Biomedical subjects
Werner Paulus
Publications and source records attributed to Werner Paulus.
Announcing the winner of the 2005 Kurt Jellinger Prize.
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Comparative genetic analysis of metachronous anaplastic oligoastrocytomas with extended recurrence-free interval.
Two metachronous anaplastic oligoastrocytomas with different cerebral locations were analyzed in a 51-year-old patient with an extended recurrence-free interval of 6 years and an a long survival of 9 years. Remarkably, the patient had not undergone adjuvant chemotherapy. Different cytogenetic and molecular techniques were performed including comparative genomic hybridization (CGH), fluorescence in situ hybridization (FISH), allelic loss analysis, sequencing of p53, p16(INK4a)/CDKN2A and p14(ARF), EGFRamplification studies, investigation of the DNA mismatch repair system as well as tumor clonality. Using CGH and FISH a profile of low accumulation of cytogenetic aberrations was found in the second tumor, with no significant increase in the percentage of hyperdiploid nuclei. Microsatellite analysis showed a common pattern of allelic losses at 1p36, 19q13 and 9p21. Both specimens were also similar in that they retained heterozygosity at 10q23-q24 and 13q14 and that they harbor neither EGFR amplification nor mutations of p53, p16(INK4a)/CDKN2A or p14(ARF). The only further alteration in the second tumor was an allelic loss at p53. The X-chromosome inactivation (HUMARA) analysis revealed a polyclonal pattern in both samples. Our data strongly suggest that the second anaplastic oligoastrocytoma developed as a distant relapse of the first tumor. Whether the paucity of accumulation of the observed genetic alterations might be associated with the unusually extended relapse-free time of the patient remains to be elucidated.
Establishment of a benign meningioma cell line by hTERT-mediated immortalization.
Meningioma represents the most common intracranial tumor, but well-characterized cell lines derived from benign meningiomas are not available. A major reason for the lack of benign tumor cell lines is senescence of nonmalignant cells in vitro, while malignant cells are often immortal. We have developed a meningioma cell line by retrovirally transducing primary cells derived from a human WHO grade I meningothelial meningioma with the human telomerase reverse transcriptase (hTERT) gene, which enables bypassing cellular senescence. Five clones have been cultured for more than 21 months so far, while corresponding nontransfected cells ceased proliferation within 3 months. Quantitative RT-PCR and a telomeric repeat amplification protocol (TRAP) assay revealed high hTERT mRNA levels and high telomerase activity in all transduced populations, while nontransduced cells were negative. The average telomere size of transduced cells was considerably longer than that of parental cells and the biopsy specimen. One clone, designated Ben-Men-1, was characterized in more detail, and exhibited typical cytological, immunocytochemical, ultrastructural and genetical features of meningioma, including whorl formation, expression of epithelial membrane antigen, desmosomes and interdigitating cell processes, as well as -22q. Following subdural transplantation into nude mice, tumor tissue with typical histological features of meningothelial meningioma was found. We conclude that Ben-Men-1 represents an immortalized yet differentiated cell line useful for biological and therapeutical studies on meningioma.
Genes associated with fast glioma cell migration in vitro and in vivo.
Identification of genes mediating glioma invasion promotes the understanding of glia motility and might result in biologically based therapeutic approaches. Most experimental studies have been performed in vitro, although glial cells typically undergo marked phenotypic change following placement into cell culture. To evaluate migration mechanisms operating in vitro versus in vivo, we used C6 rat glioblastoma cells for selecting highly migratory cells in a monolayer migration assay as well as in brains of nude mice, and analyzed in each paradigm the expression profiles of these "fast" cells versus those of the original "slow" cells using oligonucleotide microarrays comprising 8832 genes. In vitro, 516 (10.6%) of 4848 expressed genes were regulated (i.e., differentially expressed in fast versus slow cells); 916 genes were expressed only in vitro, including 142 (15.5%) regulated genes. In vivo, 245 (6.1%) of 4044 expressed genes were regulated; 112 genes were expressed only in vivo, including 25 (22.3%) regulated genes, none of them having a known relation to glioma invasion. Of 730 regulated genes, only 31 (4.2%) were regulated in parallel in vitro and in vivo, most of them having a known relation to (glioma) invasion. Our data provide new molecular entry points for identifying glioma invasion genes operating exclusively in the brain. They further suggest that genes underlying glia cell motility are strikingly different in vitro and in vivo.
