Pro-angiogenic cellular and genomic expression patterns within glioblastoma influences dynamic susceptibility weighted perfusion MRI.

AIM: To investigate whether quantitative dynamic susceptibility-weighted contrast-enhanced (DSC) perfusion magnetic resonance imaging (MRI) metrics are influenced by cellular and genomic expression patterns of glioblastoma angiogenesis. MATERIALS AND METHODS: Twenty-five stereotactic neurosurgical tissue samples were prospectively obtained from enhancing and non-enhancing tumour regions from 10 patients with treatment-naïve glioblastoma. Using monoclonal antibodies, histopathological features of angiogenesis were examined: total microvascular density, vascular morphology, and hypoxia. Angiogenic expression patterns of tissue samples were investigated using RNA microarrays. DSC perfusion MRI metrics were measured from the tissue sampling sites. MRI and histopathological variables were compared using Pearson's correlations. Microarray analysis was performed using false discovery rate (FDR) statistics. RESULTS: Thirteen enhancing and 12 non-enhancing MR image-guided tissue specimens were prospectively obtained. Enhancing tumour regions demonstrated a significant difference in DSC perfusion and histopathological metrics of angiogenesis when compared to non-enhancing regions. Four angiogenic pathways (vascular endothelial growth factor [VEGF], hypoxia inducible factor [HIF], platelet-derived growth factor [PDGF], fibroblast growth factor [FGF]; 25 individual genes) were significantly up-regulated within enhancing regions when compared to non-enhancing regions (adjusted p<0.05, FDR <0.05). A statistically significant correlation was observed between VEGF-A expression, microvascular density, microvascular morphology, and DSC perfusion MRI metrics (p<0.05). CONCLUSION: Pro-angiogenic genomic and cellular expression patterns of treatment-naïve primary glioblastoma significantly influences morphological and physiological DSC perfusion metrics suggesting that expression levels of therapeutically relevant genetic signatures can be quantified using MRI.

Neutralizing the EGF receptor in glioblastoma cells stimulates cell migration by activating uPAR-initiated cell signaling.

In glioblastoma (GBM), the EGF receptor (EGFR) and Src family kinases (SFKs) contribute to an aggressive phenotype. EGFR may be targeted therapeutically; however, resistance to EGFR-targeting drugs such as Erlotinib and Gefitinib develops quickly. In many GBMs, a truncated form of the EGFR (EGFRvIII) is expressed. Although EGFRvIII is constitutively active and promotes cancer progression, its activity is attenuated compared with EGF-ligated wild-type EGFR, suggesting that EGFRvIII may function together with other signaling receptors in cancer cells to induce an aggressive phenotype. In this study, we demonstrate that in EGFRvIII-expressing GBM cells, the urokinase receptor (uPAR) functions as a major activator of SFKs, controlling phosphorylation of downstream targets, such as p130Cas and Tyr-845 in the EGFR in vitro and in vivo. When EGFRvIII expression in GBM cells was neutralized, either genetically or by treating the cells with Gefitinib, paradoxically, the cells demonstrated increased cell migration. The increase in cell migration was explained by a compensatory increase in expression of urokinase-type plasminogen activator, which activates uPAR-dependent cell signaling. GBM cells that were selected for their ability to grow in vivo in the absence of EGFRvIII also demonstrated increased cell migration, due to activation of the uPAR signaling system. The increase in GBM cell migration, induced by genetic or pharmacologic targeting of the EGFR, was blocked by Dasatinib, highlighting the central role of SFKs in uPAR-promoted cell migration. These results suggest that compensatory activation of uPAR-dependent cell signaling, in GBM cells treated with targeted therapeutics, may adversely affect the course of the disease by promoting cell migration, which may be associated with tumor progression.

EGFRvIII promotes glioma angiogenesis and growth through the NF-κB, interleukin-8 pathway.

