Distribution, characterization and clinical significance of microglia in glioneuronal tumours from patients with chronic intractable epilepsy.

Cells of the microglia/macrophage lineage represent an important component of different brain tumours. However, there is little information about the microglia/macrophage cell system in glioneuronal tumours and its possible contribution to the high epileptogenecity of these lesions. In the present study, the distribution of cells of the microglia/macrophage lineage was studied by immunocytochemistry for CD68 and human leucocyte antigen (HLA)-DR in a group of glioneuronal tumours, including gangliogliomas (GG, n = 30), and dysembryoplastic neuroepithelial tumours (DNT, n = 17), from patients with chronic intractable epilepsy. A significant number of microglia/macrophage cells were observed in the large majority of glioneuronal tumours, both within the tumour and in the peritumoral region. Activated microglial cells positive for HLA-DR were localized around blood vessels and clustered around tumour neuronal cells. The density of activated microglial cells correlated with the duration of epilepsy, as well as with the frequency of seizures prior to surgical resection. These observations indicate that the presence of cells of the microglial/macrophage cell system is a feature of glioneuronal tumours and is functionally related to epilepsy, either directly in epileptogenesis or through activation following seizure activity.

Expression patterns of Group III metabotropic glutamate receptors mGluR4 and mGluR8 in multiple sclerosis lesions.

Recent evidence supports a role for metabotropic glutamate receptors (mGluRs) in neuroinflammatory diseases. In the present study, we have investigated whether the group III mGluR subtypes mGluR4 and mGluR8 are expressed in MS lesions at various stages of evolution. In control patient tissue and in normal-appearing MS white matter (NAWM), no microglial or astrocyte staining was detected. In contrast, in active lesions, mGluR8 immunoreactivity (IR) was detected in cells of the microglia/macrophage lineage. Fewer macrophage-like cells were positive for mGluR8 in chronic active and inactive lesions. No mGluR4 IR was detected in cells of the microglia/macrophage lineage in the MS lesions studied. In chronic active lesions, however, a population of reactive astrocytes localized in the rim of the lesions expressed both mGluR4 and mGluR8. Our results suggest a role for these receptor subtypes in the inflammatory response in MS that involves both astrocytes and cells of the microglia/macrophage lineage.

Neurotrophin receptor immunoreactivity in the hippocampus of patients with mesial temporal lobe epilepsy.

Recent evidence supports a critical role of neurotrophins in the regulation of both neuronal survival and synaptic transmission during epileptogenesis. We have examined the immunohistochemical expression of high- (tyrosine kinase receptors, trk) and low-affinity (p75) neurotrophin receptors (NTRs) in the hippocampal specimens from 18 patients with chronic temporal lobe epilepsy [TLE; 14 patients with hippocampal sclerosis (HS) and four with focal lesions (tumours) not involving the hippocampus proper]. Nonepileptic autopsy brains (n = 6) and surgical specimens from tumour patients without epilepsy (n = 3) were used as controls. Immunoreactivity (IR) for the trk receptors (trkA, trkB, trkC) was detected in normal human brain within the pyramidal neurones of hippocampal cornus ammoni (CA) regions and in the dentate gyrus. There were no detectable differences in the neuronal trk IR patterns in the hippocampus between control and TLE cases with HS, except for a decrease in neuronal density in regions where cell death had occurred (CA1, CA3 and CA4). In contrast, a consistent increase in trkA IR was observed in reactive astrocytes in CA1 and dentate gyrus. The low-affinity p75 neurotrophin receptor (p75(NTR)) was expressed in low levels in postnatal normal hippocampus. In contrast, neuronal p75(NTR) IR was detected in 10/14 cases of HS in spared neurones within the CA and hilar regions of the hippocampus. Double labelling revealed that p75(NTR)-positive neurones also contain trk receptor IR. In six cases with prominent glial activation strong p75(NTR) IR was observed in microglial cells within the sclerotic hippocampus. The present results indicate that changes in NTR expression are still detectable in the hippocampus of patients with chronic TLE and involve both glial and neuronal cells. Reactive astrocytes were immunoreactive for trkA, whereas activated microglia cells were reactive for p75(NTR), suggesting different functions for specific NTRs in the development of reactive gliosis. Moreover, the increased expression of p75(NTR) in hippocampal neurones of TLE patients may critically influence the neuronal survival during the epileptogenic process.

Immunohistochemical localization of vascular endothelial growth factor receptors-1, -2 and -3 in human spinal cord: altered expression in amyotrophic lateral sclerosis.