Juvenile psammomatoid ossifying fibroma of the neurocranium. Report of four cases.
Juvenile psammomatoid ossifying fibroma (JPOF) is a benign fibroosseous lesion predominantly arising within the paranasal sinuses in children and young adults. Neurocranial occurrence is exceedingly rare and a location within the neurocranial portion of the temporal bone has not been described. The authors report on one case of sinonasal JPOF secondarily extending into the cranial cavity and three cases primarily affecting the neurocranial bones to increase clinical awareness of this uncommon tumor, which may be easily mistaken for meningioma. Moreover, the absence of activating missense mutations of the GNAS1 gene in two cases strongly argues against a relationship between JPOF and fibrous dysplasia.
Prion protein (PrPc) promotes beta-amyloid plaque formation.
Prion protein (PrP) has been localized to amyloid-beta (Abeta) senile plaques in aging and Alzheimer disease, but it is unknown whether PrP is directly involved in plaque formation or represents a reaction to amyloid deposition. To evaluate possible functional effects of PrP in Abeta plaque formation, we analyzed bigenic mice (TgCRND8/Tg7), carrying mutant human amyloid precursor protein (APP) 695 (APP(Swed+Ind), TgCRND8) as well as the wild-type Syrian hamster prion protein gene (sHaPrP, Tg7), showing Abeta plaques at 3 months of age as well as highly increased HaPrP(c) levels. Compared to the control group, consisting of animals carrying only mutant APP, bigenic mice showed a higher number of senile plaques in the cerebral cortex, while APP transcription and Abeta40/Abeta42 levels were unchanged. Double-labelling immunofluorescence showed co-localization of Abeta and PrP in virtually all plaques in the brains of both control and experimental animals. Our data suggest that PrP promotes plaque formation, and that this hitherto unknown functional role of PrP appears to be mediated by increased Abeta aggregation rather than by altered APP transcription or processing.
Perivascular clustering in temporal lobe epilepsy: oligodendroglial cells of unknown function.
Marked perivascular clustering (PC), i.e., groups and rows of small round cells along white matter vessels, is seen in temporal lobe epilepsy (TLE) specimens obtained by surgery. This study focuses on the constituting cell types and discusses clinical significance and pathogenesis of PC, which are so far unknown. Based on a series of 59 nonlesional TLE surgical specimens, we characterized PC by immunohistochemistry and correlated the amount of PC with clinical parameters. PC cells were variably positive for galactocerebroside, myelin basic protein and S-100 protein, while glial fibrillary acidic protein, vimentin, nestin and neuronal antigens were not expressed. There was no correlation between the amount of PC and any clinical feature, including age at surgery, age at epilepsy onset, duration of epilepsy, preoperative seizure frequency, childhood febrile convulsions, family history of epilepsy, and postsurgical outcome. Our findings suggest oligodendroglial differentiation of PC, while its primary (dysplastic) versus secondary (reactive) pathogenesis remains unresolved.
Somatostatin receptor subtype 2 is expressed by supratentorial primitive neuroectodermal tumors of childhood and can be targeted for somatostatin receptor imaging.
PURPOSE: Although gliomas predominate among central nervous system (CNS) neoplasms in adulthood, embryonal tumors are the most common malignant brain tumors in children. Despite novel treatment approaches, including improved radiotherapy and high-dose chemotherapy, survival rates remain unsatisfactory. The timely diagnosis of residual or recurrent embryonal CNS tumors and thus the earliest possible time point for intervention is often hampered by inaccuracies of conventional imaging techniques. Novel and refined imaging methodologies are urgently needed. EXPERIMENTAL DESIGN: We have previously demonstrated the use of somatostatin receptor imaging (SRI) in the diagnosis of recurrent and residual medulloblastomas. Here, we evaluated somatostatin receptor type 2 (sst(2)) expression using an antibody in an array of CNS tumors of childhood. Eight high-grade gliomas, 4 atypical teratoid/rhabdoid tumors, 7 supratentorial primitive neuroectodermal tumors (stPNET), 1 medulloepithelioma (ME), and 8 ependymomas were screened. Tumors positive in vitro were additionally analyzed in vivo using SRI. RESULTS: Abundant expression of somatostatin receptor type 2 in stPNET, a ME, and ependymomas warranted in vivo imaging of 7 stPNET, 1 rhabdomyosarcoma, 3 ependymomas, 1 ME, and 1 glioblastoma. Although SRI was positive in 6/7 stPNET, 1 rhabdomyosarcoma, and 1 ME, none of the ependymomas nor the glioblastoma could be imaged using SRI. In selected cases SRI was more sensitive in the detection of relapse than conventional imaging by magnetic resonance imaging and computed tomography. CONCLUSIONS: SRI should be considered in the evaluation of residual or recurrent embryonal CNS tumors, especially stPNET. The strengths of SRI lie in the differentiation of reactive tissue changes versus residual or recurrent tumor, the detection of small lesions, and possibly in the distinction of stPNET from gliomas.
