodulators and are targets for drug therapy in depression, Parkinson's and Alzheimer's diseases. Knowledge of their distribution in the brain is essential to understand their physiological role. To study the regional distribution and abundance of monoamine oxidases A and B in human brain, pituitary and superior cervical ganglion, we used quantitative enzyme radioautography with radioligands [3H]Ro41-1049 and [3H]lazabemide, respectively. Furthermore, 35S-labelled oligonucleotides complementary to isoenzyme messengerRNAs were used to map the cellular location of the respective transcripts in adjacent sections by in situ hybridization histochemistry. A markedly different pattern of distribution of the isoenzymes was observed. Highest levels of monoamine oxidase A were measured in the superior cervical ganglion, locus coeruleus, interpeduncular nucleus and ventromedial hypothalamic nucleus. The corresponding messengerRNA was detected only in the noradrenergic neurons of the superior cervical ganglion and locus coeruleus. In contrast to rat brain, monoamine oxidase B was much more abundant in most human brain regions investigated. Highest levels were measured in the ependyma of ventricles, stria terminalis and in individual hypothalamic neurons. Monoamine oxidase B transcripts were detected in serotoninergic raphe neurons, histaminergic hypothalamic neurons and in dentate gyrus granule cells of the hippocampal formation. We conclude that [3H]Ro41-1049 and [3H]azabemide are extremely useful radioligands for high-resolution analyses of the abundance and distribution of catalytic sites of monoamine oxidases A and B, respectively, in human brain sections. From levels of messenger RNA detected, the cellular sites of synthesis of the isoenzymes are the noradrenergic neurons of the locus coeruleus (for monoamine oxidase A) and the serotoninergic and histaminergic neurons of the raphe and posterior hypothalamus, respectively (for monoamine oxidase B). The combination of quantitative enzyme radioautography with in situ hybridization histochemistry is a useful approach to study, with high resolution, both the physiology and pathophysiology of monoamine oxidases in human brain.

Localization of monoamine oxidase A and B in human pancreas, thyroid, and adrenal glands.

We studied monoamine oxidase (MAO) A and B localization in human pancreas, thyroid gland, and adrenal gland by immunohistochemistry. The primary antibodies used were mouse monoclonal anti-human MAO-A (6G11/E1) and anti-human MAO-B (3F12/G10/2E3). Samples were obtained from six routine autopsy cases and fixed in 2% paraformaldehyde. Exocrine pancreas showed a widespread distribution of MAO-A, whereas MAO-B was present only in centroacinar cells and epithelial cells of pancreatic ducts. In endocrine pancreas, MAO-A was observed in around 50% of islet cells, whereas MAO-B was less abundant and was restricted to the periphery of islets. Thyroid gland showed strong MAO-A immunoreactivity in all cell types and was MAO-B-negative. In adrenal gland, the capsule displayed MAO-A but not MAO-B immunoreactivity, whereas the cortex showed widespread MAO-A staining but was MAO-B-negative in interstitial cells. Finally, in the medulla only a few scattered cells showed either MAO-A or MAO-B immunoreactivity. To our knowledge, these data represent the first study of the cellular distribution of MAO-A and MAO-B in the three human tissues included.

Etoposide-induced PC12 cell death: apoptotic morphology without oligonucleosomal DNA fragmentation or dependency upon de novo protein synthesis.

Etoposide, a topoisomerase II inhibitor used in cancer therapy, has been shown to induce apoptosis in vitro in a variety of cell types. In the present study, we have characterized the effects of etoposide on undifferentiated rat pheochromocytoma PC12 cells. Etoposide killed PC12 cells in a time- and concentration-dependent manner. 20-24 h incubation with 10 micrograms/ml etoposide induced 25-50% cell death. Hoechst 33258 staining revealed apoptotic morphology in dying cells. No evidence was found of either oligonucleosomal DNA fragmentation, as shown by agarose gel electrophoresis, or endonuclease involvement, as shown by the inability of aurintricarboxylic acid to prevent cell death. Cycloheximide and actinomycin-D were unable to prevent etoposide cytotoxicity indicating that the process is not dependent upon de novo protein or mRNA synthesis. NGF (5 ng/ml) prevented etoposide-induced PC12 cell death. These results offer an example of how the morphological features of apoptosis are not necessarily associated with oligonucleosomal DNA fragmentation or with de novo macromolecule synthesis.

CCAAT-enhancer binding protein alpha is expressed in activated microglial cells after brain injury.