Vascular endothelial growth factor (VEGF) has recently been implicated in several neurological disorders. Apart from its prominent role in angiogenesis, VEGF has been shown to have direct effects on neuronal and glial cells through activation of different VEGF receptor (VEGFR) types. In the present study the expression patterns of VEGFR-1, -2 and -3 were investigated in the spinal cord of control and both sporadic and familial amyotrophic lateral sclerosis (ALS) patients. Immunocytochemical analysis of control human spinal cord demonstrated that VEGFR-1, but not VEGFR-2 or -3 was found to be present in blood vessels of both white and grey matter. All three VGEFRs were not detectable in resting glial cells of control tissue. Diffuse neuropil staining was observed in the control spinal cord grey matter for VEGFR-3. Regional differences in VEGFRs immunoreactivity (IR) were apparent in ALS compared to controls. In particular, VEGFR-1 expression was increased in reactive astroglial cells in both grey (ventral horn) and white matter of ALS spinal cord. In addition to the astroglial labelling, increased expression of VEGFR-1 and, to a less extent also of VEGFR-2, was observed in blood vessels of the ALS spinal cord. No changes in VEGFR-3 IR were detected in blood vessels or reactive astroglial cells, whereas VEGFR-3 neuropil expression was reduced and paralleled the distribution of neuronal loss in the ventral horn of ALS spinal cord. These findings indicate that VEGFRs have specific distribution patterns, suggesting different physiological functions in human spinal cord. Moreover, the altered expression observed in ALS supports a role for these receptors in the complex reactive processes that are associated with the progression of spinal cord damage.

Expression and cellular distribution of multidrug transporter proteins in two major causes of medically intractable epilepsy: focal cortical dysplasia and glioneuronal tumors.

The cell-specific distribution of multidrug resistance extrusion pumps was studied in developmental glioneuronal lesions, including focal cortical dysplasia (15 cases) and ganglioglioma (15 cases) from patients with medically intractable epilepsy. Lesional, perilesional, as well as normal brain regions were examined for the expression of the multidrug resistance gene 1 encoded P-glycoprotein (P-gp) and the multidrug resistance-associated protein 1 (MRP1) by immunocytochemistry. In normal brain MRP1 expression was below detection, whereas P-gp staining was present only in blood vessels. MRP1 and P-gp immunoreactivity was observed in dysplastic neurons of 11/15 cases of focal cortical dysplasia, as well as in the neuronal component of 14/15 ganglioglioma. Glial cells with astrocytic morphology within the lesion showed multidrug-resistant protein immunoreactivity (P-gp>MRP1). Moderate to strong MRP1 and P-gp immunoreactivity was observed in a population of large ballooned neuroglial cells. P-gp appeared to be most frequently expressed in glial fibrillary acidic protein-positive balloon cells (glial type), whereas MRP1 was more frequently expressed in microtubule-associated protein 2-positive balloon cells (neuronal type). In both types of lesions strong P-gp immunoreactivity was found in lesional vessels. Perilesional regions did not show increased staining in vessels or in neuronal cells compared with normal cortex. The predominant intralesional cell-specific distribution of multidrug transporter proteins supports the hypothesis of a constitutive overexpression as common mechanism underlying the intrinsic pharmaco-resistance to antiepileptic drugs of both malformative and neoplastic glioneuronal developmental lesions.

Immunoelectron microscopy on material retrieved from paraffin: accurate sampling on the basis of stained paraffin sections.

As a rule, immunoelectron microscopy (immuno-EM) is performed on fresh material processed according to specialized methods (e.g., freezing or embedding in hydrophilic resins). Paraffin-embedded tissue has only occasionally been used as a source of material for immuno-EM; this was usually as a last resort, when no fresh material was available. The authors used archival formalin-fixed, paraffin-embedded basal cell carcinomas for studying the fine-structural distribution of the cell surface molecule CD44 and its variants, as well as some other antigens. The results demonstrate, firstly, that paraffin-embedded material is far more suitable for immuno-EM than frequently assumed and, secondly, that the use of paraffin-embedded material enables highly accurate sampling on the basis of immunohistochemically or conventionally stained light microscopic sections.

The expression of CD44 glycoprotein adhesion molecules in basal cell carcinomas is related to growth pattern and invasiveness.

