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.

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.

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."

Correlation of DNA content and histology in prognosis of astrocytomas.

Thirty-two cases of astrocytoma were analyzed for DNA content and cell-cycle proliferation features by flow cytometry using paraffin-embedded tissue. The findings were correlated with histologic grading and survival. Abnormal DNA (aneuploidy or elevated G2-M fraction greater than or equal to 7%) was present in 18 cases (56%). Glioblastoma multiforme (GBM) had 11 of 16 (69%), anaplastic astrocytomas (ANA) 7 of 11 (64%), and low-grade (LG) neoplasms 0 of 5 cases with abnormal DNA content. Short-term survival (less than or equal to 26 months) occurred in all 16 patients with GBM (100%), 7 of 11 patients with ANA (64%), and 1 of 5 patients with LG neoplasms (20%). Seventeen of 18 patients (94%) with abnormal DNA content were short-term survivors (P less than 0.0002). Abnormal DNA content was found in 17 of 24 short-term survivors (71%), whereas histologic grading identified 16 of 24 such cases (67%). A combination of grading and abnormal DNA content identified 22 of 24 (92%) of the poor survival cases. DNA content was most useful in the anaplastic group. Six of seven cases (86%) with abnormal DNA content had short survival (P less than 0.055), and three of four (75%) with normal DNA content had long survival. DNA analysis combined with histologic grading improves prognosis designation.

Intradural primary chondroblastic osteosarcoma: case report.

We report a rare case of intradural primary osteosarcoma (IPOS) in a 74-year-old man with aphasia and right-sided hemiparesis. Radiologic workup revealed a large, partially calcified, left-sided frontotemporal intracranial mass lesion. At surgery, the tumor was found to be entirely intradural; it involved the brain and subarachnoid space of the left sylvian fissure. The adjacent dura was uninvolved. Neuropathologic findings confirmed the diagnosis of chondroblastic osteosarcoma. To our knowledge, this is the sixth reported case of IPOS and the first reported case of the chondroblastic subtype.

Effect of voxel position on single-voxel MR spectroscopy findings.

BACKGROUND AND PURPOSE: Single-voxel MR spectroscopy is a widely used tool for evaluating brain tumors. Although extensive data are available on the MR spectral appearance of tumors, less is known about the effect of voxel position on the accuracy of single-voxel MR spectroscopy findings. The purpose of this study was to test the hypothesis that the accuracy of single-voxel MR spectroscopy in the categorization of lesions as either tumor or not tumor is dependent on voxel position. METHODS: Fifty single-voxel MR spectra acquired with a fully automated stimulated-echo spectroscopy sequence were reviewed retrospectively in 43 patients with new or previously treated intra-axial brain tumors. Spectra were analyzed for the presence of choline, creatine, N-acetylaspartate (NAA), and lipid/lactate. Choline/creatine and NAA/creatine peak area ratios were assessed qualitatively. Lesions were grouped into one of three categories on the basis of spectral pattern: tumor, not tumor, or indeterminate. Results of MR spectroscopy were compared with the final histopathologic diagnosis. RESULTS: Histologic confirmation was obtained in 19 patients; MR spectra were interpretable in 17 of those. MR spectra correctly categorized nine of 17 lesions (six tumor, three nontumor). All eight misdiagnosed lesions were tumors. When the MR spectroscopy voxel included the enhancing edge of the lesion, the spectra correctly categorized seven of eight lesions (four of five tumors and all three cases of radiation necrosis). When the MR spectroscopy voxel was positioned centrally within the lesion, the spectra correctly reflected histologic outcome in two of nine lesions (all tumors). CONCLUSION: The reliability of single-voxel MR spectroscopy findings is dependent on voxel position. Spectra obtained from voxels at the enhancing edge of a tumor more accurately reflect lesion histopathology than do spectra obtained from the lesion center, even if the centrally placed voxels contain solidly enhancing tissue.

Prognostic significance of flow cytometry deoxyribonucleic acid analysis of human astrocytomas.

Flow cytometry was used to determine the deoxyribonucleic acid ploidy and proliferative activity of 230 astrocytomas. The relationships among survival, ploidy, proliferation, histological features, and clinical variables were analyzed. Multivariate analysis confirmed the independent prognostic significance of the S-phase fraction (P < 0.01), ploidy (P = 0.04), age at diagnosis (P < 0.001), extent of surgery (P < 0.01), and sex (P = 0.03). Three groups with significantly different survival were defined based on S-phase fraction ranges of < 3%, 3%-5.9%, and > or = 6%. The strong correlation between the S-phase fraction and survival confirmed the importance of quantitative proliferation assays in predicting tumor behavior and demonstrated that specific reference ranges can be defined for clinical application. The weaker association between ploidy and survival leaves the usefulness of the determination of ploidy with flow cytometry in doubt.

