A new EAE model of brain demyelination induced by intracerebroventricular pertussis toxin.

Experimental autoimmune encephalomyelitis (EAE) is a primary animal model of multiple sclerosis (MS). MS predominantly presents with evidence of lesions in the subcortical periventricular white matter regions of the brain. Research into the pathogenesis of the demyelinating lesions in the brain has been hampered by the fact that conventional models of EAE present with progressive ascending paralysis which recapitulates mainly the spinal cord lesions of multiple sclerosis. There is little evidence of brain involvement. Systemic administration of pertussis toxin (PTx) has been shown to induce the proinflammatory cascade of TGF-beta, IL-6, and Th17 in the central nervous system, which recently has been identified as essential in the development of EAE. To determine whether intracerebroventricular (icv) administration of PTx would result in subcortical periventricular demyelinating lesions in the brain, we examined the effect in a MOG induced EAE model. We found that icv PTx induced subcortical periventricular brain lesions that resemble the pathologic demyelinating lesions of MS. Moreover, icv PTx induced Th17 infiltration and increased expression of cytokines IL-6 and TGF-beta. We thus generated a highly reproducible model with remarkable histological similarities to the predominant demyelinating brain lesions seen in MS.

IL-21 modulates CD4+ CD25+ regulatory T-cell homeostasis in experimental autoimmune encephalomyelitis.

The homeostasis of CD4+ CD25+ regulatory T cells (Tregs) depends on the cytokine interleukin (IL)-2. As IL-21 shares sequence homology with IL-2 and the IL-21 receptors contain a gamma-chain common to IL-2, we hypothesized that IL-21 could also affect the homeostasis of Tregs. We tested this hypothesis in experimental autoimmune encephalomyelitis (EAE), an animal model of relapsing-remitting human multiple sclerosis. We show that blockade of IL-21 in SJL/J mice before and after the induction of EAE enhances the influx of inflammatory cells into the central nervous system (CNS). The blockade of IL-21 leads to proliferation of proteolipid peptide (PLP(139-151))-autoreactive CD4+ T cells, which are capable to cause severe EAE in adoptively transferred recipient mice. Conversely, Tregs from mice where IL-21 was blocked, lose their capacity to prevent EAE induced PLP(139-151)-reactive T cells. Notably, direct effects of IL-21 on Tregs are confirmed by studies of blockade of IL-21 in mice expressing a green fluorescent protein 'knocked' into a Foxp3 allele, in which a reduction of the number of Tregs and a downregulation of their frequency and expression of Foxp3 are observed. These data suggest a role of the IL-21/IL-21R axis in the homeostasis of Tregs in CNS autoimmunity.

Hypothalamic hamartomas: Correlation of MR imaging and spectroscopic findings with tumor glial content.

BACKGROUND AND PURPOSE: There is variability in the literature concerning the appearance and histology of hypothalamic hamartomas. This study correlates the MR imaging and proton MR spectroscopic properties of hypothalamic hamartomas with histopathologic findings. METHODS: Studies were performed with 3T and 1.5T scanners. Single voxel hamartoma spectra were acquired by using short-echo-time point-resolved spectroscopy sequences (PRESS). 2D PRESS chemical shift imaging (CSI) spectroscopic sequences were also obtained for comparison of tumor-derived spectra with normal gray matter of the amygdala. Sequences were used to compare choline (Cho), N-acetylaspartate (NAA), and myoinositol (mI) resonances by using a creatine (Cr) reference. Spectral ratios and T2 signal intensity ratios of the hamartomas were then compared with histopathologic findings. RESULTS: Data from single voxel spectroscopic sequences demonstrated a statistically significant decrease in NAA/Cr and an increase in mI/Cr ratios in tumor tissue when compared with values in normal gray matter of the amygdala. In addition, Cho/Cr ratios were also increased when compared with those in normal gray matter controls. Among the 14 hamartomas sampled, a spectrum of increased mI/Cr ratios was seen. Those tumors with markedly elevated mI/Cr demonstrated an increased glial component when compared with the remaining tumors. Increased glial component was also found to have a positive correlation with hyperintensity of lesions on T2-weighted images. CONCLUSION: We have identified a correlation between the glial/neuronal fraction as determined by histopathology and MR spectral and T2 hyperintensity variations among hypothalamic hamartomas.

