Epigenetic regulation of tumor necrosis factor alpha.

Tumor necrosis factor alpha (TNF-alpha) is a potent cytokine which regulates inflammation via the induction of adhesion molecules and chemokine expression. Its expression is known to be regulated in a complex manner with transcription, message turnover, message splicing, translation, and protein cleavage from the cell surface all being independently regulated. This study examined both cell lines and primary cells to understand the developmental regulation of epigenetic changes at the TNF-alpha locus. We demonstrate that epigenetic modifications of the TNF-alpha locus occur both developmentally and in response to acute stimulation and, importantly, that they actively regulate expression. DNA demethylates early in development, beginning with the hematopoietic stem cell. The TNF-alpha locus migrates from heterochromatin to euchromatin in a progressive fashion, reaching euchromatin slightly later in differentiation. Finally, histone modifications characteristic of a transcriptionally competent gene occur with myeloid differentiation and progress with differentiation. Additional histone modifications characteristic of active gene expression are acquired with stimulation. In each case, manipulation of these epigenetic variables altered the ability of the cell to express TNF-alpha. These studies demonstrate the importance of epigenetic regulation in the control of TNF-alpha expression. These findings may have relevance for inflammatory disorders in which TNF-alpha is overproduced.

Laser-excited fluorescence studies of mitochondrial function in saponin-skinned skeletal muscle fibers of patients with chronic progressive external ophthalmoplegia.

The functional behavior of mitochondria in skeletal muscle of patients with chronic progressive external ophthalmoplegia was studied by laser-excited fluorescence measurements of NAD(P)H and flavoproteins in saponin-skinned fibers. Variations in the mitochondrial content and the presence of partially respiratory chain-inhibited mitochondria can be detected using this novel method.

Immunohistochemical determination of five somatostatin receptors in meningioma reveals frequent overexpression of somatostatin receptor subtype sst2A.

Meningioma is one of a variety of human tumors that exhibit a very high density of somatostatin receptors and in many cases show a true positive somatostatin receptor scintigraphy. However, the level of expression of individual somatostatin receptor proteins in meningioma has not been investigated. We have recently developed a panel of somatostatin receptor subtype-specific antibodies that effectively stain formalin-fixed, paraffin-embedded tumor tissue (S. Schulz et al., Clin. Cancer Res., 4: 2047-2052, 1998). In the present study, we have used these antibodies to determine the somatostatin receptor status of 40 randomly selected meningiomas. Immunoreactive staining for all somatostatin receptors was clearly located at the plasma membrane of the tumor cells and completely blocked with antigenic peptide. The vast majority of tumors (29 cases; 70%) were positive for sst2A immunoreactivity; among these, 20 (69%) tumors showed high levels of sst2A immunoreactivity. In contrast, all other somatostatin receptors were only detected sporadically, and none of these cases revealed a particularly strong staining. However, it is uncertain to what extent somatostatin receptor-immunoreactive staining intensity may translate into somatostatin receptor protein expression on the tumor cells. Therefore, in a prospective study, 16 surgically removed meningiomas were collected, and the level of sst2A expression was determined using Western blot analysis. Whereas sst2A was readily detectable as a broad band migrating at Mr 70,000 in 12 (75%) of these tumors, 8 tumors (50%) showed particularly high levels of immunoreactive sst2A receptors. There was an excellent correlation (P < 0.001) between the level of sst2A protein expression detected in Western blots and the sst2A- immunoreactive staining seen in tissue sections. Thus, the frequent overexpression of the sst2A receptor may explain the high tracer uptake often observed in meningioma patients during somatostatin receptor scintigraphy. Moreover, this simple immunohistochemical method could prove useful in identifying those cases of recurrent disease that may possibly respond to therapy with sst2-selective agonists.

Heterogeneous tissue distribution of a mitochondrial DNA polymorphism in heteroplasmic subjects without mitochondrial disorders.

