Lorcaserin and metabolic disease: weight-loss dependent and independent effects.

OBJECTIVE: Weight management pharmacotherapies can improve metabolic diseases through weight-dependent and weight-independent effects. Lorcaserin is a selective 5-hydroxytryptamine 2C receptor agonist. The objective of this analysis is to quantify the relative contribution of weight loss to the treatment effects of lorcaserin 10 mg twice a day on key metabolic parameters. METHODS: This retrospective analysis evaluated 6,897 patients with overweight or obesity (with or without diabetes mellitus) across three randomized, placebo-controlled, double-blind, 52-week clinical trials that evaluated lorcaserin 10 mg twice daily (BID; NCT00395135, NCT00603902, and NCT00603291); 509 patients from only one of the studies had type 2 diabetes mellitus. A mediation analysis was applied to help rank the relative contribution of weight loss to metabolic study outcomes. RESULTS: According to this mediation analysis, lorcaserin 10 mg BID improved a spectrum of adiposopathic metabolic abnormalities with varying contributions attributable to weight loss. Improvements in waist circumference and blood pressure were almost exclusively attributable to weight loss. Less than 50% of the improvement in glucose parameters (fasting blood glucose and haemoglobin A1c) were attributable to weight loss. CONCLUSIONS: Across Phase III clinical trials, lorcaserin 10 mg BID improved multiple cardiometabolic parameters through both weight-loss dependent and independent mechanisms.

Histological findings on fetal striatal grafts in a Huntington's disease patient early after transplantation.

Cell transplantation is a promising therapeutic approach that has the potential to replace damaged host striatal neurons and, thereby, slow down or even reverse clinical signs and symptoms during the otherwise fatal course of Huntington's disease (HD). Open-labeled clinical trials with fetal neural transplantation for HD have demonstrated long-term clinical benefits for HD patients. Here we report a postmortem analysis of an individual with HD 6 months after cell transplantation and demonstrate that cells derived from grafted fetal striatal tissue had developed into graft-derived neurons expressing dopamine-receptor related phosphoprotein (32 kDa) (DARPP-32), neuronal nuclear antigen (NeuN), calretinin and somatostatin. However, a fully mature phenotype, considered by the expression of developmental markers, is not reached by engrafted neurons and not all types of interneurons are being replaced at 6 months, which is the earliest time point human fetal tissue being implanted in a human brain became available for histological analysis. Host-derived tyrosine hydroxylase (TH) fibers had already heavily innervated the transplants and formed synaptic contacts with graft-derived DARPP-32 positive striatal neurons. In parallel, the transplants contained a considerable number of immature neuroepithelial cells (doublecortin+, Sox2+, Prox-1+, ss3-tubulin+) that exhibited a pronounced migration into the surrounding host striatal tissue and considerable mitotic activity. Graft-derived astrocytes could also be found. Interestingly, the immunological host response in the grafted area showed localized increase of immunocompetent host cells within perivascular spaces without deleterious effects on engrafted cells under continuous triple immunosuppressive medication. Thus this study provides for a better understanding of the developmental processes of grafted human fetal striatal neurons in HD and, in addition, has implications for stem cell-based transplantation approaches in the CNS.

The neuropeptide NAP provides neuroprotection against retinal ganglion cell damage after retinal ischemia and optic nerve crush.