Microglial cells play important roles in brain injury and repair and are implicated in diseases such as Alzheimer's disease, Creutzfeldt-Jacob disease, multiple sclerosis, the Aids Dementia Complex and stroke. Despite their importance in neuropathology, the underlying molecular basis for the activation of microglia after brain injury is not understood. We show, using RT-PCR, in situ hybridisation, immunocytochemistry, and electrophoretic mobility shift assay, that the CCAAT-enhancer binding protein alpha (C/EBP alpha), a sequence specific DNA-binding protein, is induced in microglial cells, but not astrocytes or neurons, after hypoxic-ischemic brain injury. These results suggest that C/EBP alpha might regulate gene expression and consequentially have a role in the activation and/or proliferation of microglia following brain injury.

Molecular neuroanatomy of monoamine oxidases in human brainstem.

Specific, high-resolution techniques (quantitative enzyme radioautography and in situ hybridisation histochemistry) have revealed distribution, abundance and cellular localization of the isoenzymes MAO-A and MAO-B and their mRNAs in human post-mortem brainstem. Whereas MAO-A protein and mRNA are expressed by noradrenergic neurons of the locus coeruleus, MAO-B protein and mRNA are expressed by serotoninergic neurons of the raphé nuclei. In the substantia nigra, MAO-B was more abundant than MAO-A; the former was localized in the reticular zone and the latter in the compact zone (where melanin-containing dopaminergic neurons are found). To date, it has not been possible to detect mRNA for either MAO-A or MAO-B in the substantia nigra or in glial cells of the brain regions investigated, suggesting either that the technique has limited sensitivity, or the possible existence of MAO-A and MAO-B subtypes.

Neuroprotective effects of Gly-Pro-Glu, the N-terminal tripeptide of IGF-1, in the hippocampus in vitro.

Insulin-like growth factor 1 (IGF-1) plays a critical role in CNS development. IGF-1 can block neuronal apoptosis in vitro and in vivo. IGF-1 is thought to be cleaved into des-N-(1-3)-IGF-1 and an amino terminal glycine-proline-glutamate (GPE tripeptide). Here we report a neuroprotective role for GPE tripeptide, with enhanced survival of the CA1-2 hippocampal neurons following an excitotoxic insult in vitro. Binding and displacement studies suggest uniquely distributed sites of action within the rat including the hippocampal CA1-2, pyriform cortex, amygdala, choroid plexus, blood vessels and to a lesser extent in the cortical regions. A similar pattern of binding was seen in the human. This finding could lead to new strategies to reduce neuronal death after injury and in disease.

Alternating chemotherapy in advanced gastric cancer. A phase II study.

A phase II study with an alternating chemotherapeutic scheme, in advanced gastric cancer was conducted. Twenty-two patients were treated with cisplatin and mitomycin C on day 1 and BCNU and doxorubicin on day 28. Tegafur p.o. was given daily from the start to the end of chemotherapy. This scheme was repeated every four weeks. Response rate was 25% (2 complete and 3 partial). However, remissions were short lived, and median survival for the entire group was 7 months. Haematological and gastrointestinal toxicities were severe. In conclusion, the low response rate and the high toxicity of this regimen preclude its use for the treatment of gastric cancer.

Age-related changes on MAO in Bl/C57 mouse tissues: a quantitative radioautographic study.

The distribution of MAO-A and -B in brain and peripheral tissues of Bl/C57 mice and their changes during ageing were studied by quantitative enzyme radioautography with [3H]Ro41-1049 and [3H]Ro19-6327. In the brain, MAO-A decreased between weeks 4 and 8 and then remained unchanged until 25 months, whereas MAO-B increased for the whole period studied. Heart also showed a continuous increase in MAO-B, but not MAO-A, with ageing, and liver showed a decrease in MAO-B in the older animals. These results show marked species differences in the distribution and age-related changes of MAO and might help to elucidate the high sensitivity of Bl/C57 mouse of MPTP, which increases with age.

Monoamine oxidases: from brain maps to physiology and transgenics to pathophysiology.

The present report reviews recent advances in mapping the cellular sites of synthesis and catalytic activity, as well as age- and disease-related changes of monoamine oxidases A and B in the brain. A transgenic model of oxidative stress is also described. The relevance of these findings for the physiological and pathophysiological roles of monoamine oxidases is briefly discussed.

Localization of monoamine oxidases in human peripheral tissues.

Localization of monoamine oxidases (MAO) A and B and beta-adrenoceptors, was studied in aged human peripheral tissues (age 68-80 years) by quantitative autoradiography. The tissues analyzed were heart, lung, liver, kidney, spleen and duodenum. [3H]Ro41-1049 and [3H]lazabemide, two recently characterized selective radioligands were used to map MAO-A and MAO-B respectively. The regional pattern of distribution of MAO-A and MAO-B did not differ markedly, except in kidney and especially in duodenum. Highest levels of MAOs were measured in liver, and lowest in spleen. MAO-A was more abundant than MAO-B in lung and duodenal mucosa, and the reverse was true in myocardium. These results show marked differences in the abundance and patterns of distribution of MAOs, particularly MAO-B, in human and rodent peripheral tissues.