CONTEXT: Several maternally inherited point mutations of the mitochondrial genome cause mitochondrial disorders, but the correlation between genotype and phenotype remains obscure in many cases. The same mutation may cause various diseases, probably because of a different tissue distribution. OBJECTIVE: To assess the role of random somatic segregation in generating interperson differences by analysis of an apparently neutral polymorphism. DESIGN: Screening of 81 brain samples from subjects without mitochondrial disorders and selection of five necropsy cases showing a high level of heteroplasmy for the polymorphism. MAIN OUTCOME MEASURES: A proportion of various distinct genotypes in the mtDNA pool of the tissues, identified by fluorescent PCR products, representing a short polycytosine tract of variable length in the mitochondrial displacement loop. RESULTS: Differences were found between organs or groups of organs within subjects, pointing towards somatic segregation of mtDNA. In addition, marked differences of this organ distribution occurred between subjects, which cannot be explained by tissue specific selection. CONCLUSIONS: The observed interperson differences can be explained by somatic segregation, which occurs randomly at various developmental stages. Besides tissue specific selection, this process might participate in the distribution of pathogenic mtDNA mutations.

Short incubation with 2-methoxyestradiol kills malignant glioma cells independent of death receptor 5 upregulation.

We investigated the effects of 2-methoxyestradiol (2-ME), a promising new antitumor agent, on viable cell number and nuclear morphology of malignant glioma cells (three human and one rat glioma cell lines) and analyzed the controversial role of death recepor 5 (DR5) upregulation in 2-ME induced apoptosis. Microtiter-tetrazolium (MTT) assays showed a significant reduction of viable cells after incubation with 2 microM and 20 microM 2-ME for 48 and 72 hours in all cultures. In the 20 microM concentration, there were even significant effects in the majority of shorter incubation periods. Hoechst 33258 stains showed a substantial amount of cells with nuclear fragmentation indicating a late stage of apoptosis after 20 microM 2-ME treatments of 24 hours and more. The role of the DR5-mediated extrinsic apoptotic pathway was further studied in the three human glioma cell lines; 50 ng/ml of the DR5 ligand TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) and 2 microM 2-ME showed no synergism, as determined by MTT assays. Real-time PCR revealed no significantly increased amount of DR5 mRNA, suggesting that receptor upregulation does not play a major role for 2-ME-induced apoptosis in glioma cells, in contrast to data for a breast cancer cell line in the literature.

Expression of members of the interleukin-6 family of cytokines and their receptors in human pituitary and pituitary adenomas.

There is increasing evidence for the role of members of the Interleukin (IL)-6 family in pituitary function, particularly in the regulation of the hypothalamo-pituitary-adrenal axis. However, there is only a limited amount of data available on the expression in human normal and tumorous pituitary tissue. In this study we investigated the expression of members of the IL-6 family of cytokines and their receptors in normal human pituitaries as well as pituitary adenomas using the RT-PCR technique. Eighteen pituitary adenoma biopsies removed in transsphenoidal surgery (six corticotrophic adenomas, four nonfunctioning adenomas, four somatotrophinomas, four prolactinomas) and six normal anterior pituitaries were examined for the expression of IL-6 receptor (-R), leukemia inhibitory factor (LIF), LIF-R, IL-11, IL-11-R, oncostatin M (OSM), OSM-R, ciliary neurotrophic factor (CNTF), CNTF-R and cardiotrophin-1 (CT-1). All pituitaries and pituitary adenomas expressed OSM transcripts, whereas no expression of CT-1 was found. The expression of all other cytokines (LIF, IL-11, CNTF) and receptors (IL-6-R, LIF-R, IL-11-R, OSM-R, CNTF-R) was found in different patterns in the adenoma subtypes and normal pituitaries. However, we did not detect expression of LIF-, IL-11-, IL-6-R and CNTF-R in prolactinomas, of CNTF in normal pituitaries and of OSM-R in ACTH-secreting adenomas. In conclusion, our study provides further evidence for a role of the members of the IL-6 family of cytokines in pituitary function.

Spatial and temporal disease progression of adult-onset subacute sclerosing panencephalitis.

An adult-onset case of subacute sclerosing panencephalitis with occipitofrontal spread of the infection documented clinically and by MRI is reported. Autopsy revealed numerous intranuclear viral inclusions and widespread demyelination in both frontal lobes. In the occipital lobes where the disease started 5 years previously, inclusions were rare, but degenerative tissue changes were prominent. This case underlines the importance of measles virus migration for the progression of this fatal disorder.