BACKGROUND: NAP, an 8-amino acid peptide (NAPVSIPQ=Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln) derived from activity-dependent neuroprotective protein (ADNP), plays an important role in neuronal differentiation and the survival of neurons in different pathological situations. We already discovered that NAP increases the survival of retinal ganglion cells (RGC) in vitro, and supports neurite outgrowth in retinal explants at femtomolar concentrations. The aim of this study was to investigate the effects of NAP on RGC survival after transient retinal ischemia and optic nerve crush. METHODS: RGC of male Wistar rats were labelled retrogradely with 6 l FluoroGold injected stereotactically into both superior colliculi. Seven days later, retinal ischemia was induced by elevating the intraocular pressure to 120 mm Hg for 60 minutes or by crushing one optic nerve for 10 s after a partial orbitotomy. NAP was either injected intraperitoneally in the concentration of 100 microg/kg [corrected] 1 day before, directly after, and on the first and the second days after damage, or intravitreally (0.05 or 0.5 microg/eye) [corrected] directly after the optic nerve crush. Controls received the same concentrations of a control peptide. Densities of surviving RGC and activated microglial cells (AMC) were quantified in a masked fashion 10 days after damage by counting FluoroGold-labelled cells. RESULTS: After retinal ischemia, intraperitoneal injections of NAP increased the number of surviving RGC by 40% (p < 0.005) compared to the control group. After optic nerve crush, NAP raised the number of surviving RGC by 31% (p = 0.07) when injected intraperitoneally and by 54% (p < 0.05) when administered intravitreally. CONCLUSIONS: NAP acts neuroprotectively in vivo after retinal ischemia and optic nerve crush, and may have potential in treating optic nerve diseases.

Expression and function of cytochrome p450 in brain drug metabolism.

Cytochrome P450 (CYP, P450) is the collective term for a superfamily of heme-containing membrane proteins responsible for the metabolism of approximately 70 - 80 % of clinically used drugs. Besides the liver and other peripheral organs, P450 isoforms are expressed in glial cells and neurons of the brain. To enlighten their function and significance is a topic of high interest, as most of the neuroactive drugs used in therapy today are not only substrates, but also inducers of brain P450s with far reaching consequences. First of all, brain P450s are regulated differentially from those in liver. The availability of the prosthetic heme group appears to be essential for correct membrane insertion and enzymatic functionality of brain P450s. Furthermore, although not contributing to body's overall drug metabolism, brain P450s fulfil particular functions within specific cell types of the brain. In astrocytes of brain's border lines P450 isoforms are expressed at very high level. They form a metabolic barrier regulating drugs' influx, modulate blood-flow regulation, and act as signalling enzymes in inflammation. In neurons, however, P450s apparently have different function. In specified brain regions such as hypothalamus, hippocampus and striatum they provide signalling molecules like steroids and fatty acids necessary for neuronal outgrowth and maintenance. Induction of these P450s by neuroactive drugs can alter steroid hormone signalling directly in drug target cells, which may cause clinically relevant side effects like reproductive disorders and sexual or mental dysfunction. The understanding of brain P450 function appears to be of major interest in long-term drug mediated therapy of neurological diseases.

Predominantly neuronal expression of cytochrome P450 isoforms CYP3A11 and CYP3A13 in mouse brain.

Despite the very small amounts of cytochrome P450 enzymes expressed in different areas and cell populations of the brain as compared with the liver, there is significant evidence for their specific involvement in brain development, function, and plasticity. Nevertheless, the current discussion about occurrence and importance of cerebral cytochrome P450 isoforms is determined by controversial interpretations of their function in general and with respect to single isoforms. Continuing a series of publications about brain P450 isoforms, we now present evidence for the expression of cytochrome P450 3A11 and 3A13 in mouse brain. Immunocytochemical and non-radioactive in situ hybridization studies revealed identical distribution of their proteins and mRNAs throughout the brain especially in neuronal populations, and to some extent in astrocytes. The cerebral expression of these P450 isoforms was confirmed by Western blot and RNAse protection assay analysis. The well-known testosterone-metabolizing capacity and the inducibility of cytochrome P450 3a isoforms by xenobiotics as well as their presence in steroid hormone-sensitive areas and neurons (e.g. hippocampus) clarify the significance of these isoforms for impairment of steroid hormone actions by P450-inducing environmental substances. Therefore, investigation of inducible cerebral P450 isoforms which are able to metabolize xenobiotics as well as steroid hormones might help us to understand neuroendocrine regulation of brain's plasticity.

Oxidative hydrolysis of scoparone by cytochrome p450 CYP2C29 reveals a novel metabolite.