Sustaining a high growth rate requires tumors to exploit resources in their microenvironment. One example of this is the extensive angiogenesis that is a typical feature of high-grade gliomas. Here, we show that expression of the constitutively active mutant epidermal growth factor receptor, ΔEGFR (EGFRvIII, EGFR*, de2-7EGFR) is associated with significantly higher expression levels of the pro-angiogenic factor interleukin (IL)-8 in human glioma specimens and glioma stem cells. Furthermore, the ectopic expression of ΔEGFR in different glioma cell lines caused up to 60-fold increases in the secretion of IL-8. Xenografts of these cells exhibit increased neovascularization, which is not elicited by cells overexpressing wild-type (wt)EGFR or ΔEGFR with an additional kinase domain mutation. Analysis of the regulation of IL-8 by site-directed mutagenesis of its promoter showed that ΔEGFR regulates its expression through the transcription factors nuclear factor (NF)-κB, activator protein 1 (AP-1) and CCAAT/enhancer binding protein (C/EBP). Glioma cells overexpressing ΔEGFR showed constitutive activation and DNA binding of NF-κB, overexpression of c-Jun and activation of its upstream kinase c-Jun N-terminal kinase (JNK) and overexpression of C/EBPß. Selective pharmacological or genetic targeting of the NF-κB or AP-1 pathways efficiently blocked promoter activity and secretion of IL-8. Moreover, RNA interference-mediated knock-down of either IL-8 or the NF-κB subunit p65, in ΔEGFR-expressing cells attenuated their ability to form tumors and to induce angiogenesis when injected subcutaneously into nude mice. On the contrary, the overexpression of IL-8 in glioma cells lacking ΔEGFR potently enhanced their tumorigenicity and produced highly vascularized tumors, suggesting the importance of this cytokine and its transcription regulators in promoting glioma angiogenesis and tumor growth.

Real-time application of automated ribotyping and DNA macrorestriction analysis in the setting of a listeriosis outbreak.

A cluster of three cases of listeriosis cases occurred against a background of endemic listeriosis in Western Australia. Human and environmental isolates of Listeria monocytogenes obtained during the outbreak investigation were rapidly subtyped by automated ribotyping using an EcoRI protocol and a RiboPrinter. DNA macrorestriction analysis by pulsed-field gel electrophoresis (PFGE) was used to confirm the relatedness of isolates. Serogroup 1/2 predominated among the food samples and the four clinical isolates from the outbreak cluster were also of this serogroup. All isolates from chicken material were serogroup 1/2 and indistinguishable by ribotype pattern. PFGE subdivided strains of this ribotype into four subtypes. The preliminary analysis had an immediate impact on hypothesis generation, environmental health investigations, environmental specimen collection and initial control measures. Sufficient typing data to guide environmental health and disease control initiatives was generated in less than one week by combining automated ribotyping with PCR-based detection of L. monocytogenes in suspect foodstuffs and an L. monocytogenes DNA probe. There were no further cases of bacteriologically confirmed listeriosis in Western Australia for six months after completion of the investigation.

Control of the nuclear-cytoplasmic partitioning of annexin II by a nuclear export signal and by p11 binding.