[Use of mortality probability models (MPM II) in the evaluation of the effectiveness of care of critically ill patients. European and North American Study of Severity Systems]. / Utilización de los modelos probabilísticos de mortalidad (MPM II) para evaluar la efectividad de la atención a pacientes en estado crítico. European and North American Study of Severity Systems.

BACKGROUND: The performance of the Mortality Probability Models (MPM II) has been assessed in Intensive Care Units (ICUs) in Catalonia and the Balearic Islands. The MPM II system has been customized to that geographic area and quality performance has been evaluated in each ICU. METHODS: 1,270 adult critical patients, consecutively admitted in 16 ICUs from Catalonia and 1 from the Balearic Islands have been included. Probability of dying in the hospital has been calculated at admission in the ICU and at 24 hours using the models MPM II0 and MPM II24. Goodness-of-fit of the MPM II system in the overall group of 17 ICUs has been analyzed. Logistic regression has been used to customize the MPM II system to all the ICUs together. A Quality Performance Index (QPI) for each ICU has been obtained by dividing the number of the observed deaths by the number of deaths expected by the MPM II system. RESULTS: The overall QPI was 1.15 when using the MPM II0 and 1.17 when using the MPM II24. The QPI in the 17 ICUs ranged from 0.58 to 2.05. Three ICUs showed excess of mortality and 2 ICUs had less deaths than expected. The process of customization of MPM II to the 17 ICUs as a group improved the estimation of expected mortality. CONCLUSIONS: The use of severity indexes allows to compare the outcome of patients in the ICU and provides an indicator of quality of care. The excess of mortality observed in some ICU should produce a watchful follow-up of outcome. Risk factors for excess of mortality should be studied.

[Mortality and hospital stay adjusted for severity as indicators of effectiveness and efficiency of the care provided to critically ill patients. European and North American Study of Severity Systems]. / Mortalidad y estancia hospitalaria ajustadas por gravedad como indicadores de efectividad y eficiencia de la atención a pacientes en estado crítico. European and North American Study of Severity Systems.

BACKGROUND: Hospital mortality and length of stay, both adjusted for severity of illness, have been used as indicators of effectiveness and efficiency of health care in critical patients. PATIENTS AND METHODS: 1,270 adult critical patients, consecutively admitted in 17 intensive care units (ICU) from Catalonia and the Balearic Islands, Spain, have been included. For each hospital, effectiveness has been assessed with a quality performance index (QOI) obtained by dividing the number of observed deaths by the number of deaths expected according to the MPM system (MPM II0). Efficiency has been assessed with a resource utilization index (RUI) obtained by dividing the number of observed weighted hospital days (WHD) by the number of expected WHD. WHD is a measure of resource use which weights ICU days more heavily than non-ICU days. Expected WHD have been obtained by a regression model including severity of illness and the presence/absence of surgery. RESULTS: Ten of the 17 hospitals life within one standard deviation of the mean on both clinical and economical indices. There are 3 hospitals with optimal values on both indices. There is no evidence of association between effectiveness and resource utilization. CONCLUSIONS: Clinical and economical performance of hospitals can be quantified with simple indicators which allow to compare centers. Hospitals can be effective and efficient at the same time.

[Lung cancer in patients under 40 years of age: a different problem?]. / Cáncer de pulmón en enfermos menores de 40 años: un problema diferente?

BACKGROUND: Less than 5% of the cases of lung cancer diagnosed are found in young patients. Although there are few studies concerning this problem it has been suggested that in this age group the behavior of the disease is more aggressive and the prognosis worse. METHODS: In order to study the clinical characteristics, histology, and evolution of bronchogenic lung cancer diagnosed in patients under 40 years of age the data of 56 patients with this neoplasm and age was compared with those obtained from 500 patients, equal to or over 40 years of age, diagnosed during the same period of time. RESULTS: The proportion of males/females among the patients under 40 years of age was 4:1, while in those patients over this age it was 13:1 (p less than 0.001). Epidermal cancer was the most frequent histological variety found among the patients over 40, while microcytic cancer of the lung was more frequent in those under 40 (p less than 0.05). In the group of patients under 40, 8 patients under 30 had special characteristics: male/female proportion 1:1 (p = 0.078), histological predominance of the adenocarcinoma variety and undifferentiated carcinoma of large cells (p = 0.008) and lower number of smokers (p = 0.035). The time of evolution of symptoms, the extension of the disease at the time of diagnosis and survival of the patients over or under the age of 40 was similar. CONCLUSIONS: Despite the presentation of special characteristics in young patients with bronchogenic lung cancer, the behavior of the disease is similar to that observed in other age groups.