Basal cell carcinomas (BCCs) of the skin exhibit a wide range of histological growth patterns as well as a highly variable rate of invasiveness. A large body of experimental and clinical studies supports a role for the CD44 glycoprotein family in the latter process. In the present study, we explored the distribution and the level of expression of pan-CD44, CD44v3, CD44v5 and CD44v6 in BCCs. The use of paraffin sections, combined with an antigen retrieval procedure, yielded far more detailed data than would have been possible with frozen sections. On average, the level of expression of the four CD44 isoforms studied appeared to differ relatively little. However, tumours or tumour areas consisting of thin tumour cell strands showed a significantly stronger expression of all four isoforms than those consisting of solid tumour cell groups. Furthermore, the highest CD44 expression was frequently observed in the smallest tumour cell strands in the tumour periphery. In these strands, the label seemed to be located not only at the tumour cell-tumour cell interface, as in other tumour areas, but also on the tumour cell surfaces facing the stroma. We are presently assessing the exact localization of CD44 at the cellular level by immunoelectron microscopy. In most cases, different growth patterns with significantly different levels of CD44 expression were found side by side within individual tumours. CD44 expression is therefore not a static tumour cell characteristic but is correlated with tumour architecture and tumour-stroma interaction.

Expression of granzyme B by cytotoxic T lymphocytes in the lymph nodes of HIV-infected patients.

During HIV infection the architecture of secondary lymphoid tissues is severely disrupted. In particular the germinal centers, which play a key role in the orchestration of the secondary immune response, undergo gross phenotypic alterations, leading to a complete destruction of the germinal center microenvironment. The precise mechanisms responsible for the lymphoid tissue destruction in HIV infection are still unknown. However, the large influx of CD8+ T lymphocytes suggests an important role for T cell-mediated cytotoxicity. To establish whether the infiltrating CD8+ T lymphocytes are killing competent, we investigated the expression of granzyme B, which is known to be present in the cytotoxic granules of NK cells and "activated" CTLs with cytolytic potential. We observed a 20-fold increase in the percentage of granzyme B-expressing CD8+ T cells in both the germinal center and the interfollicular areas in HIV patients relative to HIV-negative controls. This increase was present in patients with early-stage disease (i.e., absolute CD4+ T cell count > 500/microliters) as well as in patients with intermediate and late-stage disease. Thus, from relatively early stages of HIV infection onward large numbers of killing competent T lymphocytes are present in the lymphoid tissues, a finding that supports the notion that CTL act as mediators of destruction of immune function during HIV infection.

Functional morphology of the light yellow cell and yellow cell (sodium influx-stimulating peptide) neuroendocrine systems of the pond snail Lymnaea stagnalis.

Neuroendocrine light yellow cells of the pond snail Lymnaea stagnalis express a neuropeptide gene encoding three different peptides. The morphology of the cell system has been studied by in situ hybridization, using two synthetic oligonucleotides encoding parts of light yellow cell peptides I and III, and by immunocytochemistry with antisera to synthetic light yellow cell peptide II and to two fragments of light yellow cell peptide I. One large cluster of light yellow cells was observed in the ventro-lateral protrusion of the right parietal ganglion, smaller clusters lying in the posterior dorsal part of this ganglion and in the visceral ganglion. The cells had an extended central neurohaemal area. Immunopositive axons projected into all nerves of the ganglia of the visceral complex, into the superior cervical and the nuchal nerves, and into the connective tissue surrounding the central nervous system. Axon tracts ramified between the muscle cells of the walls of the anterior aorta and of smaller blood vessels. Peripheral innervation by the light yellow cell system was only found in muscular tissue of the ureter papilla. The antisera to the two peptide fragments of light yellow cell peptide I not only stained the light yellow cells, but also the identified yellow cells, which have previously been shown to produce the sodium influx-stimulating neuropeptide. The latter cells were negative to the in situ hybridization probes and antisera specific to the light yellow cell system. It is therefore unlikely that the yellow cells express the light yellow cell neuropeptide gene. Nevertheless, the cells contain a neuropeptide sharing antigenic determinants with light yellow cell peptide I.(ABSTRACT TRUNCATED AT 250 WORDS)

Neurons in a variety of molluscs react to antibodies raised against the VD1/RPD2 alpha-neuropeptide of the pond snail Lymnaea stagnalis.