This study investigated mechanisms controlling the nuclear-cytoplasmic partitioning of annexin II (AnxII). AnxII and its ligand, p11, were localized by immunofluorescence to the cytoplasmic compartment of U1242MG cells, with minimal AnxII or p11 detected within nuclei. Similarly, GFP-AnxII and GFP-p11 chimeras localized to the endogenous proteins. Likewise, GFP-AnxII(1-22) was excluded from nuclei, whereas GFP-AnxII(23-338) and GFP alone were distributed throughout the cells. Immunoprecipitation and biochemical studies showed that GFP-AnxII did not form heteromeric complexes with endogenous p11 and AnxII. Thus, the AnxII N-tail is necessary and sufficient to cause nuclear exclusion of the GFP fusion protein but this does not involve p11 binding. A nuclear export signal consensus sequence was found in the AnxII 3-12 region. The consensus mutant GFP-AnxII(L10A/L12A) confirmed that these residues are necessary for nuclear exclusion. The nuclear exclusion of GFP-AnxII(1-22) was temperature-dependent and reversible, and the nuclear export inhibitor leptomycin B (LmB) caused GFP-AnxII or overexpressed AnxII monomer to accumulate in nuclei. Therefore, AnxII monomer can enter the nucleus and is actively exported. However, LmB had little effect on the localization of AnxII/p11 complex in U1242MG cells, indicating that the complex is sequestered in the cytoplasm. By contrast, LmB treatment of v-src-transformed fibroblasts caused endogenous AnxII to accumulate in nuclei. The LmB-induced nuclear accumulation of AnxII was accelerated by pervanadate and inhibited by genistein, suggesting that phosphorylation promotes nuclear entry of AnxII. Thus, nuclear exclusion of AnxII results from nuclear export of the monomer and sequestration of AnxII/p11 complex, and may be modulated by phosphorylation.

Mechanical trauma induces rapid astroglial activation of ERK/MAP kinase: Evidence for a paracrine signal.

Astrogliosis is a prominent and ubiquitous reaction of astrocytes to many forms of CNS injury, often implicated in the poor regenerative capacity of the adult mammalian CNS. Transmembrane signals that rapidly trigger and maintain astroglial responses to injury are largely undefined. Several candidate inducers of astrogliosis, including growth factors and neuropeptides, act via the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway. We previously observed chronically activated ERK/MAPK in human reactive astrocytes. To investigate mechanisms of pathway activation in a defined in vitro model, primary cultured astroglial monolayers were subjected to focal mechanical injury. Within 2-10 min, ERK/MAPK was activated, but only in cells near the wound edge. By 30 min, the entire monolayer showed activation, which persisted for 4 to 8 h. ERK/MAPK activation was specifically blocked by application of the MEK inhibitors, PD98059 and U0126. Cell-cell contact was not necessary for intercellular spread of ERK/MAPK activation, and ERK/MAPK-stimulating activity was found in the injury-conditioned medium. The activating factor was shown to have a native size of 50-100 kD and did not signal through the classical EGF receptor. Injury-induced signaling to ERK/MAPK required Ras, as demonstrated by specific blockade after transient transfection with a dominant negative Ha-RasN17 construct. Finally, we demonstrated that focal lesioning of adult rat cortex induces a rapid activation and spreading of astroglial ERK/MAPK, suggesting that similar mechanisms may operate in astroglial activation following acute brain injury.

Phorbol 12-myristate 13-acetate induces protein kinase ceta-specific proliferative response in astrocytic tumor cells.

Protein kinase C (PKC) activation has been implicated in cellular proliferation in neoplastic astrocytes. The roles for specific PKC isozymes in regulating this glial response, however, are not well understood. The aim of this study was to characterize the expression of PKC isozymes and the role of PKC-eta expression in regulating cellular proliferation in two well characterized astrocytic tumor cell lines (U-1242 MG and U-251 MG) with different properties of growth in cell culture. Both cell lines expressed an array of conventional (alpha, betaI, betaII, and gamma) and novel (theta and epsilon) PKC isozymes that can be activated by phorbol myristate acetate (PMA). Another novel PKC isozyme, PKC-eta, was only expressed by U-251 MG cells. In contrast, PKC-delta was readily detected in U-1242 MG cells but was present only at low levels in U-251 MG cells. PMA (100 nm) treatment for 24 h increased cell proliferation by over 2-fold in the U-251 MG cells, whereas it decreased the mitogenic response in the U-1242 MG cells by over 90%. When PKC-eta was stably transfected into U-1242 MG cells, PMA increased cell proliferation by 2.2-fold, similar to the response of U-251 MG cells. The cell proliferation induced by PMA in both the U-251 MG and U-1242-PKC-eta cells was blocked by the PKC inhibitor bisindolylmaleimide (0.5 micrometer) and the MEK inhibitor, PD 98059 (50 micrometer). Transient transfection of wild type U-251 with PKC-eta antisense oligonucleotide (1 micrometer) also blocked the PMA-induced increase in [(3)H]thymidine incorporation. The data demonstrate that two glioblastoma lines, with functionally distinct proliferative responses to PMA, express different novel PKC isozymes and that the differential expression of PKC-eta plays a determining role in the different proliferative capacity.