Expression of the tumor suppressor gene DCC in human gliomas.

Reduced expression and/or allelic loss of the putative tumor suppressor gene DCC has been demonstrated in colorectal, gastric, pancreatic, esophageal, breast, and hematological malignancies. We examined the expression of the DCC gene in 22 tissue samples from human gliomas (glioblastoma multiforme, oligodendroglioma, and mixed oligodendroglioma/astrocytoma). Seven of 8 glioblastomas multiforme (88%) had reduced or absent DCC expression, and 8 of the other 14 tumors underexpressed DCC when compared to normal brain tissue. These results demonstrate that reduced expression of DCC occurs in human malignant gliomas and may be part of a common genetic pathway leading to neoplastic transformation and/or tumor progression.

Cytogenetic and flow cytometry DNA analysis of regional heterogeneity in a low grade human glioma.

This study combined flow cytometry with standard cytogenetic analysis of first division cells to evaluate regional heterogeneity in 38 spatially mapped regions of a low grade human oligoastrocytoma. Histologically, the tumor was relatively homogeneous. In contrast, flow cytometry and cytogenetic analyses identified variable percentages of near-diploid (ND; 35 to 57 chromosomes/metaphase) and near-tetraploid (81-103 chromosomes/metaphase) populations. The largest proportion of cells in the ND population was 46,XY with normal Giemsa bands; however, four karyotypically unrelated ND clones also were identified. The development of these clonal populations centered around a region in which more than 50% of the cells contained nonclonal abnormalities and which demonstrated more histological pleomorphism than any other region. The frequency of the nonclonal karyotypes suggested that this region was genetically unstable. Three of the clonal ND populations resided in small, spatially discreet areas of the tumor. The largest and the most widely distributed clonal population, 47,XY,+7, underwent further evolution to give rise to seven additional sidelines. This investigation demonstrates that low grade gliomas have areas of genetic instability capable of generating mutant cells with the capacity to proliferate and to form cellular foci. As a result, multiple, spatially distinct clonal populations can exist in low grade gliomas, some of which are capable of further cytogenetic evolution and clonal expansion, resulting in tumor progression.

Identification and validation of P311 as a glioblastoma invasion gene using laser capture microdissection.

The mRNA expression profiles from glioblastoma cells residing at the tumor core and invasive rim of a human tumor resection were compared. From a single tumor specimen, 20,000 single cells from each region were collected by laser capture microdissection. Differential expression of 50-60 cDNA bands was detected. One of the sequences overexpressed by the invasive cells showed 99% homology to the P311 gene, the protein product of which is reported to localize at focal adhesions. Relative overexpression of P311 by invading glioblastoma cells compared with tumor core was confirmed by quantitative reverse transcription-PCR of six glioblastoma specimens after laser capture microdissection collection of rim and core cells. In vitro studies using antisense oligodeoxynucleotides and integrin activation confirmed the role of P311 in supporting migration of malignant glioma cells. Immunochemistry studies confirmed the presence of the P311 protein in tumor cells, particularly at the invasive edge of human glioblastoma specimens.

Retrospective study of the correlation between the DNA repair protein alkyltransferase and survival of brain tumor patients treated with carmustine.

We tested the hypothesis that the level of the DNA repair protein O6-alkylguanine-DNA alkyltransferase in brain tumors was correlated with resistance to carmustine (BCNU) chemotherapy. Alkyltransferase levels in individual cells in sections from 167 primary brain tumors treated with BCNU were quantitated with an immunofluorescence assay using monoclonal antibodies against human alkyltransferase. Patients with high levels of alkyltransferase had shorter time to treatment failure (P = 0.05) and death (P = 0.004) and a death rate 1.7 times greater than patients with low alkyltransferase levels. Furthermore, the size of the subpopulation of cells with high levels of alkyltransferase was correlated directly with drug resistance. For all tumors the variables most closely correlated with survival, in order of importance, were age, tumor grade, and alkyltransferase levels. For glioblastoma multiforme, survival was more strongly correlated with alkyltransferase levels than with age. These results should encourage prospective studies to evaluate alkyltransferase levels as a method, for identifying brain tumor patients with the best likelihood of response to BCNU chemotherapy.