Regioselective 7-demethylation of scoparone is regularly employed as an indicator of phenobarbital-like induction of rat liver cytochrome P450 isoform CYP2B1, e.g., by the antiepileptic drug phenytoin. After induction with phenobarbital and phenytoin, a new reaction sequence catalyzed by Cyp2c29 was identified in mouse liver microsomes. Cyp2c29-dependent 6-demethylation of scoparone resulted in the formation of isoscopoletin, an intermediate which is susceptible to further oxidation. This subsequent oxidation was also catalyzed by Cyp2c29 with a K(m) of 30,31 microM and a V(max) of 3,41 microM/min x microM P450, and resulted in the formation of the new metabolite 3-[4-methoxy-p-(3,6)-benzoquinone]-2-propenoate. This novel metabolite is the product of two consecutive oxidation reactions, proceeding over isoscopoletin to a putative lactone which is accessible to immediate hydrolysis, due to the onium character of the ring oxygen. This opening of the lactone ring corresponds to an oxidative hydrolysis. Differential oxidation of scoparone can be used as a sensitive indicator for distinguishing between different cytochrome P450 isoforms.

Expression and localization of the CYP2B subfamily predominantly in neurones of rat brain.

Despite the very small amounts of cytochrome P450 (P450, CYP) enzymes expressed in different areas and cell populations of the brain as compared with the liver, there is significant evidence for their specific involvement in brain development, function and plasticity. Nevertheless, the current discussion about occurrence and importance of cerebral cytochrome P450s is determined by inconsistent interpretations of their function in general and with respect to single isoforms. Continuing a series of publications about brain P450 isoforms, we now present evidence for the constitutive expression of CYP2B1 and CYP2B2 mRNAs in rat brain. Immunocytochemical and non-radioactive in situ hybridization studies revealed the same expression pattern throughout the brain predominantly in neuronal populations, but to some extent in astrocytes of corpus callosum and olfactory bulb. The well known testosterone-metabolizing capacity and the presence of CYP2B isoforms shown in steroid hormone-sensitive areas and neurones (e.g. hippocampus) clarify the significance of isoforms like CYP2B1 and CYP2B2 for impairment of steroid hormone actions by P450 inducing environmental substances. We argue that cerebral P450 isoforms which are induced by xenobiotics and are able to metabolize these as well as endogenous substrates help us to understand fundamental aspects of brain's functioning.

Possible function of astrocyte cytochrome P450 in control of xenobiotic phenytoin in the brain: in vitro studies on murine astrocyte primary cultures.

[4-(14)C]Phenytoin underwent a rapid cellular uptake by diffusion within 5 min when applied in a concentration of 10 microM to mouse brain astrocyte cultures. Subsequently, a slow linear increase of intracellular radioactivity indicated metabolic trapping of the drug, with final concentrations reaching 144 pmol phenytoin/mg protein in the astrocytes. Phenytoin levels from 1 to 10 microM decreased cell viability by 15%. The action of cytochrome P450 present in astrocytes in concentrations of 16-17 pmol P450/mg protein could explain these slight cytotoxic effects by generating intermediate metabolites of phenytoin. In contrast, concentrations of 50 microM strongly inhibited cell proliferation. A Cyp2c29 immunorelated P450 isoform was expressed in nearly all astrocytes in culture. Intracellular [4-(14)C]phenytoin was degraded to its major metabolites dihydrodiol, p-HPPH, and m-HPPH through a P450-dependent reaction with a specific activity of 0.66 pmol/min x mg protein, or 0.12 pmol/min x mg protein as measured in cell homogenates. These data underscore the importance of astrocytes as brain cells active in the detoxification of foreign substrates, but also in their toxification due to reactive metabolites generated during these metabolic processes. After diffusionary influx of drugs and other xenobiotics, the astrocyte P450 monooxygenases perform an essential role in the mediation of toxicity most frequently encountered in highly vulnerable neurons.

Identification, induction and localization of cytochrome P450s of the 3A-subfamily in mouse brain.