Stable antisense RNA expression neutralizes the activity of low-density lipoprotein receptor-related protein and promotes urokinase accumulation in the medium of an astrocytic tumor cell line.

Low-density lipoprotein receptor-related protein (LRP) binds and internalizes multiple ligands that are structurally and functionally diverse. However, the effects of LRP on cellular phenotype remain unclear. To study LRP in human astrocytic tumor cells, we designed LRP antisense RNA expression constructs in which the antisense cDNA fragment was expressed under the control of the cytomegalovirus (CMV) promoter. U-1242 MG astrocytic tumor cells were transfected with the antisense constructs and cloned from single cells to yield multiple cell lines with decreased LRP expression. Further studies were performed with two cell lines in which LRP antigen was completely eliminated (L(alpha)42) or substantially decreased (Lalpha47), as determined by Western blot analysis. Untransfected U-1242 MG cells and cells that were stably transfected with empty vector (pBK-CMV) bound activated alpha2-macroglobulin (alpha2M) in a specific and saturable manner. The Bmax was about 5000 receptors/cell. Lalpha42 cells did not bind alpha2M, and binding was decreased by >60% in Lalpha47 cells. Lalpha42 and Lalpha47 cells also demonstrated reduced susceptibility to the cytotoxin, Pseudomonas exotoxin A, and accumulated greatly increased levels of urokinase-type plasminogen activator (uPA) in conditioned medium. The accumulation of uPA demonstrates a major role for LRP in the catabolism of this protein in astrocytic tumor cells. The LRP-deficient cell lines, developed using antisense technology, represent a new model system for studying LRP function in astrocytes.

Epidermal growth factor differentially regulates low density lipoprotein receptor-related protein gene expression in neoplastic and fetal human astrocytes.

Low density lipoprotein receptor-related protein (LRP) is a multifunctional endocytotic receptor that may modify the biological activity of reactive astrocytes in neuroplasticity and neurodegeneration and of malignant astrocytes in brain invasion. In this study, the regulation of LRP by epidermal growth factor receptor (EGFR) ligands in both cultured human fetal astrocytes and astrocytic tumor cell lines (U-251 MG and U-1242 MG) was investigated. All astrocytic cell types expressed LRP, as determined by the binding of activated alpha2-macroglobulin (alpha2M*) on intact cells and by Western and Northern blot analyses of cell extracts. Primary cultured astrocytes expressed the highest levels of alpha2M*-binding capacity (Bmax = 30 fmol/mg protein). This was twofold higher than for the U-1242 MG astrocytoma cells (Bmax = 15 fmol/mg protein) and fourfold greater than for the glioblastoma U-251 MG cells (7.0 fmol/mg protein). Receptor affinity (K(D)) ranged from 0.25 to 0.6 nM in all the astroglial cell types. Functional LRP at the surface was down-regulated by EGF, compared with controls, as indicated by a reduction of both Bmax and LRP-mediated endocytosis by approximately 50% and 60%, respectively. In comparison, EGF treatment of primary astrocytes did not down-regulate LRP expression or LRP-mediated endocytosis. Treatment of the tumor cells with EGF or TGFalpha (25 ng/ml) significantly down-regulated total cellular LRP. Receptor-associated protein (RAP) mRNA expression was not affected by EGF in either tumor cells or primary astrocytes. The reduction of LRP in the tumor cells resulted from a specific decrease in LRP mRNA transcription, as determined by Northern blot and nuclear run-on experiments. These data suggest that EGF mediates a functional down-regulation of LRP endocytotic activity in astrocytic tumor cells and that LRP expression is differentially regulated in neoplastic and non-neoplastic astrocytes.