Flow cytometric analysis of deoxyribonucleic acid ploidy and proliferation in choroid plexus tumors.

The deoxyribonucleic acid (DNA) content of 10 choroid plexus tumors, including 4 malignant tumors and 3 normal choroid plexus controls, was analyzed by flow cytometry to determine whether a ploidy or proliferation rate is a better predictor of tumor behavior than histological features. Nine of 10 neoplasms had both diploid and aneuploid modal populations. One neoplasm and all three control cases had only a diploid peak. Among the tumors, the DNA indices of the aneuploid peaks ranged from 1.1 to 2.2. The percentage of aneuploid cells ranged from 7 to 99, and no distinction was made between benign and malignant. Proliferation rates were estimated from the combined S-phase fractions (SPF). The mean SPF of the control group was 0.7% +/- 0.15% SD. The mean SPF of the benign tumors (1.1 +/- 0.82% SD) was significantly different from the malignant group (7.0 +/- 1.25% SD; range, 5.3 to 8.6%) P = 0.0095. Low SPF fractions always correlated with favorable outcome. Higher proliferation rates were generally associated with an aggressive course. Evaluation of proliferation rates may help predict the behavior of choroid plexus tumors, particularly when histological features are equivocal. Measurement of DNA ploidy does not appear to have a role in the evaluation of choroid plexus tumors.

The prognostic significance of Ki-67 labeling indices for oligodendrogliomas.

OBJECTIVE: The prognostic significance of quantitative measurement of tumor proliferative activity was evaluated for oligodendroglial tumors. METHODS: Ki-67/MIB-1 immunochemistry was used to measure proliferative activity in 81 oligodendrogliomas and oligoastrocytomas. The relationship among survival, proliferation, histological features, and clinical variables were evaluated using a Cox proportional hazards analysis. RESULTS: After stratifying by histological grade and adjusting for age at diagnosis, there was a significant association between the Ki-67/MIB-1 labeling index (LI) (percentage of positive cells) and survival (P = 0.04). This association was illustrated further by the significantly different survival of two groups based on LI ranges of less than or equal to 5 and greater than 5 (P < 0.0001). CONCLUSION: The poor correlation between mitotic figures and survival in oligodendrogliomas that has been reported previously emphasizes the need for an accurate method to measure proliferative activity. Our study demonstrated the usefulness of the Ki-67/MIB-1 LI and demonstrated that LI ranges can be defined for clinical application.

Osteosarcoma of the temporal fossa with hemorrhagic presentation: case report.

OBJECTIVE AND IMPORTANCE: Excluding tumors of hematopoietic origin, osteosarcomas are the most common bone tumor, although involvement of the brain or cranial base is rare. CLINICAL PRESENTATION: A 16-year-old girl with an osteosarcoma of the temporal fossa presented with an intracerebral hemorrhage. The management strategy of this lesion, including the operative interventions, is described. INTERVENTION: Several modes of treatment were undertaken, including radical resection of the cranial base lesion and excision of the cavernous sinus after a cervical internal carotid artery-to-middle cerebral artery vein bypass graft. CONCLUSION: The patient was alive and without evidence of disease 11 months after presentation but died shortly thereafter of complications related to adjuvant therapies.

Prognostic significance of flow cytometry deoxyribonucleic acid analysis of human oligodendrogliomas.

Flow cytometry was used to determine the deoxyribonucleic acid ploidy and proliferative activity of 60 oligodendrogliomas and oligoastrocytomas. The relationships among survival, ploidy, proliferation, histological features, and clinical variables were analyzed. Survival was strongly associated with the S-phase fraction (P < 0.001). Three groups with significantly different survival rates were defined, based on S-phase fraction ranges of < 3%, 3 to 5.9%, and > 6%. Significant associations between survival and age at diagnosis (P < 0.001), tumor grade (P < 0.001), and extent of surgery (P < 0.01) were found also. The poor correlation between mitotic figures and survival in oligodendrogliomas that has been reported previously emphasizes the need for an accurate method to measure proliferative activity. Our study demonstrated the usefulness of the flow cytometry-determined S-phase fraction in this regard and demonstrated that specific reference ranges could be defined for clinical application. In contrast, the determination of ploidy by flow cytometry was not useful in the evaluation of oligodendrogliomas.