Chromosomal imbalances detected by comparative genomic hybridisation in atypical teratoid/rhabdoid tumours.
INTRODUCTION: Atypical teratoid/rhabdoid tumours (AT/RT) are highly malignant embryonal tumours of the brain composed of rhabdoid cells. Inactivating mutations of the hSNF5/INI-1 gene located in the chromosomal region 22q11.2 are regarded as a crucial step in their molecular pathogenesis. Apart from monosomy or deletions of chromosome 22 not much data exists on additional chromosomal aberrations. METHODS: We investigated seven primary AT/RT by comparative genomic hybridisation (CGH) and found DNA copy number changes in each case. RESULTS: These consisted of loss of 22q in 7 out of 7 (100%) and loss of 19 in 3 out of 7 (43%) patients. In 4/7 AT/RT (57%), loss of chromosome 22q was the sole aberration whereas one patient showed additional losses of 16p, 17p and 20q. CONCLUSIONS: Our CGH data suggest that apart from monosomy 22 additional genetic pathways may seem feasible for a subset of AT/RT that is yet to be defined. Furthermore, this study also emphasises the potential practical value of loss of chromosome 22 as a diagnostic marker for AT/RT.
Glioblastoma cells release factors that disrupt blood-brain barrier features.
The blood-brain barrier (BBB), mediated by endothelial tight junctions, is defective in malignant gliomas such as glioblastoma, resulting in cerebral edema and contrast enhancement upon neuroradiological examination. The mechanisms underlying BBB breakdown are essentially unknown. Since non-neoplastic astrocytes are required to induce BBB features of cerebral endothelial cells, it is conceivable that malignant astrocytes have lost this ability due to dedifferentiation. Alternatively, glioma cells might actively degrade previously intact BBB tight junctions. To examine the latter hypothesis, we have employed a transepithelial electrical resistance breakdown assay using monolayers of the C7 subclone of Madin-Darby canine kidney (MDCK-C7) cells forming tight junctions similar to those of BBB endothelial cells. We found that glioblastoma primary cells co-cultured with the MDCK-C7 monolayer (without direct contact of the two cell types) resulted in marked breakdown of electrical resistance, whereas primary cultures derived from low-grade gliomas (fibrillary astrocytoma, oligoastrocytoma) showed delayed or no effects. These results suggest that malignant gliomas have acquired the ability to actively degrade tight junctions by secreting soluble factors, eventually leading to BBB disruption within invaded brain tissue.
Potential role of Pax-2 in retinal axon navigation through the chick optic nerve stalk and optic chiasm.
The degree of fiber decussation at the optic chiasm differs between species, ranging from complete crossing in lower vertebrates to highly complex patterns of intermingling of the fibers from the two eyes seen in mammals and birds. Understanding the genetic control of fiber guidance through the chiasm is therefore important to unravel the developmental mechanisms within the visual system. Here we first report on early stages of chiasm formation, with pioneering axons from the left eye consistently arriving earlier than their counterparts from the right eye. This initial left-right asymmetry is transient and no functional significance is assigned to it yet. Secondly, we examined formation of the chiasm in relation with the expression of the transcription factor Pax-2 along the ventral eye cup and optic nerve stalk. Finally, in order to examine causal involvement of Pax-2 in chiasm formation, the gene was overexpressed along the neuraxis and in the eye cup at embryonic stages preceding the exit of axons from the eye, and hence arrival of axons at the chiasm. When studied with neuroanatomical tracing, Pax-2 overexpression resulted in visibly anomalous decussation of axons at the chiasm. A likely consequence of this perturbation was erroneous arrival of axons at the tectum, as observed by anterograde staining from the retina. These data suggest that balanced expression of Pax-2 results in the correct formation of the chick chiasm at early stages by imposing accurate pathfinding within the optic stalk and the midline.