Nuclear accumulation of fibroblast growth factor receptors in human glial cells--association with cell proliferation.

In this study we describe the presence of high affinity FGF-2 binding sites in the nuclei of U251MG glioma cells (K(d)=7 pM). Immunoprecipitation of total cell extracts with FGF receptor (FGFR) 1-4 antibodies showed that U251MG glioma cells express only FGFR1. [125I]FGF-2 cross linking to nuclear extracts followed by FGFR1 immunoprecipitation showed that FGFR1 may account for the nuclear FGF-2 binding sites. Western blot analysis demonstrated the presence of 103, 118 kDa and small amounts of 145 kDa FGFR1 isoforms in the nuclei of glioma cells. All isoforms contain both the C- and N-terminal domains. Nuclear FGFR1 retains kinase activity. Immunocytochemistry using confocal microscopy showed specific FGFR1 immunoreactivity within the nuclear interior. In continuously proliferating glioma cells, nuclear FGFR1 is constitutively expressed, independent of cell density. In contrast, in nontransformed human astrocytes, nuclear FGFR1 levels fluctuate with the proliferative state of the cell. In quiescent, confluent astrocytes nuclear FGFR1 protein was depleted. An accumulation of nuclear FGFR1 was observed following the transition to a subconfluent, proliferating state. Transfection of a pcDNA3.1-FGFR1 expression vector into glioma cells that do not express FGFR1 resulted in the nuclear accumulation of FGFR1, increased cell proliferation, and stimulated transition from the G0/G1 to the S-phase of the cell cycle. The increased proliferative rate was resistant to inhibition by the cell-impermeable FGF binding antagonist, myoinositol hexakis [dihydrogen phosphate]. Our results suggest that the constitutive nuclear presence of FGFR1 contributes to the increased proliferation of glioma cells while the transient nuclear accumulation of FGFR1 in normal astrocytes may play a role in the transition to a reactive state.

Nuclear accumulation of FGF-2 is associated with proliferation of human astrocytes and glioma cells.

FGF-2 has been implicated in the neoplastic transformation of glioma cells and in the transition of normal quiescent astrocytes to a proliferating, reactive state. In the present study we have observed that in human glial cells, levels and subcellular localization of FGF-2 are different in quiescent and proliferating cells. FGF-2 was detected in the cytoplasm of non-reactive astrocytes in human brain sections. In contrast FGF-2 was located within the cytoplasm and nuclei of reactive astrocytes in gliotic brain tissue and in neoplastic cells of glioma tumors. In vitro, FGF-2 was found predominantly in the nucleus of subconfluent proliferating astrocytes, but was detected only in the cytoplasm of density arrested quiescent astrocytes. Our results suggest that reduced cell contact stimulates nuclear accumulation of FGF-2, accompanying mitotic activation of reactive human astrocytes. FGF-2 was constitutively localized to the nucleus of continuously proliferating glioma cells independent of cell density. A role for intracellular FGF-2 was further suggested by the observation that glioma cells that are not stimulated to proliferate by extracellular FGF-2 proliferated faster when transfected with FGF-2 expressing vectors. This increased proliferation correlated with nuclear accumulation of FGF-2. Cell proliferation was attenuated by 5'-deoxy-5'-methylthioadenosine, a FGF-2 receptor tyrosine kinase inhibitor that acts within the cell, but was unaffected by myo-inositol hexakis [dihydrogen phosphate] that disrupts FGF-2 binding to plasma membrane receptors. Our results indicate that FGF-2 serves as a nuclear regulator of proliferation in astrocytic cells. In glioma cells, the constitutive presence of FGF-2 in the nucleus may promote proliferation that is insensitive to cell contact inhibition.