The VD1 and RPD2 neurons of Lymnaea stagnalis innervate other central neurons, certain skin areas, the pneumostome area, and the auricle of the heart. Recently, a set of four (delta, epsilon, alpha, beta) neuropeptides produced by these giant neurons and by certain other central neurons has been characterized. Although alternative splicing of the preprohormone of these neurons yields at least 10 different alpha neuropeptides, an affinity-purified antiserum directed against a domain common to all alpha neuropeptides has previously been shown to be highly selective in staining VD1, RPD2 and other neurons that produce the preprohormone. Since the gene encoding the neuropeptides is structurally similar to that expressed in R15 of the marine opisthobranch Aplysia californica, we have used the affinity purified antiserum as a marker for VD1/RPD2-related systems in other molluscs. Immunopositive neurons and fibers are observed in the central nervous systems of all species studied (Achatina fulica, Anodonta sp., Aplysia brasiliana, A. californica, Bulinus truncatus, Cepea sp., Eobania vermiculata, Helix aspersa, H. pomatia, Limax maximus, Mytilus edulis, Nassarius reticulatus, Viviparus viviparus). Several medium-sized and small neurons and 1-4 giant neurons are found in the pulmonates and opisthobranchs. The giant neurons in pulmonates have locations in the subesophageal ganglion, axonal branching patterns, and terminal arborizations in the auricle of the heart; all these characteristics are similar to those of VD1 and RPD2. Double-labelling (Lucifer yellow injection, immunocytochemistry) confirms that the two giant neurons in Helix pomatia are Br and Br'. The immunoreactive cells in A. fulica appear to include the VIN and PON neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential expression of four genes encoding molluscan insulin-related peptides in the central nervous system of the pond snail Lymnaea stagnalis.

In the pond snail Lymnaea stagnalis, the growth regulating system consists of (1) about 200 neuroendocrine light green cells, located in four clusters in the cerebral ganglia, and (2) the paired canopy cells, located in the lateral lobes. These cells express genes encoding the molluscan insulin-related peptides (MIPs). Six MIP genes have previously been identified. Four of these (I, II, III and V) are expressed in the light green cells and the canopy cells. The MIP-VI gene is a pseudogene. In the present in situ hybridization study, using oligonucleotide probes specific to the transcripts of MIP-I, -II, -III, -IV and -V, no signal was obtained with the MIP-IV probe, indicating that gene IV is also a pseudogene. With the other four probes, two types of light green cells were distinguished. Type-A cells express all four MIP genes, whereas type-B cells do not (or only faintly) express the MIP-I gene. Gene III is relatively strongly expressed in type-B cells. Genes II and V are moderately expressed in both cell types. Type-A cells are mainly located in the periphery of the clusters, whereas type-B cells are present in the center. The canopy cell resembles type-A light green cells. The expression levels of the MIP-II and MIP-V genes are low in the canopy cell. The expression pattern of the MIP genes correlates with the staining pattern of the anti-MIP-C antibody, which has been raised to a synthetic C-fragment shared by MIP-I, -II and -V. Type-A cells stain more intensely with the antibody than type-B cells.

Functional morphology of the neuroendocrine sodium influx-stimulating peptide system of the pond snail, Lymnaea stagnalis, studied by in situ hybridization and immunocytochemistry.

The functional morphology of the neuroendocrine system producing sodium influx-stimulating (SIS) peptide in the pond snail, Lymnaea stagnalis, was studied by in situ hybridization and immunocytochemistry. The SIS-peptide, which is 76 amino acids long, stimulates sodium uptake from the ambient medium. Two synthetic DNA probes were used for in situ hybridization. The nucleotide sequences were chosen from the cDNA structure; they encode amino acids 8-17 and 64-73, respectively. SIS-peptide sequences 10-20 and 67-76 were synthesized and antibodies were raised to them and affinity-purified. In addition to these antibodies, a monoclonal antibody raised to a bioactive, high-pressure liquid chromatography (HPLC)-purified brain extract was used for immunocytochemistry. Paraffin sections of central nervous systems and of whole snails were studied. The SIS-peptide system could be identified as the previously described yellow cell (YC) system by comparing alternate sections treated with the DNA probes, stained with the antibodies, or stained with alcian blue-alcian yellow. SIS-peptide neurons (approximately 45) occur in the ganglia of the visceral ring and in the proximal parts of visceral nerves. Axons run in the nerves of these and in several nerves of other ganglia. Numerous axon branches penetrate the perineurium forming a vast central neurohemal area. The SIS-peptide system innervates the pericardium, the nephridial gland, the reno-pericardial canal, the ureter, the spermoviduct and gonadal acini, the anterior aorta, the ventral buccal artery, and the penis protractor muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of putative egg-laying hormone containing neuronal systems in gastropod molluscs.