Several cytochrome P450 subfamilies are inducible by specific exogenous compounds like the antiepileptic drug phenytoin. Some of these P450 enzymes are involved in the metabolism of gonadal hormones also contributing to neuronal differentiation. CYP3A enzymes have the capacity to catalyze the hydroxylation of testosterone and a wide variety of therapeutic agents, but little is known about the expression and potential function of this subfamily in mouse brain. Here, we report the identification of mouse CYP3A isoforms, their induction and localization in mouse brain. Western blot analysis with anti-CYP3A1 antibodies revealed the phenytoin-inducible expression of CYP3A in brain microsomes, and also a constitutive expression of members of this subfamily in brain mitochondria. Using RT-PCR with a consensus primer pair for known mouse liver CYP3A-isoforms we could demonstrate the expression of CYP3A11 and 3A13 mRNA in mouse brain. Finally, using double immunofluorescence labeling we analyzed the histoanatomical distribution of CYP3A throughout the brain with confocal laser scanning microscopy. We found strong immunoreactivity in neurons of hippocampus and hypothalamic areas which are sensitive to steroid hormones. CYP3A immunoreactivity was apparent also in neurons of the cerebellum, the thalamus and the olfactory bulb. Non-neuronal expression of CYP3A could be found in some astrocyte populations and in vascular as well as ventricular border lines. The presence of CYP3A predominantly in neurons but also in cells contributing to the blood-brain and blood-liquor barrier suggests important roles of this subfamily in mediation of steroid hormone action in mouse brain as well as in preventing the brain from potentially cytotoxic compounds.

Different testosterone metabolism by immortalized embryonic and postnatal hippocampal neurons from C57BL/6 mice: a crucial role for androstenedione.

Steroid hormones influence the development of undifferentiated brain during ontogenesis. In the present study we investigated the metabolic pathway of testosterone in immortalized embryonic and postnatal hippocampal neurons from C57BL/6 mice. Both cell lines are capable of metabolizing testosterone to 6alpha-hydroxytestosterone, 6beta-hydroxytestosterone and androstenedione. The formation was found to correlate with protein concentration and time of incubation. These linearities were significant for all metabolites except androstenedione that was the main metabolite in embryonic hippocampal neurons and nearly absent in postnatal neurons. Moreover, only embryonic cells react to testosterone with a decrease of beta-tubulin expression, that was a typical effect indicating induced neuronal maturation. Application of androstenedione caused the same decrease of beta-tubulin expression as testosterone did before. Our results of hippocampal testosterone metabolism in vitro confirm that not only estradiol and 5alpha-dihydrotestosterone could impact neural tissue but also androstenedione is a powerful metabolite involved in prenatal neuronal differentiation.

Testosterone metabolism in rat brain is differentially enhanced by phenytoin-inducible cytochrome P450 isoforms.

Many cytochrome P450 (P450) isoforms are selectively inducible by xenobiotics, e.g. pharmaceuticals like the anti-epileptic drug phenytoin. Some of these P450 enzymes are involved in the metabolism of gonadal hormones and are of great importance, especially in early brain development. In this study, the hydroxylation of testosterone by rat brain microsomes from control and phenytoin-induced animals was examined by use of high performance liquid chromatography (HPLC) provided with a photodiode array detector (PDA). In control rats, testosterone is converted by cytochrome(s) P450 to 6alpha-hydroxytestosterone (OHT) as the main metabolite and 6beta-OHT as well as androstenedione as minor metabolites. After phenytoin treatment, brain microsomes showed a strong increase of testosterone metabolism to 2alpha-, 6beta-, 16alpha-, 16beta-OHT and androstenedione, whereby 16alpha-OHT was the main degradation product. These metabolites indicated the action of isoforms of the P450 subfamilies CYP2B, CYP2C and CYP3A. Inhibition experiments with antibodies against CYP2B1/2 and with the CYP2B specific inhibitor orphenadrine indicated the occurrence of members of this subfamily which are known to catalyse the oxidation of testosterone to 16alpha-OHT, 16beta-OHT and androstenedione. Western blots revealed the phenytoin-inducible expression of CYP2B1 and the constitutive expression of CYP3A. The latter is involved in the 6beta-hydroxylation of testosterone which was found correspondingly in control microsomes. Distinct CYP2C isoforms involved in the hydroxylation of testosterone in phenytoin-induced microsomes are not yet identified. The highly increased testosterone metabolism by phenytoin-dependent induction of specific cytochrome P450 isoforms in adult rat brain illustrates the potential influence of exogenous substances on internal regulative and metabolic pathways in the brain.