Biological and molecular analysis of a low-grade recurrence of a glioblastoma multiforme.

We and others have reported that human malignant gliomas demonstrate intratumor heterogeneity in which many regions may be benign; however, the presence of regions of increased malignancy in these same tumors is generally indicative of poor patient prognosis. These data suggested that tumor progression may be a local phenomenon, resulting in regions that progress to a more malignant type prior to the progression of the entire tumor. Implicit in this premise is the idea that molecular markers of tumor progression may be detectable prior to histological evidence of progression. This report details analyses performed on a primary and recurrent tumor obtained from the same patient in which the primary tumor was of a higher histological grade than the recurrent tumor. Results of molecular, cytogenetic, flow cytometric, and histological analyses of the primary tumor were indicative of a grade 4 glioblastoma multiforme. Standard cytogenetic and flow cytometric analyses demonstrated that the cells were near-diploid with a stem line population of 46,XX normal G-banded karyotypes. In contrast, tissue resected from the recurrent tumor 5 months later was histologically less malignant; however, the molecular, cytogenetic, and flow cytometric analyses of this sample demonstrated the presence of specific genetic abnormalities typically found in more malignant tumors. These data demonstrate that specific molecular and/or genetic changes leading to tumor progression may become detectable in a glioma prior to the appearance of histological features of a higher grade tumor.

Death-associated protein 3 (Dap-3) is overexpressed in invasive glioblastoma cells in vivo and in glioma cell lines with induced motility phenotype in vitro.

PURPOSE: To discover the genetic determinants of glioma invasion in vivo, we compared the mRNA expression profiles of glioblastoma cells residing at the tumor core versus those at the invasive rim of a human tumor resection. EXPERIMENTAL DESIGN: From a single glioblastoma specimen, 20,000 individual cells from each region (core and invasive rim) were collected by laser capture microdissection and analyzed by mRNA differential display. Differential expression of gene candidates was confirmed by laser capture microdissection and quantitative reverse transcription-PCR in additional glioblastoma multiforme specimens, and the role in migration was further evaluated in glioma cell lines in vitro. RESULTS: Reproducible overexpression the death-associated Protein 3 (Dap-3) mRNA (NM 004632, GenBank; also reported as human ionizing resistance conferring protein mRNA, HSU18321, GenBank) by invasive cells was identified. Although the full-length Dap-3 protein has been described as proapoptotic, the NH(2)-terminal fragment can act in a dominant negative way resulting in protection from programmed cell death. In glioma cell lines T98G and G112 with an induced motility phenotype, Dap-3 was up-regulated at the mRNA and protein level as assessed by quantitative reverse transcription-PCR, cDNA microarray, and Western blot analysis. These cells showed an increased resistance to undergo camptothecin-induced apoptosis, which was overcome by effective Dap-3-antisense treatment. Antisense treatment also decreased the migration ability of T98G cells. CONCLUSIONS: Dap-3 is up-regulated in invasive glioblastoma multiforme cells in vivo and in glioma cells with an induced motility phenotype in vitro. When migration is activated, Dap-3 is up-regulated and cells become resistant to apoptosis. These findings suggest that Dap-3 confers apoptosis-resistance when migration behavior is engaged.

Expression of the genes encoding myelin basic protein and proteolipid protein in human malignant gliomas.

Pathological differentiation of oligodendroglioma and mixed oligoastrocytoma from astrocytoma is difficult, relying on morphological characteristics due to the lack of reliable immunohistochemical stains. Oligodendrocytes, the presumed cell of origin of oligodendrogliomas, highly express the genes encoding myelin basic protein (MBP) and proteolipid protein (PLP). We analyzed the expression of these genes to determine whether they might be useful molecular markers of oligodendrocytic tumors. MBP and PLP were highly expressed in all oligodendrogliomas and minimally expressed in glioblastomas multiforme. MBP was highly expressed in mixed oligoastrocytomas, whereas PLP expression was minimal. The association between tumor classification and expression of the MBP and PLP genes was statistically significant. Expression of these genes may serve as a useful molecular marker for some subtypes of human gliomas.