Amniotic air insufflation during minimally invasive fetoscopic fetal cardiac interventions is safe for the fetal brain in sheep.
BACKGROUND: Amniotic air insufflation during experimental fetoscopic fetal cardiac interventions greatly improves the visualization of intra-amniotic contents. The purpose of this study was to assess any histologically discernible effects from this approach on the fetal brain after short-term studies and long-term survival in sheep. METHODS: Thirty pregnant ewes between 80 and 110 days of gestation underwent amniotic air insufflation during various fetoscopic fetal cardiac interventions. After 18 short-term and 12 long-term studies, the brains of the operated fetuses and-if available-their unoperated siblings were examined for hemorrhage, embolism, infarctions, inflammatory changes, and abnormal cortical maturation. RESULTS: Amniotic air insufflation during minimally invasive fetoscopic fetal cardiac interventions did not result in any histologically discernible damage to the brain in short-term and long-term studies in any but 2 sibling sheep. In the 2 affected siblings, a small area of chronic cortical frontal lobe infarction was observed after long-term survival. CONCLUSIONS: Amniotic air insufflation during minimally invasive percutaneous fetoscopic fetal cardiac interventions is safe for the fetal brain and does not compromise maternal hemodynamics in sheep. These findings encourage further investigation of the role this technique might play during fetoscopic fetal cardiac interventions in humans.
Age- and sex-dependent development of adrenocortical hyperactivity in a transgenic mouse model of Alzheimer's disease.
In this study, we investigated mice of the TgCRND8 line, an APP transgenic mouse model of Alzheimer's disease (AD), with respect to behavioral, endocrinological, and neuropathological parameters. Our results show that transgenic and wild-type mice did not differ in their general health status, exploratory and anxiety related behavior as well as in the activity of their sympathetic-adrenomedullary system. Significant differences, however, were found regarding body weight, amyloid plaque formation, and the activity of the hypothalamic-pituitary-adrenocortical (HPA) axis. Continuous monitoring of glucocorticoid (GC) concentrations over a period of 120 days, utilizing a noninvasive technique to measure corticosterone metabolites in fecal samples, revealed that transgenic animals showed adrenocortical hyperactivity, starting very early in males (from day 30) and later in females (around day 90). It is hypothesized that these changes in the activity of the HPA axis are linked to amyloid-beta associated pathological alterations in the hippocampus, causing degenerations in the negative feedback regulation of the HPA axis leading to hypersecretion of GC. Thus, the development of adrenocortical hyperactivity might be a key-element in the understanding of AD.
Regulators of G-protein signaling 3 and 4 (RGS3, RGS4) are associated with glioma cell motility.
Diffuse brain invasion is a major reason for poor prognosis of glioma patients. The molecular mechanisms underlying infiltration are different from those of other cancer types. To detect genes associated with glioma invasion, highly migratory clones were selected from U373MG glioma cells and from primary glioblastoma cells, and the gene expression pattern of these "fast" cells was compared with that of the original ("slow") cells using oligonucleotide microarrays comprising 12,625 genes. A total of 28 genes were differently expressed in both primary and established cell populations, including 19 genes that were upregulated and 9 that were downregulated in fast cells. Most of these genes have not been linked to glioma invasion so far. Specifically, differentially expressed genes included those encoding extracellular matrix components (COL16A1, DPT), proteases (CATD, PRSS11), cytokines (MDK, IL8), transport proteins (SLC1A3, ATP10B), cytoskeleton constituents (ACTA2, ACTSG, NEFL), DNA repair enzymes (WRN, ADPRTL2), and G-protein signaling components (GNA12, RGS3, RGS4). RGS3 and RGS4, which are homologs of the Drosophila glia gene loco, were further functionally analyzed. U373MG glioma cell clones overexpressing RGS3 or RGS4 showed an increase of both adhesion and migration. These findings expand the spectrum of possible molecular pathways underlying the invasion of neoplastic astrocytes. Specifically, they suggest that RGS proteins and G-protein-mediated signal transduction are evolutionary conserved functional players.