Effect of phenytoin on cytochrome P450 2B mRNA expression in primary rat astrocyte cultures.

Studies on cytochrome P450 2B (CYP2B) in the brain have essentially been focused on protein characterization and regional distribution. Due to the high sequence homology between the closely related CYP2B1 and 2B2 isoforms and the low amounts of the corresponding mRNAs few efforts have been made to analyze the expression, regulation, and inducibility of these P450 genes in a specific cell type. In the present study, we investigated CYP2B mRNA expression in primary rat astrocyte cultures under the influence of the anti-epileptic drug phenytoin, which is known to be a CYP2B inducing agent in liver. In situ hybridization with a digoxigenin (DIG)-labeled cRNA probe demonstrated that 30-40% of the astrocytes strongly expressed a CYP2B mRNA-specific signal within the first week of cultivation. With increasing age (> 14 days) a greater percentage of cells (>90%) expressed mRNA for P450 2B. However, the level of transcriptional activity was substantially lower than in younger cultures. To discriminate between the 2B1 and 2B2 isoforms the reverse transcription/polymerase chain reaction (RT/PCR) procedures were proved for rat hepatic mRNA as a control assay. Subsequently, the application of this method on cultured astrocytes confirmed that these brain cells may express CYP2B1 mRNA. CYP2B2 mRNA could not be detected in astrocyte cultures at any age examined. Phenytoin led to the down regulation of CYP2B1 mRNA, which contrasts with the drug inducing effect on hepatic CYP2B1 and 2B2 levels. After 4 hr of exposure of phenytoin to the astrocytes no amplification product could be detected at all. Phenytoin did not induce either CYP2B1 or 2B2 expression.

Phenytoin alters Purkinje cell axon morphology and targeting in vitro.

In adult mice, administration of the anticonvulsive drug phenytoin caused focal swellings along the Purkinje cell axon correlated with ataxia and incoordination of movements. In our model, we used murine cerebellar slice cultures to study the influence of phenytoin on postnatal Purkinje cell axon differentiation. Almost all of our untreated cultures developed to mature-like cerebellar tissue. Immunohistochemistry with anti-calbindin-D28k or UCHTI (anti-CD3) antibodies revealed numerous Purkinje cell axons in the white matter. In the area of the deep cerebellar nuclei, immunolabelled axons formed a large axonal plexus. The few neurofilament-positive neurons in this area were densely covered with Purkinje cell axon terminals. The synaptophysin immunoreactivity revealed connections between the terminals and the neurons of the deep cerebellar nuclei. Treatment of cerebellar slice cultures with phenytoin (10-80 microM) for 10-16 days resulted in focal swellings of different size along the axon. The number of swellings increased with an increasing dosage. At concentrations of 40 microM phenytoin, Purkinje cell axons seemed to be unable to invade the deep cerebellar nuclei, but numerous aberrant, recurrent collaterals could be detected immunohistochemically with the two specific Purkinje cell antibodies. Possible cytotoxic effects after treatment, such as dendritic degeneration and a decrease in the number of immunolabelled Purkinje cells, were observed above 40 microM phenytoin. These data suggest that the response of juvenile Purkinje cells is dependent upon the dosage of the antiepileptic drug because of morphological alterations as well as a misrouting of previously established connections.

Possible role of cytochrome P450 in inactivation of testosterone in immortalized hippocampal neurons.