Allogeneic astrocytoma in immune competent dogs.

We have induced in canines long-term immune tolerance to an allogeneic cell line derived from a spontaneous canine astrocytoma. Allogeneic astrocytoma cells were implanted endoscopically into the subcutaneous space of fetal dogs before the onset of immune competency (< 40th gestational day). At adulthood, dogs rendered tolerant successfully serve as recipients of intracranial transplants of their growing allogeneic, subcutaneous tumor. Transplanted dogs subsequently develop a solid brain tumor with histological features similar to the original astrocytoma. This model may allow rapid development and evaluation of new therapies for brain tumors, as well as afford tumor biology studies that are untenable in smaller, immune incompetent, or inbred animals harboring less representative tumors.

Regional heterogeneity in the proliferative activity of human gliomas as measured by the Ki-67 labeling index.

The effects of regional heterogeneity on the accuracy of histological grading of gliomas are well known, but little has been reported about its implications for other diagnostic modalities. This study investigated the relationships of regional heterogeneity in tumor proliferative activity, measured by Ki-67 labeling indices (LI), and histological grades for 16 regionally sampled glioma resections. There was a strong correlation between histological grades and Ki-67 LI in individual regions (p < 0.001), and both methods demonstrated comparable heterogeneity. Heterogeneity increased with tumor grade, probably as an expression of the increased genetic instability that accompanies tumor progression. Similarly, regions with comparable proliferative activity tended to cluster, paralleling clonal expansion. Thus, both histological grading and Ki-67 LI are subject to heterogeneity-induced sampling errors that limit their diagnostic accuracy, particularly in small biopsies. However, fewer grading errors occurred when using both methods together than when using either method alone, suggesting that the use of multiple techniques may reduce the adverse effects of regional heterogeneity on diagnostic accuracy. Regional heterogeneity appears to be a ubiquitous feature of gliomas: it also has been reported in karyotype, p53 oncogene mutations, and PDGF and EGFR expression. The effects of regional heterogeneity on new methods for studying gliomas need to be considered.

Choroid plexus carcinoma in siblings: a study by light and electron microscopy with Ki-67 immunocytochemistry.

Choroid plexus carcinomas in siblings aged 11 months and 10 years were examined by light microscopy and immunocytochemistry. One case was studied by electron microscopy. Choroid plexus carcinoma is rare, with approximately 24 reported cases in children. Predicting the behavior of choroid plexus tumors from the histology can be difficult. Neither mitoses nor necrosis were seen in one case, but evaluation of proliferation using Ki-67 monoclonal antibody showed 9% of the cells to be in proliferative phases of the cell cycle, a high value for a glial-derived neoplasm. Ki-67 activity may be a more sensitive measure of proliferation in malignant choroid plexus tumors than the presence of mitoses and necrosis, and additional studies may establish its role in distinguishing between choroid plexus carcinoma and papilloma when histologic classification is equivocal. Both tumors were immunoreactive for keratin, which confirmed previous studies. Both were nonreactive for glial fibrillary acidic protein, S-100, and carcinoembryonic antigen (CEA), unlike a previous study which reported that choroid plexus carcinoma, compared to papilloma, was uniquely S-100-negative and CEA-positive. Choroid plexus carcinoma in siblings has not been reported. Chance occurrence in siblings is extremely unlikely; thus a genetic basis for the neoplasms is likely, although environmental influences cannot be excluded.

Anterior communicating artery aneurysm paraparesis syndrome: clinical manifestations and pathologic correlates.