Insulin-like growth factor-1 content and pattern of expression correlates with histopathologic grade in diffusely infiltrating astrocytomas.

Studies of experimental tumorigenesis have strongly implicated signaling of the insulin-like growth factor 1 (IGF-1) as a key component in astrocytic neoplasia; however, its role in the growth of low-grade and malignant human tumors is not well understood. Correlative analyses of IGF-1, p53, and Ki-67 (MIB-1) immunohistochemistry and IGF-1 receptor (IGF-1R) mRNA expression were performed to examine the cellular pattern of IGF-1 signaling in 39 cases of astrocytoma (World Health Organization grades II-IV). Tumor cells expressing IGF-1 and IGF-1R were present in all tumor grades. The proportion of tumor cells that expressed IGF-1 correlated with both histopathologic grade and Ki-67 labeling indices, while expression of IGF-1R mRNA correlated with Ki-67 indices. In cases where stereotactic tissue sampling could be identified with a specific tumor area by neuroimaging features, the numbers of IGF-1 immunoreactive cells correlated with the tumor zones of highest cellularity and Ki-67 labeling. In glioblastomas, the localization of IGF-1 immunoreactivity was notable for several features: frequent accentuation in the perivascular tumor cells surrounding microvascular hyperplasia; increased levels in reactive astrocytes at the margins of tumor infiltration; and selective expression in microvascular cells exhibiting endothelial/pericytic hyperplasia. IGF-1R expression was particularly prominent in tumor cells adjacent to both microvascular hyperplasia and palisading necrosis. These data suggest that IGF-1 signaling occurs early in astroglial tumorigenesis in the setting of cell proliferation. The distinctive correlative patterns of IGF-1 and IGF-1R expression in glioblastomas also suggest that IGF-1 signaling has an association with the development of malignant phenotypes related to aberrant angiogenesis and invasive tumor interactions with reactive brain.

ERK/MAP kinase is chronically activated in human reactive astrocytes.

Reactive astrogliosis is the most prominent macroglial response to diverse forms of CNS injury. We assessed a potential role for the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway because it represents a common effector for several major families of transmembrane receptors implicated in astrogliosis. Immunohistochemical detection of activated ERK/MAPK in a series of human neurosurgical specimens utilizing phosphorylation state-dependent antibodies consistently revealed intense immunoreactivity in reactive astrocytes in both subacute and chronic lesions, including infarct, mechanical trauma, chronic epilepsy, and progressive multifocal leukoencephalopathy. Neurons, oligodendroglia, and most inflammatory cells showed little or no detectable activation. These observations suggest a testable hypothesis: activation of the ERK/MAPK pathway is an obligatory step for the triggering and/or persistence of reactive astrogliosis.

In situ visualization of intratumor growth factor signaling: immunohistochemical localization of activated ERK/MAP kinase in glial neoplasms.

Abnormal growth factor signaling is implicated in the pathogenesis of gliomas. The extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway is a likely target, linking receptor tyrosine kinase activation to downstream serine/threonine phosphorylation events regulating proliferation and differentiation. Signaling within heterogeneous cell populations of gliomas cannot be adequately assessed by traditional biochemical enzyme assays. Immunohistochemical detection of doubly phosphorylated (activated) ERK/MAPK permitted visualization of spatially discrete cellular patterns of ERK/MAPK activation, compared with the relatively uniform expression of total ERK/MAPK protein. The astrocytic tumors, regardless of grade, had the highest overall degree of enzyme activation, whereas oligodendrogliomas had the least. Anaplastic progression in oligodendrogliomas resulted in a larger number of cells with active ERK/MAPK. Within glioblastomas, microvascular hyperplasia and necrosis were associated with ERK/MAPK activation in adjacent tumor cells. In addition to spatial patterns of intratumor paracrine signaling, a possible cell-cycle-associated regulation was detected: mitotic and actively cycling tumor cells showed diminished activation relative to cells in G0. Although ERK/MAPK activation was not restricted to neoplastic glia, consistent patterns of selective activation in tumor cells suggests that sustained activation may contribute to the neoplastic glial phenotype.