The hippocampus as part of the limbic system is sensitive to gonadal hormones. The time-dependent expression of steroid receptors and the testosterone converting enzyme aromatase (CYP19) is well studied. In contrast, little is known about other cytochrome P450 enzymes in hippocampus which inactivate the gonadal hormones. For investigation of the total cytochrome P450 content and the expression of testosterone degrading CYP2B10 we used embryonic (E18) in comparison to postnatal (P21) immortalized hippocampal neurons. These embryonic neurons were demonstrated to react to hormones according a 'critical period' of sexual differentiation: testosterone treatment (1 microM to 5 microM in the culture medium) resulted in a decrease of beta-tubulin, as showed by immunocytochemistry and Western blotting. Measurements with reduced CO-difference spectrum elucidated that the P450 concentration in the embryonic neurons (10.2 pmol/mg protein; S.D. +/- 1.9) was twice as high as in the postnatal ones (5.2 pmol/mg protein; S.D. +/- 1.0). Correspondingly, a high value of the mitochondrial subfraction of approx. 141 pmol P450/mg protein was found in the embryonic neurons relative to the mitochondrial value of 37.7 pmol P450/mg protein in the postnatal neurons. Our results suggest a differential expression of cytochrome P450 during development. CYP2B10 was proved by electron microscopy and hormone degrading activity.

Localization and characterization of cytochrome P450 in the brain. In vivo and in vitro investigations on phenytoin- and phenobarbital-inducible isoforms.

The antiepileptic drug phenytoin is known to be substrate as well as inducer of cytochrome P450 (P450) in the mammalian liver. We were able to show the expression of P450 species immunorelated to the main phenytoin-induced hepatic isoforms in mice (CYP2C29) and rats (CYP2B1,2) also in the central and peripheral nervous system and primary cultures of cell types from the brain. The 2B1,2 related protein showed only a weak constitutive expression in vivo and in vitro analyzed by immunocytochemistry, in situ hybridization, Northern blot and RT/polymerase chain reaction (PCR). Contrary, the CYP2C29 related form is inducible by phenytoin at about 1.5-fold starting from an already higher constitutive level. This protein is characterized by a remarkable tendency to dissociate from the endomembranes during tissue homogenization. The supernatant of microsomal pellet is able to metabolize phenytoin in a reconstitutive system.

Intermittent ethanol exposure of adult rats: hippocampal cell loss after one month of treatment.

The effects of intermittent intraperitoneal ethanol exposure (3 g/kg body weight twice daily) on hippocampal cells of adult rats were examined after a 1-month exposure period. Continuous oral exposure to ethanol in the drinking water (20%) over the same time period was used for comparison. Pyramidal cells from the CA3 region and granule cells from the dentate gyrus were also qualitatively assessed using electron microscopy. One month of intermittent, peaking, exposure induced a significant loss of CA2-CA3 pyramidal cells and an increase in the number of lipofuscin granules in the remaining cells. A significant reduction in thickness of the dentate gyrus granule cell layer was also seen in the same animals. No significant reduction in pyramidal cell number or granule cell layer thickness was seen in continuously orally exposed animals in spite of a higher total ethanol intake. These studies thus demonstrate the harmful effect of intermittent high ethanol peaks and repeated withdrawal phases on hippocampal cells of the mammalian brain and emphasize the importance of considering the differential effects of different ethanol exposure patterns when assessing ethanol-induced cellular damage.

Cytochrome P450 in rat astrocytes in vivo and in vitro: intracellular localization and induction by phenytoin.

Cytochrome P450IIB1,2 (nomenclature according to Nelson et al., DNA Cell Biol 12:1-51, 1993 and Volk et al., Neuroscience 42:215-235, 1991) immunoreactivity (P450-IR) is associated with astrocytes both in vivo and in vitro. Although they are unevenly distributed throughout the brain with a preference for phylogenetically elder parts, no significant differences between astrocytes prepared from different brain regions were observed in astrocyte cultures. The percentage of strongly immunoreactive astrocytes decreased from 40% after 7 days in culture to 15% after 21 days. Essentially all astrocytes have a low but significant P450-IR within this interval. Preembedding immunoelectron microscopy revealed peroxidase reaction products on the endoplasmic reticulum and on the outer membranes of mitochondrial and nuclear envelopes. Phenytoin (1 microM) added to the medium for 7 days significantly (1.22-fold) increased the amount of total P450 in astrocyte homogenates as measured by spectrophotometry. Considerably more immunoreactive cells (1.5-fold) were found in treated cultures than in controls.

Long-term effects of intermittent versus continuous ethanol exposure on hippocampal synapses of the rat.