OBJECTIVE: Clinicopathologic evaluation of patients with lower extremity paraparesis/-plegia following rupture and repair of anterior communicating artery (ACoA) aneurysms. DESIGN: Institution-based retrospective review. SETTING: A tertiary neurologic referral center. PATIENTS, PARTICIPANTS: Seven of 101 patients with subarachnoid hemorrhage from ruptured ACoA aneurysms treated between January 1987 and December 1992. MAIN OUTCOME MEASURES: Neurologic status at latest follow-up examination. RESULTS: All patients presented with severe hemorrhage, poor clinical grade, and intracranial hypertension. Motor deficits developed within 7 days of aneurysm rupture and persisted for a mean duration of 39 days. Angiographic evidence of vasospasm in the anterior cerebral artery (ACA) distribution was documented in all cases, and paraparesis persisted beyond the angiographic resolution of vasospasm. All patients had evidence of frontal lobe dysfunction throughout their postoperative courses, and deep venous thrombosis and pulmonary emboli were common causes of morbidity and mortality. Autopsy data supported regional microvascular ischemia within the ACA distribution as the etiology of these motor deficits. CONCLUSIONS: The combination of vasospasm in the ACA distribution and lower extremity weakness associated with cognitive and affective impairment that resolves with time is common in patients with ACoA aneurysms. We propose that this constellation of clinical, radiographic, and pathologic findings be referred to as the "ACoA aneurysm paraparesis syndrome."

Development of sleep-wake patterns and non-rapid eye movement sleep stages during the first six months of life in normal infants.

Thirty-one normal infants were selected for 24-hour polygraphic monitoring at different ages during the first six months of life. The development of sleep-wake distribution patterns during day and night was observed. Qualitative changes in non-rapid eye movement (NREM) sleep as it becomes differentiated in stages 1, 2, and 3-4 were measured. Sustained periods of wake are present by 6 weeks of age. After 3 months of age, wake is predictably distributed in late afternoon and early evening. REM sleep is disproportionately distributed within sleep in 24 hours, presenting a higher percent of total sleep during the night. At 4.5 and 6 months of age, stages 2 and 3-4 NREM are coincident during the nocturnal hours and 3-4 NREM sleep peaks in the early period of the night. The decreasing proportion of REM sleep, particularly in its daytime distribution, suggests a reciprocal relationship to the developing of wakefulness.

Mixed and obstructive sleep apnea and near miss for sudden infant death syndrome: 2. Comparison of near miss and normal control infants by age.

Twenty-nine full-term near miss for sudden infant death syndrome (SIDS) and 30 normal control infants underwent 24-hour polygraphic monitoring. Several types of respiratory events during sleep (eg, central, mixed, and obstructive apnea, periodic breathing) were defined and tabulated. Analysis of these respiratory variables and comparison of groups of near miss and control infants indicated that between 3 weeks and 4 1/2 months of age only one variable was consistently different at a statistically significant level: the number of mixed and obstructive apnea greater than 3 seconds during total sleep time. This study also showed an increase in mixed and obstructive respiratory events during sleep at 6 weeks of age in control as well as in near miss infants.

Near-miss sudden infant death syndrome in eight infants with sleep apnea-related cardiac arrhythmias.

Eight full-term infants between 3 and 8 weeks of age, who had had 24 to 48 hours of vague problems involving a congested upper airway and/or irritability, had a life-threatening respiratory episode at home, called a near-miss for sudden infant death syndrome event. Polygraphically monitored mixed apneas occurred in clusters during non-rapid eye movement (NREM) sleep with significant cardiac arrhythmias, predominantly sinus arrest. The cardiorespiratory problems continued during sleep in the following 8 to 12 weeks but did not recur after that time, and there were no long-term sequelae when the children were studied again 4 to 7 years later.

Sleep parameters and respiratory variables in "near miss' sudden infant death syndrome infants.

Twenty-nine near miss for sudden infant death syndrome and thirty normal infants between the ages of 3 weeks and 6 months were monitored polygraphically for 24 hours. The distribution of sleep and abnormal respiratory events were analyzed for both groups. On the basis of mixed and obstructive apnea, 12-hour nocturnal segments (8 PM to 8 AM) consistently distinguished near miss from normal infant groups between the ages of 3 weeks and 4.5 months. Daytime naps do not provide statistical differences sufficient to differentiate between the two groups. During sleep, abnormal respiratory events are more likely to occur between 1 AM and 6 AM, at least 40 minutes after sleep onset. Respiratory pauses show a significant increase just prior to waking (a strong respiratory stimulus). Any impairment of the arousal threshold during sleep will place near miss infants at increased risk.