Of gastropod molluscs, only in the Aplysiidae and the Lymnaeidae have the genes encoding the respective egg-laying hormones been cloned and the neurons controlling egg laying and egg-laying behavior been identified. Immunocytochemistry, using antibodies raised against alpha-CDCP (one of the neuropeptides encoded on the egg-laying hormone gene of Lymnaea stagnalis), identified neurons in various species of gastropods. In the basommatophoran snail, Biomphalaria glabrata, large and small neurons were observed in areas of the central nervous system similar to where immunoreactive cells exist in L. stagnalis, i.e., in the cerebral and pleural ganglia. In the stylommatophoran snail (Helix aspersa) and the slug (Limax maximus), large immunopositive neurons occur in the visceral and right parietal ganglia. In L. maximus, small immunoreactive neurons were found in the cerebral ganglia while in H. aspersa similar cells were observed intermingled with the large cells in the visceral and right parietal ganglia. Similar to the situation in L. stagnalis, in the female part of the reproductive tract of B. glabrata, L. maximus, and A. californica, but not in H. aspersa, neurons and/or fiber tracts are present. The results indicate that egg-laying hormone precursor molecules of gastropod molluscs are phylogenetically closely related. The alpha-CDCP antiserum may allow the identification of hitherto unknown egg-laying regulating systems of gastropod molluscs.

The VD1/RPD2 neuronal system in the central nervous system of the pond snail Lymnaea stagnalis studied by in situ hybridization and immunocytochemistry.

VD1 and RPD2 are two giant neuropeptidergic neurons in the central nervous system (CNS) of the pond snail Lymnaea stagnalis. We wished to determine whether other central neurons in the CNS of L. stagnalis express the VD1/RPD2 gene. To this end, in situ hybridization with the cDNA probe of the VD1/RPD2 gene and immunocytochemistry with antisera specific to VD1 and RPD2 (the alpha 1-antiserum, Mab4H5 and ALMA 6) and to R15 (the alpha 1 and 16-mer antisera) were performed on alternate tissue sections. A VD1/RPD2 neuronal system comprising three classes of neurons (A1-A3) was found. All neurons of the system express the gene. Division into classes is based on immunocytochemical characteristics. Class A1 neurons (VD1 and RPD2) immunoreact with the alpha 1-antiserum, Mab4H5 and ALMA 6. Class A2 neurons (1-5 small and 1-5 medium sized neurons in the visceral and right parietal ganglion, and two clusters of small neurons and 5 medium-sized neurons in the cerebral ganglia) immunoreact with the alpha 1-antiserum and Mab4H5, but not with ALMA 6. Class A3 neurons (3-4 medium-sized neurons and a cluster of 4-5 small neurons located in the pedal ganglion) immunoreact with the alpha 1-antiserum only. All neurons of the system are immunonegative to the R15 antisera. The observations suggest that the neurons of the VD1/RPD2 system produce different sets of neuropeptides. A group of approximately 15 neurons (class B), scattered in the ganglia, immunostained with one or more of the antisera, but did not react with the cDNA probe in in situ hybridization.

Axonal mapping of the giant peptidergic neurons VD1 and RPD2 located in the CNS of the pond snail Lymnaea stagnalis, with particular reference to the innervation of the auricle of the heart.

VD1 and RPD2 are two giant neuropeptidergic neurons located respectively in the visceral and right parietal ganglion of the central nervous system (CNS) of the pond snail Lymnaea stagnalis. They are the most prominent representatives of a system of neurons expressing a gene that is similar to the gene expressed in R15 of Aplysia californica. Both neuronal systems are involved in the regulation of cardio-respiratory phenomena. In the present study the axonal branches of VD1 and RPD2 were mapped using immunocytochemical and tracer studies. To this end the alpha 1-antiserum (directed to one of the VD1/RPD2 neuropeptides) was used in combination with Lucifer yellow (LY) and Ni-lys tracers. In whole mount preparations of the CNS, immunostained axons of VD1 and RPD2 were observed to run to the pleural, cerebral and pedal ganglia and in several nerves. Upon LY injection of VD1 thin axon branches were observed in the internal right parietal nerve. These run to the skin in the mantle area near the pneumostome and osphradium. The skin of the lips appeared to receive a similar innervation via the lip nerves. Thick LY filled axons of VD1 and RPD2 were observed in the intestinal nerve. They could be traced to the heart region. The pericardial branch of the intestinal nerve innervates the pericardium and heart (Ni-lys tracing). Immunocytochemically, using the alpha 1-antiserum, it was demonstrated that this nerve branch carries the axons of VD1 and RPD2 to the venous side of the auricle, where they enter the pericardial cavity and ramify in the auricle (but not in the ventricle).(ABSTRACT TRUNCATED AT 250 WORDS)