The hippocampus is known to be very sensitive to a large spectrum of different neurotoxins including ethanol. Ethanol administered continuously or intermittently may affect the hippocampus in different ways. Intermittent administration of ethanol has many features in common with the low level electrical stimulation protocols which lead to the functional changes associated with the phenomenon of kindling. In this study, the differential effects of intermittent intraperitoneal ethanol injections (3 g/kg twice daily) and continuously administered ethanol in drinking water (20%) on hippocampal synapses in the rat were studied using ethanolic phosphotungstic acid staining and electron microscopy. After 1 month of intermittent exposure a significant reduction (18%) of synapses was seen in the stratum lucidum of the CA3 region. Continuously treated animals showed no significant change over this time despite a higher total ethanol intake. In the dentate gyrus, a compensatory increase in supragranular synaptic number was seen only in continuously treated animals. These findings demonstrate the sensitivity of synapses of the hippocampus to the presence of ethanol and the larger effects of peaking ethanol concentrations compared to more constant levels. These results emphasize the need to consider the differential effects of various types of ethanol consumption also on the human brain.

Cytoplasmic expression of the leu-4 (CD3) antigen in developing Purkinje cells in the rat cerebellum.

Although commonly known to represent a T cell receptor (CD3) associated polypeptide, the leu-4 (CD3) antigen occurs in cerebellar Purkinje cells (PCs) of many species. The monoclonal pan T lymphocyte marker anti-leu-4 (CD3) recognizes both the lymphocytic and the Purkinje cell type of this antigen [22]. To obtain more information about the merit of anti-leu-4 (CD3) as an investigational tool, we evaluated the expression of leu-4 (CD3) in PCs of the developing rat cerebellum (in situ) by light microscopy. Positive anti-leu-4 (CD3) immunoreaction of PCs did not occur prior to post-natal day (D) 4. The analysis of immunostaining during cell differentiation revealed three major phases of post-natal PC maturation including antigenic development of cell somata (phase 1: until D6), dendrites (phase 2: D7-D11), and axons (phase 3: D12-D14). A massive post-weaning expansion of the dendritic arborization led then to the mature PC architecture. Additionally, the leu-4 (CD3) antigen was observed in ectopic PC dendrites (D10) and in ectopic (mature) PCs. Throughout post-natal development as well as in mature PCs, the leu-4 (CD3) antigen was found to be cytoplasmic. Due to its labile nature, neither an ultrastructural localization nor molecular characterization could be achieved. For the same reason, its application is basically restricted to cryo-fixed cerebellar tissue. However, at the level of light microscopy, the monoclonal human T cell marker anti-leu-4 (CD3) proved to be a useful tool for specific and sensitive labelling of differentiated cerebellar PCs in the rat.

A clinicopathologic and immunomorphologic study of 13 cases of ganglioglioma.

The authors present the clinical, histopathologic, and immunomorphologic data of 13 intracranial gangliogliomas. Preoperative computed tomography scans showed a commonly cystic tumor of variable density. Six tumors were completely excised and seven were subtotally resected. After a mean follow-up of 4.5 +/- 2.6 years, 11 patients are asymptomatic or only slightly incapacitated. All tumors were examined with a panel of neuronal and neuroendocrine markers. Immunoreactivity (IR) to anti-neurofilament polypeptide (clone 2F11) was observed in neuronal processes in ten cases and in neuronal perikarya in five. With anti-synaptophysin (clone SY38), IR was present along the lining of ganglion cell perikarya and processes in 11 tumors whereas staining of the perinuclear cytoplasm was prominent in two. IR to anti-chromogranin A (clone LK2H10) was observed within the neuronal perikarya in eight cases. Only one ganglioglioma of the brain stem showed IR for tyrosine-hydroxylase (clone 2/40/15) and dopamine-beta-hydroxylase in some neoplastic ganglion cells. In this study, synaptophysin was the most reliable neuronal marker. For immunocytochemical identification of neoplastic neurons in ganglioglioma as well as other tumors with neuronal differentiation the authors propose a panel of well-characterized monoclonal antibodies against neurofilament polypeptides, synaptophysin, and chromogranin A to support the histomorphologic diagnoses.