Comparative genomic hybridization in central and peripheral nervous system tumors of childhood and adolescence.
Brain tumors amount to less than 2% of all malignant neoplasms. However, they account for approximately 20% of all childhood cancers and are the leading cause of cancer mortality among children. Recently, enormous progress has been achieved in the field of pediatric neuro-oncology regarding the classification of children's brain tumors, as well as the understanding of the genetic events involved in their pathogenesis; thus leading to an emerging role of molecular diagnostic approaches using novel tools. Comparative genomic hybridization (CGH) is a technique that has revolutionized cytogenetic knowledge in the past decade. It permits the detection of chromosomal copy number changes without the need for cell culturing and gives a global overview of chromosomal gains and losses throughout the whole genome of a tumor. A survey of CGH-related publications on central and peripheral nervous system tumors in the pediatric and adolescent population revealed 884 cases. The CNS tumor groups most frequently examined by CGH were embryonal tumors (268 cases/30.3%) and ependymomas (241/27.2%), followed by astrocytic (163/18.4%), peripheral nerve (73/8.2%), choroid plexus tumors (56/6.3%), and craniopharyngiomas (38/4.3%). The most common CNS tumor entities were medulloblastomas (238/26.9%), classic ependymomas (160/18.1%), anaplastic ependymomas (70/7.9%), pleomorphic xanthoastrocytomas (53/6.0%), and pilocytic astrocytomas (50/5.6%). This article provides a short review of the CGH technique and its pitfalls, summarizes the current CGH-related data on pediatric brain tumors and muses on the future of CGH.
Gene expression profiling in the intact and injured brain following environmental enrichment.
An enriched environment promotes structural changes in both injured and intact brain and improves behavioral performance. In 2 different experimental approaches, the effects of enriched surroundings were analyzed utilizing DNA microarrays. First, gene expression patterns of the sensorimotor cortex and the hippocampus of noninjured adult rats with enriched housing were compared with analogous regions of rats kept in standard cages. Second, circumscribed infarcts affecting the forelimb area of the sensorimotor cortex were induced, and gene expression patterns of the non-necrotic ipsilesional as well as the contralesional homotopic cortex of rats (postlesionally enriched housing versus standard) were analyzed. In the intact brain, the hippocampus, which had 43 upregulations and 15 downregulations showed more changes than the sensorimotor cortex, which had 13 upregulations and 4 downregulations, indicating a greater responsiveness of the hippocampus to environmental stimuli. In the injured brain, enrichment led ipsilesionally to 28 downregulations and 14 upregulations, while in the contralesional cortex, upregulations prevailed with 46 upregulations and 13 downregulations. The larger number of genes responsive to enrichment in the contralesional cortex (59 gene regulations) as compared to the analogous area (i.e. sensorimotor cortex) of the intact brain (17 gene regulations) likely reflects increased susceptibility for plastic changes due to injury. With the exception of the perilesional cortex, similar functional groups of genes were differentially regulated in different brain regions/paradigms, suggesting basically similar molecular cascades being involved in reorganizing the brain following external stimuli. Many of the genes detected here correspond to molecular pathways known to be involved in neuroplasticity, whereas others provide new and hitherto unrecognized entry points.
Angiomatous meningioma: a clinicopathologic study of 38 cases.
To characterize histopathological and clinical features of angiomatous meningioma, 38 cases of angiomatous meningioma, ie, meningiomas whose vascular component exceeded 50% of the total tumor area, are reported. In addition to histologic examinations, clinical characteristics as well as follow-up data were compiled. Angiomatous meningiomas constituted 2.1% of all meningiomas. Histologic signs of atypia or anaplasia were not observed in any tumor. The mean MIB-1/Ki67 proliferation index was 2.4%. Based on vessel size, two distinct histologic subtypes were identified, which differed in localization but not with regards to sex, age, presence of peritumoral edema, MIB-1/Ki67 proliferation index, or progesterone receptor status. In patients with gross tumor resection, no recurrences occurred. To conclude, angiomatous meningiomas share histologic and clinical features of benign meningiomas. Since all angiomatous meningiomas examined here were grade 1 tumors, the diagnosis of angiomatous meningioma may have prognostic implications. Therefore, the existence of this rare subgroup of meningioma appears justified.