Annexins I and II bind to lipid A: a possible role in the inhibition of endotoxins.

Annexins are Ca2+-dependent phospholipid-binding proteins with anti-inflammatory properties that are present on the surfaces of, and released from, certain cell types, such as leukocytes and secretory epithelia. The present study investigated the possibility that annexins may bind directly to bacterial endotoxin, inhibiting its interactions with cellular receptors or accessory binding proteins. An enzyme-linked immunoassay demonstrated calcium-dependent binding of low nanomolar concentrations of annexin-I and annexin-II p36/p11 heterotetramer to lipid A. In contrast, little or no annexin binding to lipopolysaccharide (LPS) was detected under similar conditions. LPS-binding protein binding to lipid A was blocked by annexin-I, and the annexins inhibited nitrite generation in RAW 264.7 cells induced by lipid A but not that induced by LPS. The data suggest that direct binding of annexins to lipid A may represent a mechanism for suppressing cellular and systemic responses to endotoxin.

Efficacy of adenoidectomy in relieving symptoms of chronic sinusitis in children.

OBJECTIVE: To determine the efficacy of adenoidectomy in relieving symptoms of chronic sinusitis in children. DESIGN: Retrospective case series. SETTING: Tertiary care center. PATIENTS: Symptoms of chronic sinusitis were studied in 48 consecutive patients who underwent adenoidectomy or adenotonsillectomy between October 1993 and May 1995. Children with cystic fibrosis or those who underwent concurrent endoscopic sinus surgery were excluded. Patient ages ranged from 1 to 12 years. Four patients were unavailable for follow-up and did not complete the study. MAIN OUTCOME MEASURES: Patients' charts were reviewed for the presence of preoperative symptoms, including rhinorrhea, nasal congestion, headache, postnasal drainage, cough, halitosis, and irritability. Also recorded were mouth breathing, fevers, and frequent antibiotic use. Telephone interviews with the patients' caregivers were conducted to collect information following the surgery regarding the presence of the same symptoms as well as an estimate of overall improvement. Follow-up ranged from 5 months to 2 years. RESULTS: The most frequently reported symptoms before surgery were rhinorrhea, nasal congestion, mouth breathing, and frequent antibiotic use (35, 37, 34, and 38 patients, respectively). These numbers decreased following surgery to 18, 20, 11, and 10 patients, respectively. The average number of symptoms experienced by each patient decreased from 5.3 to 2.9. Complete or near symptom resolution was reported in 25 (58%) of 43 patients. Some improvement was reported in another 9 patients (21%). Minimal or no improvement was reported in 9 patients (21%). To date, only 3 patients have gone on to have endoscopic sinus surgery. CONCLUSION: In the majority of cases, symptoms of chronic sinusitis in children are relieved by adenoidectomy.

Quantitative chromatin pattern description in Feulgen-stained nuclei as a diagnostic tool to characterize the oligodendroglial and astroglial components in mixed oligo-astrocytomas.