Effects of phospholipase Cgamma on Polo-like kinase 1 expression in human glioma cells.

PURPOSE: The precise regulation of the mammalian PLK-1 gene, involved in progression of the G2/M transition, remains to be clarified. Phospholipase Cgamma, associated with cell proliferation and invasion in human gliomas, could be one of the important candidates for the modulation of PLK-1 expression. Therefore, we studied the correlation between PLCgamma activity and PLK-1 expression and determined cell size and proliferation after PLCgamma inhibition in two glioma cell lines and a primary culture of a glioblastoma. METHODS: The glioma cells were investigated with highly specific chemical and antisense inhibition of PLCgamma. The effects of the inhibition were checked by means of morphometrical, semiquantitative immunohistochemical methods, and MTT-assays. RESULTS: The chemical and antisense inhibition of PLCgamma resulted in decreased expression of PLK-1 and reduced cell density in glioma cell lines as well as in a primary culture of a glioblastoma. These findings were verified by MTT-assays. CONCLUSION: The activity of PLCgamma seems to modulate the expression of PLK-1. In further experiments serum-depleted cultures, stimulated by different growth factors, should be used after inhibitor pretreatment to study the in vivo conditions.

Long-term survival of glioblastoma multiforme: importance of histopathological reevaluation.

The overall prognosis for patients with glioblastoma multiforme is extremely poor. However, a small proportion of patients enjoy prolonged survival. This study investigated retrospectively the extent to which erroneous histopathological classification may contribute to long-term survival of patients initially diagnosed with "glioblastoma multiforme." We compared two age- and gender-matched patient groups with different postoperative time to tumor progression (TTP), defined as "short-term" for TTP of less than 6 months (n = 54), and "long-term" for TTP of more than 12 months (n = 52). Histological specimens of the corresponding tumors, all primarily diagnosed as glioblastoma multiforme, were reevaluated according to the current World Health Organization (WHO) classification of central nervous system tumors, with the investigators being blinded to clinical outcome. Among the tumors from short-term TTP patients, one tumor (2%) was reclassified as anaplastic oligoastrocytoma (WHO grade III) while the remaining 53 were confirmed as glioblastoma multiforme. In contrast, 13 tumors (25%) from the long-term TTP patients were reclassified, mostly as anaplastic oligodendroglioma (WHO grade III; n = 7) or anaplastic oligoastrocytoma (WHO grade III, n = 2), respectively. In addition, three were reclassified as anaplastic astrocytoma (WHO grade III), and one was identified as anaplastic pilocytic astrocytoma (WHO grade III). Our data indicate that a sizable proportion of glioblastoma patients with long-term survival actually carry malignant gliomas with oligodendroglial features. The correct histopathological recognition of these tumors has not only prognostic but also therapeutic implications, since oligodendroglial tumors are more likely to respond favorably to chemotherapy.

Correlation of TGF-alpha and EGF-receptor expression with proliferative activity in human astrocytic gliomas.

Fifty-nine paraffin-embedded astrocytic gliomas (four WHO grade 1, 21 WHO grade 2, 17 WHO grade 3 and 17 glioblastomas, WHO grade 4) were immunohistochemically investigated for expression of transforming growth factor-alpha (TGF-alpha), epidermal growth factor receptor (EGF-R) and oncoprotein c-erbB-2 by semiquantitative assessment. Proliferative activity was simultaneously analyzed by using the antibody Ki-67 (MIB-1). Immunostaining in neoplastic cells was quantified by image analysis. Concerning the antibodies used, the percentage of immunoreactive cells increased with histologic malignancy. There was no expression of EGF-R and c-erbB-2 in the majority of low-grade astrocytomas. However, small focal expressions of TGF-alpha and EGF-R were observed in several low-grade astrocytomas (11/25), suggesting an early stimulation of malignant transformation. With regard to percentage, a strong positive correlation between TGF-alpha and EGF-R-stained cells was found, indicating an autocrine stimulation of the mitogenic pathway of the TGF-alpha/EGF-R system. Likewise, indices of EGF-R and c-erbB-2 positive cells correlated significantly. Less significant correlations were also seen between EGF-R, c-erbB-2 frequencies and the Ki-67 labeling index. However, there was no correlation between TGF-alpha and Ki-67 indices. The results suggest that TGF-alpha expression is not directly related to the proliferative potential as judged by the Ki-67 labeling index. Furthermore, besides EGF-R and c-erbB-2, other growth factors and their receptors or mutant EGF-R might participate in the proliferative activity of gliomas.

Expression of the plasminogen activator system and the inhibitors PAI-1 and PAI-2 in posttraumatic lesions of the CNS and brain injuries following dramatic circulatory arrests: an immunohistochemical study.

Plasminogen activators as inducible extracellular serine proteases are involved in a variety of processes, such as the degradation of brain structures. In regions of brain degradation, an increase in the expression of genes encoding cytokines and proteinases has recently been demonstrated. We tested the hypothesis, whether the plasminogen activator system as well as the plasminogen activator inhibitors are expressed and possibly involved in a proteolytic cascade that breaks down the extracellular matrix as a result of ischemic or posttraumatic brain destructions. To study this supposition, we investigated immunohistochemically the expression of tPA, uPA and its receptor, the plasminogen activator inhibitors PAI-1 and PAI-2, tetranectin as well as the laminin breakdown as an event of secondary brain injury. Brain tissue from 21 autopsy cases with severe brain injuries, material from 14 ischemic infarcts and 11 controls with acute hypoxia were used. All components of the plasminogen activator system studied were over-expressed immunohistochemically in reactive astrocytes, microglia and endothelial cells around the lesion zone. Tetranectin showed an analogous distribution to the plasminogen activator system. A reduced immunoreactivity of laminin within the identical region of destruction was detected concomitant with laminin remnants in perivascular macrophages, so that a remarkable role of the plasmin cascade in the degradation of extracellular matrix proteins in the brain is taken into consideration.

Immunohistochemical detection of vascular growth factors in angiomatous and atypical meningiomas, as well as hemangiopericytomas.

The arachnoideal compartment provides the vascular sources for three different tumor types rich in vessels: angiomatous meningioma, some atypical meningioma with high vascularity and meningeal hemangiopericytoma. We investigated immunohistochemically the expression and distribution of vascular mitogenes in 7 angiomatous meningiomas, 8 atypical meningiomas with high vasculature and 4 hemangiopericytomas. On the one hand it should be studied which vascular growth factors such as VPF/VEGF-1, VPF/VEGF-2, bFGF, PDGF and TGF-alpha could be responsible for the close meshwork of vessels within the tumors. On the other hand we were interested in whether or not there are differences in vascular mitogens between slowly growing angiomatous meningiomas and both other types with their increased tendency to recur. PDGF and TGF-alpha were extensively expressed in the endothelium and smooth muscle cells of the vessels, as well as tumor cells. VEGF-2 could only be found in endothelial cells of all three tumor entities. bFGF was localized in some vessels of angiomatous meningiomas and VEGF-1 revealed a very low expression with a localization comparable with VEGF-2. Moreover, uPAR was diffusely expressed in nearly all tumor cells and endothelial cells. The fact that tumor cells of hemangiopericytomas and meningiomas did not show any immunohistochemical reaction with VEGF's could indicate a lower priority of these growth factors for neovascularization in this type of neoplasm. A different expression of vascular mitogens between benign angiomatous meningiomas and atypical meningiomas as well as hemangiopericytomas with their tendency for recurrence could not be observed. The morphological evidence for extravasates of IgG-proteins, Fibrin and Fibronectin due to VPF-effects seems not to be a renouncable condition for neoangiogenesis in the tumors investigated.

Dendritic spines and immunoreactivity of synaptophysin in the frontal cortex of humans with infantile brain damage. A correlative study.

Some neurodegenerative disorders of adults involving mental retardation are associated with reduction of dendritic spines and number of synapses. A study was performed to prove the changes in the density of dendritic spines and synaptophysin-immunoreactive granules in the frontal cortex of cases with infantile brain damage and a possible correlation between them. Specimen from cortex of children and adolescents with severe mental retardation, aged 3 to 24 years and a group of non-psychiatric age-matched controls were processed according to the Golgi-Kopsch method. Density of dendritic spines on the apical dendrites of layer V pyramidal neurons (Brodman area 10) was analysed in correlation to the density of synaptophysin-reactive granules (counts/microns 2) in the same region. Both density of spines and number of immunoreactive granules were reduced in brains with infantile damages, but there was no correlation between spine density and the number of immunoreactive granules against synaptophysin.