The oligoastrocytoma, as a mixed glioma, represents a nosologic dilemma with respect to precisely defining the oligodendroglial and astroglial phenotypes that constitute the neoplastic cell lineages of these tumors. In this study, cell image analysis with Feulgen-stained nuclei was used to distinguish between oligodendroglial and astrocytic phenotypes in oligodendrogliomas and astrocytomas and then applied to mixed oligoastrocytomas. Quantitative features with respect to chromatin pattern (30 variables) and DNA ploidy (8 variables) were evaluated on Feulgen-stained nuclei in a series of 71 gliomas using computer-assisted microscopy. These included 32 oligodendrogliomas (OLG group: 24 grade II and 8 grade III tumors according to the WHO classification), 32 astrocytomas (AST group: 13 grade II and 19 grade III tumors), and 7 oligoastrocytomas (OLGAST group). Initially, image analysis with multivariate statistical analyses (Discriminant Analysis) could identify each glial tumor group. Highly significant statistical differences were obtained distinguishing the morphonuclear features of oligodendrogliomas from those of astrocytomas, regardless of their histological grade. When compared with the 7 mixed oligoastrocytomas under study, 5 exhibited DNA ploidy and chromatin pattern characteristics similar to grade II oligodendrogliomas, I to grade III oligodendrogliomas, and I to grade II astrocytomas. Using multifactorial statistical analyses (Discriminant Analysis combined with Principal Component Analysis). It was possible to quantify the proportion of "typical" glial cell phenotypes that compose grade II and III oligodendrogliomas and grade II and III astrocytomas in each mixed glioma. Cytometric image analysis may be an important adjunct to routine histopathology for the reproducible identification of neoplasms containing a mixture of oligodendroglial and astrocytic phenotypes.

Ganglioglioma: an ultrastructural and immunohistochemical study.

BACKGROUND: Ganglioglioma is a rare, mixed neuronal-glial neoplasm of the central nervous system that occurs in young patients and has a benign clinical course. METHODS: To define the immunophenotypic and morphologic features of ganglioglioma precisely, 27 specimens were studied by routine histochemistry, 21 specimens by immunochemistry, and 14 specimens were examined at the ultrastructural level. RESULTS: The age of the 27 patients, 14 males and 13 females, ranged from 3 to 52 years (mean, 22 years). The most commonly affected site was the temporal lobe (13 patients). Three patients experienced a local recurrence. Microscopically, the tumors were comprised of well differentiated, somewhat abnormal neurons as well as glial cells, the latter including astrocytes of fibrillary (59%) and pilocytic (41%) type. Scant mitotic activity was observed in 2 tumors (7%). Glial cells of all tumors were immunoreactive for glial fibrillary acidic protein, S-100 protein, and vimentin. Ki-67 labeling indices (LI) ranged from 0.6 to 10.5% (mean, 2.7%) and p53 LI from 1.1 to 42.4% (mean, 15.6%). Ki-67 and p53 LI in recurrent tumors were significantly higher than those of nonrecurrent ones (P = 0.036 and 0.026, respectively). No examples of anaplastic transformation were encountered. Immunohistochemically, many neuronal cells were positive for synaptophysin (100%), Class 3 beta-tubulin (100%), neurofilament protein (90%), and chromogranin A (86%), in addition to S-100 protein (71%) and, occasionally, vimentin (24%). Ultrastructural characteristics of neuronal cells included the presence of numerous, 100-230-nanometer dense core granules within both perikarya and cell processes, well developed rough endoplasmic reticulum, microtubules within cell processes, and synapses associated with clear vesicles. Astrocytic cells usually contained abundant intermediate filaments; their cell membranes, when abutting the stroma, were covered by basal lamina. CONCLUSIONS: Gangliogliomas are comprised of well differentiated neuronal cells and glial cells that are very often of pilocytic type. No cells with features intermediate between neurons and glia were observed. Neuronal cells are characterized by prominent neurosecretory features distinct from those of normal neurons in the central nervous system. Higher Ki-67 and p53 LI may indicate more aggressive behavior.

Low density lipoprotein receptor-related protein is expressed early and becomes restricted to a somatodendritic domain during neuronal differentiation in culture.