Altered dendritic maturation and loss of GABA immunoreactivity of hippocampal neurons after prenatal exposure to chloroquine.

The aim of the study was to analyze the intrauterine and postpartal effects of Chloroquine on the dendritic maturation in the hippocampus and on the expression of GABA within hippocampal interneurons of the stratum moleculare. Fifty-nine brains of rat fetus on day 22 of gestation and 37 brains from rats from postnatal day P7 were examined. We found changes in the cytoarchitecture of hippocampal CA3 neurons on P7 day. Chloroquine treatment resulted in a significant increase of the length of the apical shafts, apical dendrites and basal dendrites of the CA3 neurons (p < 0.05) under doses comparable to serum levels reached during long-term therapy. Furthermore, an early reduction of GABA-expressing interneurons of the hippocampal striatum moleculare was observed.

Mdr1 mRNA expression differs between grade III astrocytomas and glioblastomas.

104 N2-frozen samples from 33 astrocytic tumors previously untreated with cytotoxic drugs have been analyzed for the expression of p-glycoprotein transcripts (mdrl) by RT-PCR using beta2-microglobulin as an internal control. A remarkable variation was observed even within a single tumor in 50% of the cases. Nevertheless, a difference became visible between the groups of anaplastic astrocytomas and glioblastomas. While 78% of the grade 3 astrocytomas contained at least a minimum of 1 sample with a very low mdr1 expression, this was the case only in 23% of the glioblastomas. This supports an earlier observation revealing a positive correlation between tumor grading and the tumor cell fraction stained with the monoclonal antibody JSB1. On the other hand, no major differences were found between the histological groups when the samples with the highest mdr1 expression were selected to represent the individual tumors. Those samples are less informative. They might be derived from tumor regions in which capillaries deliver a larger fraction of the total mRNA pool. No induction of mdr1 was observed in some early astrocytoma or glioblastoma cell cultures even after administration of high concentrations of the drugs ACNU and VM26, often used in glioma therapy.

Stiff-man syndrome: possible autoimmune etiology targeted against GABA-ergic cells.

We report the case of a female patient, who died at the age of 66 years. Besides an insulin-dependent diabetes mellitus (IDDM) she had developed the clinical symptoms of stiff-man-syndrome (SMS) and harbored autoantibodies against glutamate-decarboxylase (GAD) in blood and liquor. GAD catalyzes the biosynthesis of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). The autopsy revealed typical alterations observed in diabetes mellitus including an incomplete fibrosis of pancreatic Langerhans islets. A decrease of GABA-ergic cells in the cerebellar cortex was observed, and a size reduction of Renshaw cells in the spinal cord. Furthermore, a dilution series of a polyclonal GABA antibody delivered a reduced immunofluorescence in the cerebellum. In skeletal muscle a neurogenic atrophy was observed. As described in literature, the clinical symptoms decayed following clonazepam administration. We suggest that this case including GAD autoantibodies, dramatic loss of GAD-expressing pancreatic cells, and loss or atrophy of GABA secretory neurons, supports the hypothesis that SMS may be an autoimmune disease directed against GABA-ergic cells. Furthermore, we suggest a neuronal hypersensitivity at the spinal cord level caused by the atrophic Renshaw cells.

MGMT- and P450 3A-inhibitors do not sensitize glioblastoma cell cultures against nitrosoureas.