Low density lipoprotein receptor-related protein (LRP) is a multi-functional receptor which mediates the endocytotic uptake of several ligands implicated in neuronal pathophysiology. In this study, LRP expression and localization, in cultured hippocampal neurons from 18-day-old rats, were examined by immunofluorescence microscopy. LRP was restricted to the cell bodies and dendrites of mature neurons, where it was uniformly distributed on both dendritic shafts and spines. Immunoreactive protein was detected within the first 24 h of culture and acquired a polarized distribution by the end of the first week. Expression of LRP mRNA by the cultured neurons was demonstrated by Northern blot analysis. Binding studies with the LRP ligand, activated alpha2-macroglobulin, confirmed that LRP was present and functional on the hippocampal neuron cell surface. These studies demonstrate that neuronal LRP undergoes selective compartmentation during neuronal maturation and suggest that LRP-mediated endocytosis is largely restricted to the somatodendritic compartment.

Maturation of intracranial immature teratomas. Report of two cases.

Immature teratomas arising within the central neuraxis are rare neoplasms. These tumors contain diverse cell lineages that retain an embryonal character and display phenotypic differentiation attributed to the three classic germ layers. The clinical management of these lesions is unclear, due in part to their low incidence and to an incomplete understanding of their natural history. Although the potential for phenotypic differentiation and cellular maturation within immature teratomas arising in the gonads is well documented, this has not been described in the intracranial tumors. In the present report, the authors describe two cases of intracranial immature teratomas, one involving the pineal region and the other involving the left frontotemporal lobes, which underwent cellular differentiation and maturation. At initial resection, the tumors from both cases were composed predominantly of primitive neuroepithelial tissue that was admixed with immature and differentiating mesenchymal and epithelial structures. No foci of germinoma, endodermal sinus, choriocarcinoma, or embryonal carcinoma tissue were present. Subsequent resections in both cases revealed an absence of immature tissue. The tumor in Case 1 contained only differentiated epithelial and mesenchymal tissue with no neuroepithelial component, whereas the tumor in Case 2 demonstrated abundant mature neuronal and glial tissue. These two cases from different intracranial sites suggest that spontaneous maturation may be a significant aspect of the natural history of intracranial immature teratomas.

Transcriptional regulation of LDL receptor-related protein by IFN-gamma and the antagonistic activity of TGF-beta(1) in the RAW 264.7 macrophage-like cell line.

Low density lipoprotein receptor-related protein (LRP) is a major receptor for multiple ligands, including chylomicron and VLDL remnants, bacterial toxins, viruses, proteinases, lipoprotein lipase, and activated alpha2-macroglobulin (alpha2M). In this study, we used Northern blot analyses and nuclear run-on experiments to demonstrate that interferon-gamma (IFN-gamma) causes a concentration-dependent decrease in steady-state LRP mRNA expression and gene transcription rate in RAW 264.7 cells. IFN-gamma also markedly increased expression of inducible nitric oxide synthase (NOS), as expected; however, the increase in nitric oxide was not responsible for the down-regulation of LRP expression since the NOS inhibitor, N(G)-monomethyl-L-arginine, did not preserve LRP expression in IFN-gamma-treated cells. Transforming growth factor-beta1 (TGF-beta1; 2.5 ng/mL) had no independent effect on LRP expression and did not modify the response to IFN-gamma when the two cytokines were added simultaneously to cultures. When TGF-beta1 was added 24 h prior to IFN-gamma, the extent of LRP down-regulation was significantly reduced. Specific binding of the LRP ligand, activated (125)I-alpha2M, was decreased by 76 +/- 5% in cells treated with 100 U/mL IFN-gamma, but only by 45 +/- 7% in cells treated with 100 U/mL IFN-gamma after TGF-beta1-pretreatment. The antagonistic activity of TGF-beta1 on the IFN-gamma response in RAW 264.7 cells did not result from a change in LRP mRNA stability or IFN-gamma receptor expression, as determined by Northern blot analyses and (125)I-IFN-gamma binding experiments. The studies presented here suggest that the balance between IFN-gamma and TGF-beta1 may be critical in determining LRP expression at sites of infection and inflammation.