UNLABELLED: AIM, MATERIAL AND METHODS: Using RT-PCR and immunohistochemistry the expression of cytochrome P450 was evaluated in a series of 22 glioblastomas and 4 anaplastic astrocytomas (WHO grade III). Since rat liver P450 can catalyze the denitrosation of the nitrosourea compound BCNU in vitro, cell culture experiments were performed to test a possible sensitizing effect of P450 3A inhibitors (tiamulin and ketoconazole) in BCNU treatment of glial tumor cells. O6-benzylguanine (BG), an inhibitor of the DNA repair enzyme O6-methylguanine-DNA-methyltransferase (MGMT), was used in parallel experiments, since MGMT is discussed as a main mechanism in nitrosourea resistance. RESULTS: RT-PCR reactions with primers designed according to the sequences for CYP1A1 and CYP2E1 were always positive, while those for CYP1A2 and CYP2D6 were negative. The strongest PCR products were detected with CYP3A primers, but CYP3A expression was heterogeneous within the tumor samples. Antibodies to human liver CYP3A4 stained a subfraction of tumor cells (18% of the cells in glioblastomas and 14% in grade III astrocytomas) and to some extend neurons in normal brain areas, while astrocytes were negative. For cell culture experiments with P450 3A and MGMT inhibitors, early passages of 3 glioblastomas, a late passage of an immortalized cell line derived from a reoccurring glioblastoma, and the human glioblastoma line LN405 were used. The sensitivity of the tumor cells for both nitrosourea compounds was very low, when low concentrations were applied (comparable to the achievable blood concentrations in glioma patients). Strong effects did only occur when the concentrations were raised 9-fold or 27-fold. CONCLUSION: In no case could a significant sensitizing effect of P450 3A- and MGMT inhibitors be demonstrated.

Expression of matrix metalloproteinases in a series of 12 meningiomas.

The expression of metalloproteinases was evaluated in a series of 12 meningiomas of various histological subtypes including 3 meningotheliomatous, 3 fibroblastic, 4 transitional and one psammomatous meningioma (WHO grade I) as well as one anaplastic meningioma (WHO grade III). No gelatinolytic activity could be detected in all tumor samples pointing towards no or very low activity of both MMP-2 and MMP-9. At least MMP-2 mRNA could be found in 10 out of 12 tumor samples by the reverse transcription PCR method (RT-PCR) followed by electrophoresis on silver-stained polyacrylamide gels, which allows the detection even of small traces of a specific mRNA. The PCR products were identified as MMP-2 sequences without introns (mRNA-derived) by direct sequencing, thereby demonstrating a low transcriptional activity of the gene. The translation of these mRNAs, however, did not result in amounts of protein detectable by immunohistochemistry or Western blotting. Therefore, neither MMP-2 nor MMP-9 should play a major role for tumor growth within dura mater or bone structures or for brain infiltration in our tumor series. Therefore, other mechanisms must be responsible for extracellular matrix degradation at least in a fraction of meningiomas.

Primary Intracranial Manifestation of CD7/CD56-Positive Acute Myelogenous Leukemia.

BACKGROUND: CD56 which is considered as a marker of natural killer cells is also expressed in some cases of acute myelogenous leukemia (AML) and is involved in cell adhesion mediating extramedullary leukemic infiltration. CD7/CD56 coexpression has been suggested to be a distinct biological and clinical entity of AML. PATIENT: This is a report of a 53-year-old woman who developed CD7/CD56-positive AML with primary manifestation as intracranial tumor. The patient reported of neurological impairment (impairment of visus and occurrence of double pictures). Cranial computed tomography showed an intracranial tumor, and histological examination exhibited myeloid blast cells. Peripheral leukocyte count at admission was within the normal range (5,32 Gpt/l), and percentage frequency of blasts in the blood smears was 54%. Cytological bone marrow examination showed diffuse infiltration by the same myeloid blast cells. The immunophenotype was CD7/CD13/CD33/CD38/CD56/ HLA-DR-positive. The blast cells were myeloperoxidase-positive but lactoferrin-negative. Thus, diagnosis of acute myeloid leukemia (M2 FAB) was established. Treatment consists of chemotherapy (Ara-C and anthracycline) and local radiation of the intracranial tumor. After treatment patient achieved a complete remission. CONCLUSION: With regard to the literature CD7/CD56-positive AML have a high incidence of central nervous system involvement which should be kept in mind and may be associated to CD56 expression. Copyright 2000 S. Karger GmbH, Freiburg

Sudden progression of a glioblastoma in partial remission?

There are only sparse data on viral CNS infections in patients with malignant glioma. We report a case of fatal herpes encephalitis in a patient with glioblastoma in partial remission and provide a short review of the literature.